Indian Agricultural

Research Institute, New Delhi

I.A.R.I. 6.

GIPN— 84—3 IARI/56~28-5-57— 1000.

McGRAW-HiLL PUBLICATIONS IN THE
BOTANICAL SCIENCES
EDMUND W, SINNOTT, Consulting Editor

PLANT MICROTECHNIQUE
 Selected Titles From

McGRAW-HILL PUBLICATIONS IN THE

BOTANICAL SCIENCES
Edmund W. Sinnott, Consulting Editor

Babcock and Clausen —Genetics —

Lutman Microbiology
—The Use of the Microscope
Belling —
Maximov Plant Physiology
Boysen Jensen—Growth Hormones Miller —Plant Physiology
Pool— Flowers and Flowering Plants
in Plants

Sass —^Elements of Botanical Micro-

Braun-Blanquet and Fuller and Con-
—
ard Plant Sociology
technique
—
Curtis The Translocation of Solutes
Seifriz—Protoplasm
in Plants
Sharp— Introduction to Cytology
Eames— Morphology of Vascular
Plants Sharp— Fundamentals Cytology
of
Barnes and MacDaniels—Plant Sinnott — Botany
Anatomy Sinnott and Dunn—Genetics
—^The Lower Fungi
Fitepatrick Smith —Cryptogamic Botany
Odutnann and Do^e—Comparative Vol. I, Algae and Fungi
Morphology of Fungi VoL II, Bryophytes and
Haupt—An Introduction to Botany Pteridophytes
Haupt —^Laboratory Manual of Ele- Smith —Fresh-water Algae of the
mentary Botany U. 8.

HiU—Economic Botany Swingle—Systematic Botan^*^

HiUf OverhoUSf and Popp— Botany Weaver— of Field
Johansen — Plant Microtechnique Crops
Loomis and Shull— Methods in Plant Weaver and Bruner— Root Develop-
Physiology ment of Vegetable Crops
Loomis and Shull— Experiments in Weaver and Clements — Plant Ecology
Plant Physiology Wodehouse — Pollen Grains

There are also the related series of McGraw-Hill Publications in the Zoologi-
cal Sciences, of which A. Franklin Shull is Consulting Editor, and in the
Agricultural Sciences, of which Leon J. Cole is Consulting Editor.
 PLANT
MICROTECHNIQUE

BY

DONALD ALEXANDER JOHANSEN

First Edition
Third Impression

McGRAW-HILL BOOK COMPANY, Inc.

NEW YORK AND LONDON

1940
 Copyright, 1940, by the
McGraw-Hill liooK Company, Inc.

PRINTED IN THE UNITED HTATEH OF AMERICA

All rights reserved. This hook, or

parts thereofy
may not be reproduced
in any form without permission of
the publishers.
 PREFACE
Four (ionsiderations prompted the preparation of the present text:
(1) the acute necessity for a sifting and synthesis of the hundreds of
methods and procedures that have been proposed during the past ten
years of rapid development in microtechnique; (2) the need for a manual
of modern botanical technique methods by botanists lacking special
training in that field but who must prepare slides as part of their work in
other fields; (3) to bring together the accumulated and mostly unpub-
lished results of some sixteen years of extensive personal experience in
collecting materials and preparing slides of plants from over the entire
range of the plant kingdom and (4) to provide an answer to the increas-
;

ingly numerous enquiries received by the author concerning the methods

he employs in routine and research work.
The main purpose of the book is to acquaint the user with the prin-
ciples and procedures of all phases of botanical microtechnique. The
specific aim is to enable elementary and advanced students, instructors
and r(\s(^arch investigators to prepare their own microscope slides of plant
materials.
The text is in no sense an encyclopedia of botanical microtechniques
UK'thods. Manyproposed fixing fluids, staining and dehydration
methods, etc., have been omitted because it was concluded after thorough
trial that they had not demonstrated their superiority over accepted

l)rocedures. With but few exceptions, every procedure cited has been
carefully tested by the author or by students under his immediate super-
vision. Methods which are questionable under certain circumstances are
so indicated. Such minor changes as might be required to adapt older
schedules to modern conditions have been incorporated. In general, if a
procedure or formula has not been credited to a specified person, the
author may be held responsible.
The text has been divided, for reasons of convenience and experience,
into two sections.
The first section describes the apparatus, reagents, dyes, etc., and
the general methods universally employed by botanical technicians.
Partiality in the selection of methods for presentation has been avoided,
but must be confessed that experience has engendered a preference for
it

the paraffin over the celloidin method. Each method, however, has been
described in sufficient detail to permit mastery in all its various phases.
Inessentials have been eliminated and procedures made as concise and
explicit as possible. The chapters on Whole-mount Methods, Smear
Methods, and Cytgl^^ Methods constitute features that have never
before been inr]url||||pyir '
viii PREFACE

The second section takes up all plant phyla in phylogenetic order and
gives as detailed directions or suggestions as are available for the treat-
ment of specdfic groups in e^ach phylum. The plants described are mainly
those occurring naturally in the United States and Canada. The chap-
terson the algal phyla are particularly comprehensive. At the beginning
of each chapter are given l)oth general and specific suggestions regarding
the collection, preservation, cultivation and manipulation of each phylum
as an entirety, following whi(di the orders and families are taken up in
succession and more d(‘tailed directions are cited for genera and species.
Innumerable procedures are described for the first time. Whenever
references to other sourcc^s are not mentioned, the techni(;al treatment
recommended for a particular plant or group of plants is the one which
has been found prefe'rahle from the author\s personal experience.
Refercviu^es to the literature are intended primarily for the guidance
of those who may wish to pursue a topic further, and secondarily to
indicate the sources of sta((*ments or for more detailed descriptions of
procedures. Only and books cited in the text, therefore, are
article's

include^d in the bibliography at the end of the second section.

A chapter on photomicrography has not been included because of the
large number of excellent texts on the subject now available. Paleo-
botanical methods are omitted since suitable material is not available
to most botanists and because the author has had insufficient personal
experitmc;e in this fi(^ld.

The figures have in general been selected to illustrate the results of a

given tectinical treatment on the material concerned. Except as other-
wise noted, all slides fr om which the photomicrographs have been made
were prepared by the author. The photographs are practically all the
work of a former student, Mr. John D. Poindexter.
For assistance
in solving difficult technique problems and for a
criticalreading of portions of the first section, the author is deeply
indebted to Miss lilnid A. Larson. Portions of the second section have
been read, and numerous suggestions made, by Dr. G. M. Smith. The
author must also acknowledge the advice and assistance, particularly in
connection with the (commercial aspects of microtechnique, rendered by
Dr. George H. Conant over a period of many years.
Botanical microtechnique a science in which such rapid progress
is

is being made at present that for a person, without access

it is difficult

to a large botanical lil)rary which receives all current journals, to keep

abreast with proposed new f)rocedures and changes in older methods.
The author, therefore, would appreciate it if users of the text would favor
him with reprints of their articles, or would otherwise keep him informed
concerning new or improved methods.
Stanford University, Calif., Donald A. Johansen.
May, 1939.
viii PREFACE

The second section takes up all plant phyla in phylogenetic order and
gives as detailed directions or suggestions as are available for the treat-
ment of specdfic groups in e^ach phylum. The plants described are mainly
those occurring naturally in the United States and Canada. The chap-
terson the algal phyla are particularly comprehensive. At the beginning
of each chapter are given l)oth general and specific suggestions regarding
the collection, preservation, cultivation and manipulation of each phylum
as an entirety, following whi(di the orders and families are taken up in
succession and more d(‘tailed directions are cited for genera and species.
Innumerable procedures are described for the first time. Whenever
references to other sourcc^s are not mentioned, the techni(;al treatment
recommended for a particular plant or group of plants is the one which
has been found prefe'rahle from the author\s personal experience.
Refercviu^es to the literature are intended primarily for the guidance
of those who may wish to pursue a topic further, and secondarily to
indicate the sources of sta((*ments or for more detailed descriptions of
procedures. Only and books cited in the text, therefore, are
article's

include^d in the bibliography at the end of the second section.

A chapter on photomicrography has not been included because of the
large number of excellent texts on the subject now available. Paleo-
botanical methods are omitted since suitable material is not available
to most botanists and because the author has had insufficient personal
experitmc;e in this fi(^ld.

The figures have in general been selected to illustrate the results of a

given tectinical treatment on the material concerned. Except as other-
wise noted, all slides fr om which the photomicrographs have been made
were prepared by the author. The photographs are practically all the
work of a former student, Mr. John D. Poindexter.
For assistance
in solving difficult technique problems and for a
criticalreading of portions of the first section, the author is deeply
indebted to Miss lilnid A. Larson. Portions of the second section have
been read, and numerous suggestions made, by Dr. G. M. Smith. The
author must also acknowledge the advice and assistance, particularly in
connection with the (commercial aspects of microtechnique, rendered by
Dr. George H. Conant over a period of many years.
Botanical microtechnique a science in which such rapid progress
is

is being made at present that for a person, without access

it is difficult

to a large botanical lil)rary which receives all current journals, to keep

abreast with proposed new f)rocedures and changes in older methods.
The author, therefore, would appreciate it if users of the text would favor
him with reprints of their articles, or would otherwise keep him informed
concerning new or improved methods.
Stanford University, Calif., Donald A. Johansen.
May, 1939.
 CONTENTS
Pa(;k
PllKFACE vii

SECTION I
GKNERAI. METHODS
(CHAPTER T

Introduction 3

(Miapit:h II

Lahoratory Rules G

UHAPTEK HI
Apparatus 8

CHAPTER TV
Reagents 15

( JIAPTER. V
Killing and Fixation 27

(CHAPTER VI
Stains 49

cmAPTER VII
Staining Procedures G5

CHAPTER VIII
Special Methods 95

CHAPTER IX
Whole-mount Methods 110

(TIAPTER X
The Glycerin Method 119

CHAPTER XI
Celloidin Methods 121

(CHAPTER XII
Paraffin Methods 126
ix
X CONTENTS
Paqb
CHAPTER XIII
Smear Methodb 155

CHAPTER XIV
Cytologic AL Methods 170

CHAPTER XV
Microchemical Methods 182

(UIAPTER XVI
Sources of Materials 204

SECTION II

SPECIAL METHODS FOR THE VARIOUS PHYLA

CHAPTER XVII
SCHIZOPHYTA 211

C^HAPTER XVIII
Chlorophyta 227

CHAPTER XIX
Euglenophyta 252

CHAPTER XX
Pyrrophyta 255

CHAPTER XXI
Chrysophyta 256

CHAPTER XXII
Phaeophyta 262

CHAPTER XXIII
Cyanophyta 283

CHAPTER XXIV
Rhodophyta 291

CHAPTER XXV
Myxothallophyta 312

CHAPTER XXVI
Mycophyta (Eumycetae) 318

CHAPTER XXVII
Bryophyta 360
 CONTENTS xi
Page
CHAPTER XXVIII
Pteridophyta 381

CHAPTER XXIX
Cycadophyta 414

CHAPTER XXX
CONIFEROPHYTA 420

CHAPTER XXXI
Anthophyta 448

Bibliography 493

Index 503
 SECTION I

CENERAL METOODS
 CHAPTER I

INTRODUCTION
Immense have been made during the past decade in all aspects
strides
of microscopi(*al technique, and even greater progress has been made in
revealing the life histories of plants and their phylogenetic relationships.
There has thus been rendered imperative an attempt to correlate the
inffnmation provided by these two sources and to digest it in such a
manner that it may be of the optimum service to workers in all the
various botanical sciences. The present text constitutes an attempt at
such a correlation.
Modern synthetic chemistry has made available innumerable reagents
with which both botanical and zoological technicians have recently begun
to experiment. Many of th(‘ attempts turned out to have been founded
upon overoptirnism : there seems to be currently prevalent a delusion
that there exists such a thing as a single foolproof cure-all for every one
of the difficulties with which technicians are continually confronted.
Dioxan may be cited as an example: expectations at first were very
high, but this reagent soon turned out to have only a limited, though
valuable, application. Experiments with other reagents are continuing,
and every encouragement should be extended to such efforts. However,
statements with regard to the applicability of a new reagent should
always be made with due caution: it would be well continually to bear
in mind that the innately complex structural differences among plants
will always prevent any one reagent or method from being of universal
application.
Although the older methods and ideas in vogue among botanical
technicians often verged on superstition, there are nevertheless many
sound conceptions which have been evolved and have survived all
attempts at obliteration. The more prominent of the older superstitions
were that absolute ethyl alcohol must always be used to ensure complete
dehydration and that a clearing agent must be used to render the tissues
perfectly transparent before they were in a fit condition to be infiltrated
with paraffin. On the other hand, methods immediately became more
refined when botanical technicians got rid of a procedure, apparently
borrowed from clinical laboratories, in which time is the essence of any
method, and began to use a graduated series of reagents for both dehydra-
tion and infiltration. None of the newer reagents provided by modern
3
4 GENERAL METHODS

chemistry iias demonstrated its ability to work successfully at a given

single unitary concentration, and it is extremely doubtful if any ever will,
for the nature of plants cannot be changed any more than can that of
human beings.
Eachspecaes of plants must be treated as individualistically as are
specimens of Homo sapiens. The days when both plants and people
were treated en masse have passed. To deal suca^essfully with a given
species, the technicaaii must know something of the life history, the
manner through which each stage is passed, the physical structure and
chemical properties of the various tissues composing the organism and
each of its parts, and the probable reactions of the latter to the reagents
which proposed to use upon them. To this must be combined an
it is

essential knowledge of the physical and chemical characteristics of each

reagent and of dyes and stains, their interactions with one another, and
their general effects upon plant tissues.
The amazing manual skill of the older technicians has been supplanted
by procedures whi(;h are partly physical but mainly chemical. In place
of learning how to wield a razor and how to keep it sharp, tlu^ present-day
technician must learn how to handle chemic^als. Probal^ly no other
profession so abounds with ^M-ricks^’ peculiar to it, as does microtechnique.
A technician becjomes successful only to the extent that he masters these
strategems, in addition to inventing a few more of his own. It is not
possible to describe all the artifi(*es. After a thorough mastery of
methods, one instinctively learns about them and recognizes the moment
when it becomes ncHjessary to apply one of them. Some small or rela-
tively insignificant matter very frecpiently determines the success or
failure of a procedure.Accidents and near accidents 0(u*ur all the time:
the mark avoid them and to plan
of the technician is his ability to
detours around obstacles placed in the path by the plants themselves.
Experience showed that there was more than one way of killing a cat;
likewise, there arc different methods with obdurate plant
of dealing
tissues. The only trouble is that the other and correct method
is not

always immediately plain; more or less experimentation may be required,

but this occasionally gives one the chance to devise a new method which
would be of great service to other technicians. As a matter of fact,
very many methods have resulted because some one could not get the
customary procedure to work as it supposedly should.
The beginning technician and those who are proceeding for the first
time should always follow directions exactly as given. It is highly
presumptuous to imagine that one knows more about the procedure, even
before attempting to use it, than did the originator. One is not at
liberty to proceed differently until and unless one is convinced that the
nature of the material demands a digression. The formulae of most
 INTRODUCTION 5

reagents have been determined by carefully controlled experiments;

the scdiedules for all the principal staining methods generally represent
(he combined experience of many competent
workers over long periods
and with all under a variety of conditions. The
sorts of plant tissues
iron-acetocarmin method, for example, as dtwised by Belling, has been
standardized in its basic principles, and one should first learn to obtain
exa(;tly the same results as do other workers before oik^ starts in to make
modifications. Others arc prevented from understanding and inter-
preting one’s results if undesirable or unnecessary modifications are made
in familiar procedures.
A word of encouragement is in order. Many occasions will arise when
one experiences a feeling of helplessness when confronted with the task
of dealing with an unfamiliar plant. Even experienced technicians often
find themselves in such situations. Their procedure is to employ a killing
and fixing fluid with whose reactions they are familiar, then to use
dehydration, infiltration and staining methods with which they have had
successful previous experience, and finally to observe the results in the
completed preparation. A littk' study will cpiickly rc'veal where improve-
ments should be made when the rest of the material is to be worked up.
This is only a sort of trial-and-error method, of course, but if each st(^p
is carried out after due consideration, the result is generally fairly satis-
factory, and only minor changes need to be made afterward. The
beginner should follow the same procedure. If the plant concerned is
not specifically described in the text, then the methods recommended for
the most closely reflated forms should l)e used for guidanc*e.
Persons who happen to be afflicted with color blindness are under
a handicap in microteciiniquc since the ability to judge variations in

stain reactions is one of the prerequisites for successful work.

 CHAPTER II

LABORATORY RULES
These rules are the self-imposed regulations of all competent techni-
cians; by following them, the beginning technician will avoid considerable
trouble.
1. First and foremost: Keep everything clean.
2. Know what you are doing. If in doubt, stop at once, and orient
yourself before proceeding further. Do not try to rush things: there is
no more certain way of courting grief. There will, of course, be frequent
occasions for becoming exasperated with the proverbial innate perversity
of inanimate things, but one should never permit such irritations to
(ixhaust his i^atience.
3. Keep your desk or table in order. Have a definite place for every
object. Label all (Lemicals, reagents, and solutions; do not trust your
memory or senses to recognize them.
4. Use only clean glass vessels in preparing reagents, except in such
rare cases as when special containers might be indicated. Always clean
the glassware while still damp, or place it temporarily in a dishpan with
running water.
5. Keep your hands clean and dry, especially when mounting paraffin

ribbons on slides. Be careful not to leave traces of poisonous substances

{e.g.^ mercuric chloride and phenol) on your hands or clothes. It would
be wise to wear a rubber apron in order to protect the clothing.
6. Use acids with great caution. The fumes of most acids are
extremely irritating. Always pour acids into water, never water into
acids. If heat is evolved, add small quantities at a time and cool between
additions.
7. Keep containers holding anhydrous solutions tightly corked or
stoppered. Do
not use the dregs, as of absolute alcohol, for completing
dehydration, for such remnants are no longer anhydrous. Use vaseline
or petrolatum on the covers and edges of all stenders and coplins contain-
ing volatile or hygroscopic fluids, if they are to be leftstanding for more
than a day or two.
8. Keep card or other simplified records, as accurately as possible, of
all materials. Record on each card all data concerning the manipulation
of the material. Do not leave anything of consequence to memory.
9. In weighing solid chemicals, take care to avoid contamination.

Protect the pans of scales or balances with paper.

6
 LABORATORY RULES 7

10. Do not throw solids or celloidin solutions into the sink. Flush
the sink with water when pouring acids or stains into it.

11. Do not use pipettes indiscriminately. Have one for each type of
alcohol, one for xylol, one for stains, another for acids, etc.
12. Take extreme care to avoid contamination of osmic acid solutions:
they are very expensive. Never breathe the fumes of osmic acid, nor
permit them to come near the eyes.
VfS. Keep balsam containers out of the light. The balsam might
become acid and is then ruinous to stains.
14. In collecting material, remember that changes in the colls 0 (n;ur
rapidly after removal from the plant, from water, etc. Shorten the time
elapsing between removal and placing into the killing fluid as much as
possible. It should not exceed more than a few seconds. Avoid crush-
ing. Remove all superfluous tissue and cut into as small pieces as
joracticable.
15. Before starting to kill and fix tissues, be sure you have selected
what you consider to be tlu^ proper solution. Better still, use several
different fluids at the first trial, and lat(‘r select for future collocations the
one giving the best fixation. Make up the solutions and have ready for
use before starting work on tlu^ plants.
16. Study staining schedules carefully before starting to use one with
which you are unfamiliar. Make certain that all the reagcaits called for
are at hand.
17. Budget your time both a day and several days ahead. Plan
future operations far enough ahead to ensure that the most may be made
of the available time. Experienced technicians frequently have as many
as a dozen operations proceeding simultaneously.
18. Remember not to leave tissues too long in killing solutions, in
the dehydrating and in the paraffin oven.
fluids,

19. Examine all your preparations critic^ally and also, whenever

possible, obtain the opinions of competent specialists. Never be satisfied
with mediocre results. Judge on the basis of killing and fixing, infiltra-
tion, microtoming, staining, and mounting. Be honest in your judg-
ment, even if you feel that poor results were the fault of the material
or the schedule followed. You will be ^ better technician if you blame
yourself first and your materials and schedulers afterward.
20. Finally, under no circumstancers become discouraged if your first
efforts culminate miserably, but try again. Seek for the cause or causes
of the failure. As Bolles-Lee, one of the greatest of zoological techni-
cians, has truly said, even the most experienced technicians often turn
out perfectly atrocious preparations on their preliminary trials.
 CHAPTER III

APPARATUS

It is presumed that a person (;omm(mcing the study of plant micro-

technique intends to familiarize himself, to some extent at least, with
the majority of the different methods. On this basis, each student
should provide himself with the supplies noted in the following summa-
rized list. Some of the items will be discussed at greater length.
One or more boxes of slides of the standard size, 25 X 75 mm. (1X3 inches).
Coverslips: A medium thickness, generally known as No. 1, is preferable. As a
start, a half ounce each of the following sizes will serve: 22 mm. squares, 18 or
22 mm. circles, and 22 X 40 mm. rectangles.
100-cc. graduate (also 50-cc. and 500-cc. sizes if desired).
A dozen ordinary pipettes.
Giant pipet te.
Large all-steel scalpel, or sharp pocketknife.
Scalpel with ebony handle and long, thin, straight blade; intended primarily for
trimming paraffin Idocks for microtoming.
-^Several needles, in holders.
2 or more camers-hair brushes, assorted sizes.
. 2 pair of scissors, small and large.
. Forceps: a strong one for handling slides, and a narrow-pointed one for use with
coverslips.
Waterproof India ink and fine-pointed pen.
12 or inore, solid (Syracuse) watch glasses.
10 or rpore Coplin jars.
8 or more Stender dishes.
Several flat staining dishes.
8 or more bottles of 100-cc. capacity.
Several bottles of 500-cc. capacity.
Alcohol lamp: (Use only (dean ethyl alcohol; traces of xylol, etc., will cause cover-
slips to become smudged.)
Balsam bottle, with glass-rod dropper.
6 or more square Petri dishes (for smears).
Suction pump for water faucet.
Grease or china-marking pencil (red).

Microscopes. —A microscope is,of course, necessary, but all that

is required in microtechnique is an inexpensive one with a low-power
objective and a single ocular. A microscope intended for research
purposes emphatically should not be employed, as the chances are that
it will soon be somewhat damaged by chemicals and minor accidents.

The stage of the microscope should be protected by a glass plate; a large

8
 APPARATUS 9

lantern-slide cover serves the purpose admirably. It should not be

fastened to the stage, as it is frecpiently easier to move the glass than a
wet slide placed upon the glass. Also one might wish to examine a
completed preparation without getting it wet, for which purpose it is
merely necessary to withdraw the glass plate.
If the student has not had previous experience in tlu' manipulation
of the microscope, lie should first inform himself fully upon the sub-
ject. The optical companies usually provide small handbooks to
a(!(;ompany their own instruments. One can pursue with great profit
Simon H. Gage’s ‘‘The Microscope” (Comstock Publishing Company,
Ithaca, N. Y., Ifith (^d., 1932).
—
Microtomes. Although not n^quirc'd at the beginning, a microtome
soon becomes an absolute iK'cessity. The modern microtomes are, on
th(^ whole, very efficient precision machines. Iln^y are of two general
types, the sliding and the rotary. Techni(‘ians are not in agreement
as to which type' is the liest; the choice of either type appanmtly depends
upon the possession of “mechaiii(;al ability.” Those who have a feeling
for mechanical skill will do their best work with the sliding microtome;
beginners, and evem those with little or no aptitude in the use of machines,
should place their main reliance' uj)on the rotary mi(*rotome.
Th(^ rotary microtome should be used only for sectioning material
embedded in paraffin; in all other methods requiring the cutting of
sections, a sliding microtome is indicated.
Many students find it desirable to begin with a simple form of the
sliding microtome, and to progress to a larger and somewhat more
(‘omplicated as well as more accurate machine as skill increases.
There are two tyj)es of rotary microtomes. In the older type, as
exemplified by the Minot microtome manufactured by the Bausch and
Lomb OpticalCompany, the forward movement is directly related to
the up-and-down movement. For optimum results, the two movements
should be separated, but satisfactory sections can be cut on the Minot
models if due care is exercised. In the new Spencer No. 820 microtome,
the horizontal and vertical movements are wholly independent, giving
greater stability and precision. The universal joint clamp for holding
the object in this microtome, though of simple and rigid construction, is a
beginners to manage.
little difficult for

—
Knives. Microtome knives are easily available, not too expensive,
and those intended for rotary machines are interchangeable. Each
student will find it more convenient to purchase his own knife, rather
than to depend upon a laboratory knife which is liable to have been
handled by careless students. Some sliding microtome knives have
special two-pronged handles for clamping into the sliding block. The
personal possession of such a knife is at the student’s option; if he pre-
10 GENERAL METHODS

fers the sliding to the rotary microtome for cutting paraffin sections, it
would be better to secure a knife intended for use in the former. Knives
intended for rotary machines may also be used in some types of sliding
microtomes.
The student should learn how to sharpen his own microtome knives,
as a sharp knife is absolutely essential if perfect sections are to be cut.
Microtome knives should not be trusted to the ^^key maker and knife
grinder^’ in a dingy basement shop, nor to those who sharpen knives for
surgeons. Most such persons will only ruin the knife. If the edge has
a few bad nicks, it is better to send the knife to the manufacturer for
resharpening. Two hones should be available: a yellow Belgian hone
for the preliminary stages and a fine carborundum hone for the finishing,
but many people use only the Belgian hone.
Place the back on the knife and screw in the handle. Flood the
Belgian hone with (‘lean water and place on the table so that one end is
toward you. Place the knife at the far end of the hone, as close to the
handle as possible and with the edge of the knife pointed in your direction,
then draw the knife diagonally toward you in such a manner that the
end opposite the handle reaches the near end of the hone. Do not exert
any pressure on the knife; its own weight is all that should be allowed.
Next turn the knife over so that it points toward the far end of the hone
and with the handle end again close to the hone. Move diagonally
toward the far end of the hone, then turn over and repeat the entire
process for al)Out 10 minutes. To detect nicks in the knife, draw the
nail of the thumb cautiously over the edge (turning the finger over far
enough to avoid (nitting the ball), holding the knife pointed away from
you and moving the nail in the same direction; the nail will be momen-
tarily stopped by even tiny nicks. If any nicks are encountered, con-
tinue honing until they have disappeared.
There are a number of automatic sharpeners on the market. The
necessary skill required for manipulating these sharpeners is much less
than that required for stone honing, and is quickly acquired if the
manufacturer's directions are followed.
In stropping, use a stiff leather stn^p, or one mounted on a wooden
block, not one which is so soft as to bend the edge of the knife. The
honing back should also be placed on the knife when stropping the final
edge. Knives are stropped in the reverse of the directions employed
during honing (otherwise, of course, the strop will be cut), and move-
ments must also be diagonal. Never strike the knife against the strop
with the grand flourish that barbers affect.
Any particular portion of the knife-edge should be used only once,
and not too large a number of sections should be cut at this point. As
soon as the edge shows signs of becoming dull, which can readily be told
 APPARATUS 11

when the sections begin to become rough and torn, move to a new portion,
or i!»emove, clean, and resharpen or Always keep the back (the
strop).

paraffin or object side) of the knife clean. Learn to do this with the ball
of the forefinger or little finger, quickly, automatically and frequently.
—
Safety Razor Blade Itolders. The optical companies advertise
clamps designed to hold a blade of the Gillette safety razor type, but
none of these holders has any great merit. Tlu^ new Craig-Wilson
holder is superior to all others. This holder is so constructed that ice
water may be run through it when very thin sections are required;
or if sections 2b^x thick or thicker are wantc^d, lukewarm water can be run
through and long ribbons obtained. When using this clamp, it must be
kept in mind that the blade is not straight, as is the microtome knife, but
is bent into a curve. The holder should not be inclined toward the
paraffin block as far as a knife is inclined, but should be as nearly vertical
as possible without permitting the paraffin block to scrape against any
part of the holder.
—
Safety Razor Blades. There is much difference of opinion as to
which brand of safety-razor blades is best for use in microtoming. As
all who use safety blade's for shaving know only too w^ell, there is a

very great lack of uniformity in all brands on the market. Manu-

facturers^ statements are so unre ‘liable as to be meaningless. The only
thing one (;an do is to get any brand of the Gillette typ)e, but to avoid
those which are so thin as to b(^ flimsy, and try one brand after another
until a suitable one is encountered. The writer prefers Dublekeen
blades. It is advisable to purchase a good quality Twinplex stropper and
strop the blades occasionally, first being sure to remove any adhering
paraffin by wiping the blade with xylol.
Paraffin Embedding Ovens. —
It is now rc'latively easy to procure a
satisfactory paraffin bath or oven. The various forms range all the way
from an electric globe (carbon filament) suspended over a tumbler, in
which the paraffin is only partially melted, to the large Lillie water bath
with as many as 16 separate compartments. In localities where the
daily and yearly ranges in temperature are slight, a simple form of oven is
preferable, but in other places an oven witii double, insulated walls w ill
be necessary. The writer has used for many years a simple Thelco
Electric Oven (manufactured by the Thermo Ele(dTic Instrument Com-
pany, Newark, N. J.). In large laboratories, where the number of
students is considerable, one or more ovens of greater capacity are neces-
sary. Excellent vacuum ovens have recently appeared on the market.
Staining Dishes. —Two types of staining dishes are available for
staining sections mounted on slides: the Stender dish and the Coplin jar
(familiarly known as ^‘stenders^’ and “coplins’^). The latter is difficult
to clean because of its construction but has the advantage over the
12 GENERAL METHODS

Stender dish in that the slides are always in position. Some technicians
prefer one type, some the other. As a general rule, coplins are used to
hold stains and for the series of alcohols used in staining processes while
stenders are used for washes, mordants, etc. In the more precise work,
only a few slides are handled at a time and this is most conveniently done
in coplins. If a larg(i number of slides are to be stained by a standardized
procedure and if the slides do not need to be handled individually until
the process has been completed, small battery jars, each with a capacity
of about 1 liter of solution, may be used. With such a container, some
form of slide holder is necessary. One or two have rec^ently appeared on
the market, but it is just as easy to devise a suitable holder. One of the
author\s students removed the wire coil from inside the roller of an old-
style window shade and cut it into convenient lengths. Or stainless steel
wire of the proper gage (about 0.06 inch in diameter) can be wound
around a broom handle; the coil should be about inch in diameter.
The slides are insertcHi between the coils. A mi^tallic rack holding 50
slides has been us(m1 for years by the writer; it should not be used in any
—
solutions other than plain water, alcohol, or xylol -never in staining
solutions and mordants.
—
Slides. Many brands of slides now on the market are worthless.
Slides of American manufacture an^ usually more dependaVjle than those
imported from other countries. It is well known that slides of European,
and particularly of German, manufacture sold in the United States are
generally those remaining over after th(^ perfect ones have been picked out
for home consumption. Dealers who furnish high-quality slides are
listed in the chapter on Sources of Materials.
The standard thickness of slides is exactly 1 mm. this is a rather thin
;

slide more suitable for critical cytological work than for general purposes.
Some brands appear slightly greenish when viewed edgewise, but there is
no particular virtue residing in the claim that slides should appear per-
fectly white. Greenish slides arc less prone to corrosion.
—
Depression Slides. Depression slides (usually called culture slides
in most catalogues), in which to mount bulky objects permanently, are
expensive and for some purposes are unsatisfactory because the cavities
are of a standard depth and width. The use of the dental engine and
suitable abrasives mounted on mandrels has been suggested for making
cavities of the exact dimensions required. The dental engine has its
drawbacks, but there are now on the market a number of small electric
grinders or hand pieces which arc more easily manipulated and which
can be utilized to grind cavities of any dimensions, provided mounted
abrasive whe(‘ls of the proper shapes are used. The Handee grinder
(manufactured by the Chicago Wheel and Manufacturing Company, 1101
West Monroe Street, Chicago, 111.) has been used by the writer with very
satisfactory results. Use the green mounted wheels, which contain
 :

APPARATUS 13

silicon or carbide abrasives, and always lubricate the site with a saturated
solution of gum camphor in oil of turpentine. There is always consider-
able wheel wear when grinding glass; consequently the wheels should not
be pressed too hard into the glass. Fairly thick slides should be selected.
The ground portion has a very rough appearance when the grinding is
completed, but after the cavity is filled with balsam the rough edges
almost completely disappear and do not interferes with observations under
the microscope.
Coverslips. — The usual run of (soverslips (or cover-glasses) are no
better than most slides. It is an anomaly that some of the most exten-
sively advertised brands are, in the opinion of numerous compe^tent critics,
the poorest of all in quality. Coverslips of Japanese manufacture have,
in the past, been of fairly uniform quality, relatively chea]), and satisfac-
tory for general purpose's. Coverslips that are of strictly Ameri(‘an
manufacture are somc'what high in pri(*e but are usually to be preferred.
Certain manufacturers who claim that their covc'rslips are of American
manufacture are nevertheless practicing a deception: in order to take^
advantage of lower import duties, the glass is imported in sheets and cut
up in this country. Coverslips cut. from this kind of glass are usually
brittle and an^ difficult to clean, not to mt'iitiori theii* tendency to
corrode,
Coverslips come in four widely variabk^ groups of thicknesses, desig-
nated by the Nos. 0, 1, 2, and 3. Numb('r 1 which should
coverslips,
be about 0.17 mm. in thickness, arc Squares and circles
the standard.
come in the following standard siz(‘s: 15, 18, 22, and 25 mm.; rectangles
are either 22 or 24 mm. widi' and 30, 40, 50, 60, or 70 mm. in length.
Covcrslif)S of other dimensions may be purchasc'd on spe(‘ial ordei*.
Unless the coverslips arc obtained from a thoroughly reliable firm, th(‘
designated thickness should never be tak(m for granted but it should be
checked by measuring with a cover-glavss gauge. If an immersion lens is
to be used on the completed preparation, the coverslips should be as large
as possible and No. 0 is the preferable thickness. Round coverslips an^
indicated only with materials such as are run up by whole-mount meth-
ods, freehand sections, when the mounts are to be sealed on a turntable,
and in similar cases. The large coverslips and slides used by zoologists
and pathologists for very large sections are so rarely employed in botanical
technique that they need not be discussed here.
—
Cleaning Slides and Glassware. One should never believe state-
ments of the manufacturers that slides and coverslips are ready for use
but should always clean them, preferably in an acid cleaning fluid. Slides
on which paraffin sections are to be mounted or on which smears are to bo
made should always be chemically clean, otherwise the sections are certain
to be washed off. The fluid which is most generally used for cleaning
glassware consists of
 14 GENERAL METHODS
Potassium diohromate 20 g.

Water 100 cc.

Concoiitrat(Hl sulphuric acid 100 cc.

Dissolve the dirhromate in the water, add the acid cautiously in small
amounts, and cool the mixture between each addition of acid. The
mixturci must be stored in and may be used repeatedly
glass containers
until it becomes too dark. Immerse the glassware for a few hours, and
wash thoroughly in running water, finally rinsing with distilled water.
Another mixture^ consists of 1 part of concentrated nitric; acid and 4 parts
of (concentrated hydrochloric; acid. It does not keep so long, and the;
fumccs from the* acids may be annoying. Immerse glassware for several
hours, then wash in running water.
If it is iutc'nded that photomicrographs are later to be made, the slides

and, (coverslips should never have b(*en uschI before. If the slides have

/ previously had sections attacchcnl to thc‘m with adhccsive, the images of the;
(earlier sections are rat lier ccertain to ap{)ear on the negative, particularly

when the c'X])osure is over a long period.

Slide Containers. — Containers for finishced preparations are available;
in sev(‘ral difftaent ty])e‘s. Cheap, flimsy (cardboard box('S should b(‘
avoided. Wooden rack boxes with slots for 25 slides are more commonly
used than any other type. They are convenient, permanent, and easily
handled. There are two tyf)es: one with the cover fitting over shoulders
on the base portion, and the other with the cover snapping into a deprecs-
sion cut. in the; inner side of the* Ijase. The latter type is used by com-
mercial concerns since* the; sliders are more easily held in position for
shipping, t)ut the former type is otherwise precferable. Similar boxecs
holding 12 slides c'ach are also available, as arc hinged wooden or com-
bination wood-cardboard boxes with a capaecity of 100 slides each. All
such boxes should be stood on end so that the slides lie flat.
Slides should alwaysbe stored flat, particularly if they are whole-
mount whole mounts are stored on end or on one side,
preparations. If
the material tends to flow toward tlie lower end, or even from under the
(coverslip. If the balsam is not thoroughly solidified under the coverslip,
air pockets may also appear. Conseepiently, certain types of slide
holders, which carry the slides upright, cannot be recommended. These
holders, which may be large affairs constructed of metal, also generally
lack easy portability and are difficult to keep in order. There is no
really good (;ontainer on the market for large numbers of slides.
—
Giant Pipette. This useful tool is made by purchasing a rubber bulb
about 8.5 cm. in length at a drugstore and attaching to it a piece of glass
tubing of suitable diameter and about 25 cm. in length.
A giant pipette may be used for removing unicelhilar and colonial
algae from pools, making changes of dehydrating fluids on materials to be
mounted entire, and for countless other purposes.
 CHAPTER IV
REAGENTS^
GENERAL REAGENTS
The alcohols have long been the most- important of the gv*nL.'al

reagents used in microtechnique. No other reagents liave su(*h diversi-

fied uses. There are a large number of different kinds of alcohols, ))ut
only ethyl alcohol can be classific^d as a general reagent. Formalin (‘oines
next as the most widely used reagcait.
Ethyl Alcohol .
—
Whenever the term “alcohol’^ is used, whether in
the present text or elsewhere, ethyl alcohol is invariably nu^ant: if any
other kind of alcohol is to be used, the exact kind is, and should always
be, mentioTK'd.
Ihire ethyl al(*ohol iscommonly called or ‘^absolute alcohol,^’
and in th(‘ trad(‘ it is known as 200-proof
^
^ (abbrc^viated, rathcM* con-
fuvsingly, to 200%). sometimes rather expensive' and not- always
It is
easily obtainable'. It may be ru'ce'ssary for oik' to undc'rtake
oce^asionally
the U'dious process of making his own, but in siu'h instances oik' must b(‘
careful not to violate government regulations. The most satisfactory
method is distillation over calcium chloride. Shake up tlu' lower alcohol
with the chloride and allow to stand for vsoine time. Jhit some of the'
mixture, toge'ther with some fn'sh chlorides, in a glass still. H(‘at with
an electric; hot plate and distill over the alcohol slowly. The wat('r will
iH'inain in (‘ombination with the chloride. A second method involvt'S the
use of cupric sulphate. He'at some of the crystals until only a whit-('
powder remains. Add this anhydrous sulphate to a quant ity of ordinary
95% ethyl alcohol. The water is immediately absorbed by the sulphate,
which n'turns to the original l)lue color. Continue adding (‘alcined
sulphate until it no longer turns blue. Finally filter (pnckly into a dry,
tightly stoppered bottle. Some workers keep a liltU' bag of the anhy-
drous sulphate in the bottle to insure its freedom from water.
A very sensitive test for the detection of traces of water in absolute
alcohol is to add a few drops of the suspected fluid to a solution of liquid
paraffin in anhydrous chloroform: if any moisture is present, it causes an
immediate turbidity.
Ethyl alcohol is more commonly used at 95% or 96% strength, which
is few special purposes. Most botanical
sufficiently strong for all except a
te(;hnicians have overemphasized the use of absolute' ale*ohol. Some
claim that tissues and slides must be passed through several changes of
absolute alcohol to ensure a complete dehydration. The writer's experi-
15
16 GENERAL METHODS

ence, as well as that of many others, proves that this expensive process is

unnecessary. In the tertiary butyl alcohol dehydration method, absolute

alcohol is necessary at only one brief stage; a great saving of time and
money is thus secured in comparison to the expensive and tedious older
xylol method. In staining slides, if a clove oil counterstain is to be used,
95% alcohol dehydrates sufficiently, but 100% alcohol must be added to
the first (;love oil wash to remove traces of moisture that might be carried
over.
The stock dilutions of alcohol are 15, 35, 50, 70, and 85%, although, of
(course, the scries may be as extended as desired. These dilutions are
made from 95% ethyl alcohol (never 100%) and distilled water. The
quickest method of obtaining a given percentage of alcohol is to fill a
100-cc. graduate to the percentage require^d (as expressed in cubic centi-
meters) with the alcohol to be diluted, and then fill up to the percentage
of the latter with distilled water. For example, if 35% alcohol is to be
made from 85%, fill the graduate to the 35-cc. mark with 85% alcohol,
then the rest of the way up to the 85-cc. mark with water. In the average
laboratory it would be better to multiply the amounts just given by five,
and to use 500-c(;. bottles as containers.
—
Methyl Alcohol. This poisonous fluid is also known as ^^wood
alcohol.^’ It is cheaper than ethyl alcohol but is of about 90% strength.
It is scarcely ever specified in botanical microtechnique but may be used
with caution if ethyl alcohol is not available. The ‘^methylated spirits”
frequently mentioned in English papers is ethyl alcohol containing about
10% methyl alcohol.
Formalin. —Formalin is also known as formol and forniolose and in
commerce is a 36% to 40% aqueous solution of formaldehyde. When, for
instance, a 5% formalin solution is specified, what is meant is 5 cc. of
(iommercial formalin to 95 cc. of water, alcohol, or other fluid. The
commercial formalin generally used is certain to contain some formic acid,
but it is ordinarily unnecessary to remove the acid. In fact the presence
of the acid issometimes a distinct advantage, but if neutral formalin is
specifically one must resort to distillation. Add sodium
indicated,
bicarbonate to a flask of formalin, and distill only enough for immediate
needs since the formic acid will soon reappear.
If the formalin has partially or wholly decomposed, which is indicated
by the presence of a white precipitate, it is useless for critical work.
It has been (claimed that the addition of a little glycerin retards
decomposition.

DEHYDRATING REAGENTS
Reagents wdiich possess hygroscopic properties are those most com-
monly used for dehydrating tissues. The perfect dehydrating fluid is
 REAGENTS 17

one which mixes equally well with water, ethyl alcoliol, balsam, and
paraffi:^! a nd which does not produce desiccation of the tissues (Johansen
1935):" At the present time only two such fluids are known, viz., dioxan
and ter tia i^: butyl alcohol, although other fluids are commonly used for
dehydrating. Tlu' latter fluids are not miscible with paraffin and balsam,
consequently another fluid which does mix with the two latter substaiuies
must follow.
Acetone. — Acetone a satisfactory and safe dehydrating fluid but is
is

not a paraffin solvent. Secondary or tertiary butyl alcohol, chloroform,

or benzene may be used after acetone and preceding infiltration. Af*etone
dissolves fats, resins, waxes, and oils, and it precipitates albumins.
Dioxan (1 ,4-di()xan) is diethylene oxide Or dehydrated diethylene
gly(‘ol (GraupiKT and Weissl)urger 1931). With the exception of cc'rtain
Rhodo[)hyta il normally does not harden or shrink plant tissues. Tlu'
principal disach anl-age inherent in using dioxan is that it is considerably
h(\avier than iiK'lted ]3araffin. It is then'fore necessary to take all pr('-
cautions to nnnove c‘V(‘ry trace of dioxan Ix'fore (‘mlxalding can l)e don(\
J'his difficulty can in fact be (‘ircurnvented by using a fluid lighter than
melted paraffin (such as chloroform, benzol, or sec'ondary butyl alcohol)
to wash out most of tlu^ dioxan before proceeding to the infiltration with
})araffin. Th(‘ usc‘ of calcium chloride t-o (msure perfect dehydration is
advisabl(‘, espf‘cially in damp climates, but there is consid(U*able danger of
the chloride' jK'iiet rating tlu' tissue's.

Thc‘ Eastman Kodak ( 'e)mpany prexluct (#2144) is the only e)ne whie*h
at present can ])e* unre*se'rveelly re'commendeel. Other brands contain
e'onsiderable water and other impurities which will e'ause trouble'.
Ethyl Alcohol. — Eoime'rly
ethyl alce)hed was the only elehydrating
fluid in general use', but as it has e’ome to be suspected of shrinking and
hardening tissues, it is now replaced by other fluids. However, one may
safely go as far as 50% alcohol and t hen transfer to some other fluid wliich
is miscible neither with w^ateij: ne)r with grades of alcohol lower than 75%

or 80% but is miscible with paraffin or balsam.

Hygrobutol." -This is a special type of tertiary butyl alcohol (‘sj>e'cially
prepared for dehydration of materials intended for mounting entire in
balsam (Johansen 1937-1938). Although somewdiat- more expensive, it
may be substituted for the plain tertiary butyl alcohol in paraffin infiltra-
tion schedules.
—
Iso-propyl Alcohol. This alcohol may be substitut ed for ethyl alcohol,
except in killing fluids, with equally satisfactory results. Materials are
said not to be hardened so much as by ethyl alcohol.
Methylal. — Methylal is so like dioxan in all its characteristics that
there is no apparent difference between the two for microtechnique pur-
poses, Methylal is several times as expensive as dioxan.
"18 GENERAL METHODS

—
Normai Butyl Alcohol. This type of butyl alcohol has a different
molecular structure from tertiary butyl alcohol, lacks the soapy feeling
of the latter, has a slight shrinking effect on many tissues, and frequently
tends to harden others. Commercial normal butyl alcohol usually
contains around 10% water; it may be made absolute by standing over
calcium chloride until 24 hours after gas evolution has ceased. A pure,
anhydrous product is on the market. It is soluble in water to the extent
of only 8% by volume.
Normal Propyl Alcohol. —
The possibilities of normal propyl alcohol
in plant microtechnique have not been sufficiently investigated. Animal
tissues have been run directly from water through three changes of normal
propyl alcohol into paraffin. The fluid is claimed not to harden or shrink
animal tissues.
Tertiary Butyl Alcohol. —Probably the safest dehydrating fluid for
the beginner in technique is tertiary butyl alcohol; it is also the least
expensive of all. It is superior to dioxan in that it is lighter than melted
paraffin, otlu^rwise the two fluids are somewhat alike in their microtech-
nical properties. The product derived from petroleum is mor(» satisfac-
tory than other types and more d(‘pendable for the best results. Like
dioxan, tertiary butyl alcohol will mix with all reagents in common labora-
tory use.

CLEARING REAGENTS
The majority of clearing reagents are not miscible with water; tissues
must therefore be completely dehydrated before a clearing agent can be
employed.
—
Beechwood Creosote. In order to avoid cxc(\ssive hardening by the
higher alcohols when dehydrating certain typ)es of pilant materials (suf*h
as fern prothallia), which are unusually fragile' and liable to become
damaged by pressure, the material may be carried as far as 80% alcohol
and then transferred to beechwood creosote to compflete the dehydration.
Two changes of the creosote usually suffice. It may then be removed by
any balsam solvent (dioxan, hygrobutol, benzol, or toluol), the materials
transferred to balsam diluted at least five times with the solvent, and the
latter evaporated gradually.
Some counterstains are soluble in beechwood creosote. Fern pro-
thallia, for example, may first be stained with Harris' hematoxylin, run
up to 80% alcohol, transferred to clear creosote, placed in creosote con-
taining about 0.5% fast green dye, cleared with another change of creo-
sote, and finally infiltrated with balsam.
The only brand of beechwood creosote that will give satisfactory
results is Hartmann and Hauer's. Some brands are synthetic and wholly
useless.
 REAGENTS 19

Benzol, Toluol. —
These fluids work almost as well as xylol. Great
care must be taken when using these fluids because of their explosive
character.
Bergamot Oil.
— ^This was a One
favorite with the older botanists.
may begin with the material in 95%
by adding a drop at a
alcohol and,
time, gradually replace the alcohol with the oil. Some oil will remain in
the material after embedding, but this is sometimes more advantageous
than otherwise. Bergamot oil does not affect coal-tar dyes if used in the
clearing of stained preparations.

^ —
Cedar Oil. Good cedar oil is not easy to obtain. There is one type
for immersion lenses and another for clearing. Most of the cedar oil on
the market has been adulterated with xylol or other solvents. If there
is much danger of the material becoming too brittle when other fluids

are used, resort to cedar oil. Use like xylol, but a less close series of
percentages may be used. Clearing is rather slow. Tissues may be left
in cedar oil indefinitely without appreciable damage.
—
Chloroform, Chloroform is the indicated clearing reagent for some
kinds of material and occasionally had to be used in place of xylol before
the butyl alcohols came into use. In the final stages of infiltration it is
more easily removed than xylol but not so readily as the butyl alcohols.
Equal parts of chloroform and carbon bisulphide sometimes cjonstitute
an excellent clearing agent. Chloroform hardens celloidin. It has been
combinations, consequently one should
aciciised of spoiling delicate stain
avoid using it on stained slides.
^ Clove Oil. —
The reagent most commonly used for clearing sections
on the slide before mounting in balsam is clove oil. Before the mounting
is done, all removed by washing the slide in
traces of the oil should be
xylol, otherwise the stains are apt to fade. Clove oil renders tissues brit-
tle if they remain in it for any length of time. Clove oil contains about
82% eugenol While no data are on record, eugenol may conceivably be
.

substituted for clove oil if necessary, but it is far more expensive.

Terpineol. —Terpineol is a constituent of many essential oils and is of
general value, although more appreciated by zoologists than botanists
for clearing materials embedded in celloidin. It may be used as a sub-
stitute for absolute ethyl alcohol whenever this fluid is called for (Wetzel
1931) and in bringing slides from water to xylol without using alcohol
(Volkmann 1933). Terpineol is harmless to most stains. In the writer's
experience, the use of this fluid is fraught with danger because it appears
to shrink tissues excessively.
^Trichloroethylene. —This fluid is an excellent substitute for xylol.
When used as a clearing agent, extraction of stains has not been observed.
Paraffin is completely soluble in it, as is balsam. It has been claimed
lhat slides cleared in trichloroethylene and mounted in balsam dissolved
20 GENEUAL METHODS

in this solvent dry far more quickly than those mounted with the balsam
dissolved in xylol (Oltman 1935).
Xylol. —
Most tochnieians use the term ^'xyloP' in place of the chem-
ically more accurate designation ^‘xylene.’’ Tissues must be completely
dehydrated before' being gradually brought into pure xylol. This is a
time-consuming and expensive procedure as xylol is generally conside^red
to be miscible with absolute ethyl alcohol only (this is not strictly true, as
traces of 95% alcohol will mix with xylol). A very (*losc series of xylol
and absolute alcohol is required. Several hours in each mixture are
necessary, and the pure xylol needs to be changed sc'veral times. The
transfer from xylol to paraffin must also be gradual and is just as tedious
a process. Every trace of xylol must be removed from the tissues before
the latter are embeddc'd, otherwise the paraffin will crystallize. Tissues
tend to become excess! v('ly hardened if left too long in xylol.
Xylol is still the prime reagent for clearing sections previous to mount-
ing in balsam or other resinous jnedium .-
Xylol should always be fre(i from water and acids. To test a sample
for freedom from water, add some of it to paraffin oil. If a cloudiness
appears, the presence of water is indicated.

ADHESIVES

In l)otanical microtechnique, adhesives are fluids or substances us(‘d

for affixing paraffin sections to slides.
Haupt^s Adhesive. —This isby far the best fluid yet devised for the
affixing of sections to the slide and may also be used for affixing unicellular
and many colonial algae. To make it, dissolve 1 g. plain Knox gelatin
(use the type that comes four envelopes to the package) or other pure,
finely divided gelatin in 100 cc. distilled water at a temperature of 30°C.
When completely dissolved, add 2 g. phenol crystals and 15 cc. c.p.
glycerin. Stir well, then filter. A 3 to 4% aqueous solution of formalin
is used for floating sections. Place a drop of the adhesive on the clean
slide, smear evenly so as to leave a barely perceptible layer, then imme-

diately add the formalin solution by means of a pipette. Place the

sections on the formalin solution, and put the slide on a warming table
(at a temperature of between 40 and 43°C. for paraffin with a melting
point around 58 to 60°C.) until the sections have straightened out.
Remove the slide from the warming table, and set aside until the water
becomes cool then drain off excess water, arrange the sections as desired,
;

and set the slide aside to dry.

By putting mounted but still wet slides in a drying oven together with
a watch glass or two of full strength formalin, the fumes of the formalin
will further assist in coagulating the gelatin of the adhesive.
 REAGENTS 21

—
Mayer’s Adhesive. This is the older standard adhesive. To the
white of a fresh egg add about an equal quantity of c.p. glycerin and 1 g.
of sodium salicylate or a crushed crystal of thymol. Shake well, and
filter through sterile cotton or two or three thicknesses of sterile cheese-

cloth. Use clean tap water for floating sections. While it will keep for
as long as 6 months, solutions more than a month old lose their adhesive
quality. If eggs are cheap and easily available, one may use filtered
white of egg alone. Mayer’s adhesive possesses less holding quality than
Haupt’s and also has the annoying property of absorbing coal-tar dyes.
Celloidin. —Thick woody sections and serial sections of certain marine
algae which are retained on the slide with the utmost difficulty may be
coated with a 1 to 2% solution of celloidin (in equal parts of absolute
alcohol and ether) after first having been affixed with Haupt’s adhesive
and thoroughly dried. Remove the paraffin with carbol-xylol (10%
phenol in xylol) and the latter with 95% ethyl alcohol. Under ordinary
circumstances the interpolation of a 1% solution of celloidin in equal
parts of absolute alcohol and ether in the staining schedule between the
absolute al(;ohol-xylol and 95% al(*.ohol stages will make the majority of
sections adhere firmly to the slides.
Ullrich’s Adhesive. — To 100 cc. of distilled water add 1 (a;, of standard
water-glass solution and 1 cc. of concentrated ammonia. Dry thoroughly
in the air after stretching the sections with the aid of heat. Dissolve th(^

paraffin with xylol as usual and bring down the series of alcohols to 70%.
To this percentage add a trace of hydrochloric acid.

BLEACHING REAGENTS
Bleaching methods which are of general application are cited below,
although other methods are being discussed elsewhere. In using
bleachers, always watch the progress of the action, and stop it as soon as
complete, otherwise the continued action will hydrolyze or macerate the
tissues.
Hydrogen Peroxide. —The peroxide as it usually comes is of 2 to 3 %
strength. may be used full strength or diluted to as high as 50% with
It
either water or 50 or 70% ethyl alcohol. Merck’s Superoxol is a peroxide
of 30% strength and may be recommended where a powerful oxidizing
bleacher is required.
Peroxide -Ammonia. Ammonia water — will accelerate the bleaching
action of hydrogen peroxide.
Hydrogen peroxide (10% strength) 10 cc.
Water 200 cc.
Ammonia water 1 cc.

Transfer the material or mounted slides from water, and wash thor-
oughly in water after bleaching has been accomplished.
22 GENERAL METHODS

EMBEDDING MEDIA
—
The problem of obtaining a satisfactory quality of paraffin
Paraffin.
for embedding purposes has long been a vexing one. Most paraffins on
the market are worthless. There are many so-called grades'^ offered,
designated by the temperature at which they are supposed to melt and
which, illogically, governs the selling price. One should never trust the
statements which appear on the wrappers. The majority commonly
crystallize readily after being cooled, despite all precautions. The
consistency of a paraffin a matter of greater practical importance than
is

its melting point. It is either prepare his own

recommended that one
embedding paraffin or procure a guaranteed product from one of the
reputable supply concerns.
For the infiltrating, ordinary Parowax serves satisfactorily. The
Standard Oil Company of Indiana brand is superior to others.
Rubber can be mixed with paraffin to improve the consistency of the
latter and thus make micro toming easier. A stock solution of crude
rubber in Parowax should first be prepared. Obtain some crude Ceylon
rubber in the form of thin rolled sheets (but do not use dental rubber).
A rubber which melts at the melting point of the Parowax is worthless;
what is needed is one that melts very slowly just below the smoking point
of the Parowax. Cut up the rubber into as small pieces as possible with
a stout pair of scissors. Heat the Parowax in a suitable vessel until it
is very hot but not actually smoking, then adjust the heat so that this
temperature is retained, and add the rubber in small quantities at a time,
stirring the mixture occasionally. Depending somewhat on the size of
the pieces of rubber, it should require many hours to melt the rubber thor-
oughly and completely. Finally pour the mixture into a clean tin can,
(;ool, and later cut away the tin and remove the mass. Approximately
20 g. of rubber can be dissolved in 100 g. of Parowax. The embedding
mixture consists of: crude mixture, 3 to 5 g. Parowax, 100 g., ceresin wax,
;

3 to 5 g. These proportions may be varied slightly to meet special needs.

The same embedding mixture is on the market under the trade name
Parlax.
Celloidin, Colloidin, Parlodion. —These are forms of nitrocellulose,
highly inflammable but nonexplosive. They come either in the form of
tablets or as shreds. The shredded form is stored in water, and should be
prepared acjcording to the directions on the label. Various brands are on
the market and most of them are unduly expensive. No particular brand
appears to be generally preferred over others.
—
Glycol Stearate. This is a water-soluble wax melting at 48.5°C.
Small portions of tissue may be placed directly into the melted wax (in a
paraffin oven) from water. Leave the material in the wax for about 48
 REAGENTS 23

hours and give six successive changes of wax during this period. Solidify
at room temperature. Sections may be cut very thin with a sliding
microtome. Dissolve out the wax with chloroform, pass to absolutes
alcohol, and thence to the staining process.

MOUNTING MEDIA
Dammar Balsam. —Gum dammar, as this balsam is sometimes also
called, is considered to be superior to Canada balsam by many micro-
scopists. It is not readily obtainable in a condition suitable for imme-
diate use; the crude gum may have to be purchased and prepared for use.
It comes in the form of lumps of various sizes mixed with more or less
powdered material and debris. Pick out the lumps, melt over a hot
flame in a suitable container and jiour the melted balsam into the desired
solvent, which should preferably be benzol as it dries more quickly than
xylol. N(^xt filter through coarse filter paper placed in a ridge^d funnel,
and filter again through finer filter paper if the first filtrate shows some*
cloudiness due to dust or finer specks of debris. Preparations mounted
in dammar balsam do not fade around the periphery as frequently do
those mounted in Canada balsam.
—
Canada Balsam. Pormerly one had to make up his own balsam from
the solid, dry substane(*, but this has now been obviated and neutral,
filtered balsam licpiid form or dissolved in xylol or benzol may be
in the
readily purclias(‘d.For freehand sections, the solution as it comes is of
about the right eonsistency, but it will sometimes need to be diluted
slightly for paraffin sections. Xylol, dioxan, or trichloroethylene may be
used as diluent. Balsam should never, under any circumstances, be
heated to melt it. If the balsam comes from the jobber as a very dilute,
darkly colored liquid, it has been spoilt by the application of heat and the
use of too miudi solvent. It is highly essential that the balsam be kept
from becoming acid. Keep the bottle in the dark when not in use, or
paint it with black Duco. It would be well, as an added precaution, to
keep a piece of clean marble in the bottle, replacing it occasionally with a
fresh one. Always mount sections in Canada balsam from xylol, benzol,
or trichloroethylene.
Euparal. — This mixture, popular with English technicians, is a com-
bination of camsal, sandarac, eucalyptol, and paraldehyde, with a much
higher index of refraction than Canada balsam. It has the property of
intensifying hematoxylin stains, and it is often the practice to use this
medium on preparations stained with hematoxylin. Mount from 95%
alcohol.
Euparal has a slight solvent action on celloidin, but this may be taken
advantage of to make curled or too stiff sections unroll and flatten out
24 GENERAL METHODS

before adding the cover-glass. If the cuparal gets cloudy before mounting
can be completed, warm the slide gently until the cloudiness disappears,
—
Diaphane. The American equivalent of euparal is diaphane, but it
has a slightly lower refractive index. Mounting may be from either 95%
or absolute alcohol, but one should work quickly to avoid, clouding.
Diaphane is said to be nonoxidant and to preserve delicate stains well.
Styrax, —Sty rax is a synthetic mounting resin with a very high
refractive index, commonly employed for the mounting of diatoms to
bring out the s(‘ulpturing of their ^^shells.^^ It is best diluted, if neces-
sary, with benzol. Mount from xylol or benzol.
Hyrax. — This medium is similar to styrax and is used for th(‘ same
purpose. Fine details in smears stained with brazilin are well brought
out. Mount from xylol.
Cedar Oil. —
The immersion type of cedar oil hardens only along the
periphery of covc'rslips, remaining liquid inside. Mount preferably from
xylol. Stains seem to be well preserved.
—
Venetian Turpentine. The resin of Larix europaea yic^lds Venetian
Turpentine, known also as Venice Turpentine and Turpentine Venetian.
It is used ojily in the so-called Venetian Turpentine (whole mount)
method. It usually has the fault of crystallizing in time and thus spoiling
the preparations.
Lactophenol. —As a mounting medium, lactophenol (consists of equal
parts of phenol crystals, lactic acid, glycerin, and distilled water. Some-
times 2 parts of glycerin are added to 1 part each of the other reagents.
For special purposes other ingredient/S are sometimes added, such as stains
to color otherwise colorless tissues and copper acetate to preserve greeii
pigments.
Karo. — Karo an excellent substitute for glycerin jelly when
is

(employed for the same purposes. The blue-label type is preferable.

Karo is a mixture of dextrose, dextrin, and maltose, but the sugars do not
crystallize on drying. Materials may be mounted directly in Karo from
water or the lower alcohols, but for more delicate forms, the Karo should
be diluted with water or weak alcohol and concentrated by evaporation
to a mounting consistency. When hard, Karo is as firm as balsam and
ringing of the coverslips is not necessary, except in very moist climates.
S3mtlietic Resins. —
During the last few years a large number of
synthetic resins have appeared upon the market, and several have already
been shown to have excellent possibilities as substitutes for Canada
balsam. The one which seems to be the most satisfactory is Clarite
(formerly known as Nevillite V); it is colorless, strictly neutral, inert,
homogeneous and dries quickly. An 80% solution in xylol is to be pre-
ferred for mounting plant specimens, but a 60% solution in toluol has
been recommended for thin sections of animal tissues. The resin is
 REAGENTS 25

intolerant of water; consequently it cannot be substituted for balsam

in the hygrobutol method. There are other synthetic resins which are
soluble in water but insoluble in hydrocarbons (such as Abopon and
Stacol) and which form quick-drying, colorless, and nonhygroscopic
mounting media. The pH of these resins varies from 6.65 to 8.0, there-
fore further investigation of their effect upon stain preservation is

required.

Solubility of Various Reagents in Water, Absolute Ethyl Alcohol, Paraffin,

AND CeLLOIDIN

Absolute B.P.,
alcohol, Cel- degrees
Water Paraffin
or lower loidin Centi-
percentage grade

Acetone X X X 56
Alcohol, benzyl - 90 X - 204.7
Alcohol, absolute ethyl X X - X 78
Alcohol, iso-propyl X X - - 82-83
Alcohol, normal butyl 8.3 parts X X - 117
Alcohol, propyl X X X ~ 97.4
Alcohol, secondary butyl 29 pa i ts 70 X - 99.8
Alcohol, tertiary butyl X X X 82.9
Anilin oil - 90 X - 245
Beechwood creosote; Very slight 80 — 200-250
Benzol (benzene) X X 80
Bergamot oil 80 X 183
Carbon bisulphide - X X - 46
Carbon tetrachloride; - X X - 76.5
Cedar oil - 95 X - 237
Chloroform - X X - 61
Clove oil — 90 — X 250
Dioxan X At 56° Slightly 102
Ether 12:1 X X X 34
Methyl benzoate — 90 - X 199
Methylal Verj^ sliglit j
X Slightly 46.5
Oil of origanum - i
90 Slightly (?)
Petroleum ether :
X X - :
30-50
~
1

T£*-pineol 90 218
Toluci (toluene) — X i
X - no
Trichloi .^ethylene - X X - 87
__ 140
Xylol (Xvl'>nc) X X !

1
1
1

X “
mixes or dNsolves.
— a» does not mix or dissolve,
* Mixes with ceJloidin dissolved in absolute alcohol-ether.

—
Glycerin Jelly. Dissolve 1 part by weight of a high-quality gelatin
in 6 parts by weight of distilled water for 2 hours or longer. Vext add ]

7 parts glycerin, and to ofi-ch 100 g. a dd 1 g. phenol crystals. iVarm for

26 GENERAL METHODS

15 minutes, stirring continually until the flakes produced by the addition

of the phenol disappear. While still warm, filter through two or three
thicknesses of cheesecloth into a convenient bottle. The mixture solidi-
fies on cooling. The whole may be remelted by heating gently, but it is
preferable to cut out small portions and melt these since the mixture
deteriorates on continual remelting.
 CHAPTER V
KILLING AND FIXATION

The killing and be made into permanent

fixing of the material to
[)rcparations are important processes, and the student should constantly
bear this in mind. The day when a piece of tissue was dropped into any
fluid which happened to be at hand has passed. Today the most com-
petent technicians are exceedingly critical of the methods employed both
by themselves and by others, and very rightly so. The student who
expects recognition of his published w'ork by others must devote the most
careful and critical experimentation to secure the proper fixation of the
material with which he may be working. These preliminary processes,
in fact, are the most important of all, since everything else depends
upon them.
NATURE OF KILLING AND FIXATION
Although the action of the substances used in killing and fixing fluids
has been studied rather exhaustively, habit and prejudice rather than
reasoned choice often determine what reagent will be used. It should be
remembered that mixing togedher diverse reagents will not combine th(‘,
excellent properties of each, but will often result in difficulties. Certain
reagents which afford fine fixation when used alone or in certain combina-
tions are mutually antagonistic when used in other combinations.
Killing and fixing are two distinct processes, but both are usually
obtained by means of a single fluid which in turn is commonly a mixture
of various chemical reagents.
The term killing means the sudden and permanent termination of th(»
life processes. It does not apply solely to an entire organism, but to th('

individual cells of which the organism or tissues are composed. As long

as a single cell capable of reproducing itself remains alive and receives
nourishment, the organism is not yet dead. Any reagent used for
purposes of killing must, therefore, reach every cell in the organism or
piece of detached tissue if it is to do its work completely and effectively.
Killing invariably precedes fixation since the reagents which do the
killing penetrate tissues faster than those upon which responsibility for
fixation rests. Alcohol, for example, is primarily a killing reagent.
Fixation is a process very difficult to describe in terms that are easily
understood. It is commonly said to be the pres ervation of all cellular

and structural elements in as nearly the natural living condition as possi-

---27
 28 GENERAL METHODS

ble. Or, to put it a little differently, a g ood fixative is one that changes
the c ell ch emistry the least and greserves the cell structure the best
'(^chiTler 1930). What one actually sees after fixation is always a picture
entirely different from the picture during the living condition, as it is

utterly impossible to preserve anything in the exact condition in which it

existed during life. Fixation is thus an entirely empirical process. The

success or failure of fixation is judged by the quality of its usefulness when
the completed preparation is examined. That is, it is good if the tech-
nician finds what he desires to be revealed. Many technicians do not
need exact fixation; they are satisfied with a remote approximation.
For example, cytologists who spend most of their time counting chromo-
somes ask only that the chromosomes be spread out to facilitate count-
ing, that the chromosomes be darkly stained, and that the cytoplasm be
so fixed (or partially dissolved) that it stains lightly or not at all. Accu-
rate preservation of internal chromosomal structure and of the cytoplasm
is not necessary for them.
?
Fixation, cojise^quentlyj^is sgm
that can be judged in the subjec-
tive sense only, since there is no objective standard for the judgment of
the quality of fixation. Judgment is based solely on the personal attitude
of the observer. Opinions, obviously, are about as numerous as the
individuals expressing them. If one of these individuals were to bo
asked to explain the reasons for his opinion and why it clashes with the
opinions of other technicians, he is generally at a loss for a satisfactory
and unbiased explanation. This is not surprising since many technicians
labor under the mistaken assumption that ‘^perfect fixation^’ actually
exists.
/Fixation is required in order that structures, which are obscured or
entirely invisiblewhen cells are observed in the living condition, may
be seen more distinctly, and in order that soft structures may be hard-
ened sufficiently for further treatment. This explanation is the one
most commonly given. The point at issue is the fact that the evidence
for the existence of many so-called structures'^ not visible in living cells
is almost wholly ot)lique. To understand fixation more fully, it becomes
necessary to know exactly what technicians mean by structures. There is
a sharp difference in the final picture of the effects of fixatives on fixed
structures and on the nonfixed structural elements of protoplasm.
V' There are three concomitant criteria for the definition of a structure:
(1) A structure is something that can easily be seen and recognized at all

times in the living condition. Lignified plant cells, the nucleus, and
chromosomes are examples. (2) A structure is always accompanied
by an investing membrane, or may itself be made up of membranes.
Structures (joming under this classification are not always easily visible
in the living condition and are more characteristic of plant cells than of
 KILLING AND FIXATION .29

animal cells. Vacuoles are an example, (3) One test of the reality of
the structures seen in fixed material is under a diversity
their appearance
of conditions and wide variety of fixing fluids. Spindle
after the use of a
fibers are considered to be an example of such structures.
Originally the term fixation was applied to the grosser structures
which can be preserved without any relation to the change of the colloidal
state which is the fundamental structure of protoplasm. These struc-
tures are not ordinarily affected by fixatives; at least, not in the violent
manner that cytoplasm is acted upon. The earlier plant morphologists
were interested more in definite cell regions or elements than in cellular
contents. They were therefore not disturbed by the difficult artifact
question, since what they saw in their preparations could also be seen in
the living tissues. They fixed materials so that they could stain things
seen in living cells and thereby be enabled to make more precise observa-
tions. To them fixation nu‘ant merely the preservation of structure,
and from view it is possible to describe fixation as being good
this point of
or bad. This idea has been earned over into cytology, often with
unfortunate results.
If the existence of a structure (‘.annot be demonstrated by vital
observation, it has been contended that such a structure cannot bo other
than some colloidal artifact since it becomes visible only after the living
colloidal structure has undergone an irreversil)le change of condition as a
result of fixation. The appearance of this structure in a fixed and stained
preparation is said to be merely some one phase of thc^ images occurring
during necrobiosis, the result of the toxic aedion of the fixing reagents
(Yamaha 1927).
Most technicians now grant that the fundamental structure of proto-
plasm is colloidal. To state the case more fully, each protoplasmic
structure, such as nucleus, plastids,and cytoplasm, is an intricate col-
and the phase boundary where each (;olloidal system meets,
loidal system,
as at nuclear membranes and the tonoplast, is still another colloidal
system. These colloidal systems are all believed to belong to the hydroph-
ilous colloid group. This, it should be emphasized, is the case with
protoplasm in the living condition only. When the reagents in a fixative
come into contact with protoplasm, the colloidal system undergoes an
irreversible change of state. As a consequence, the state of dispersion,
the grade of hydration, and every other physicochemical property which
accompanies these properties, are subject to marked and sometimes
violent changes. What actually happens is that, when protoplasm is
acted upon by a fixing fluid, it goes through various colloidal changes
before it finally attains the irreversible state. This has been demon-
strated time and again by the fact that the instant when the action of the
fixing fluid was terminated has a direct bearing on the appearance of the
30 GENERAL METHODS

protoplasmic structure in the completed preparation. If the time of

action was brief, the final picture is very different from the one presented
when the time of action was too prolonged. This means that all the
structural changes preceding the final irreversible coagulation of proto-
plasm can easily be followed out. We can, for example, observe the
formation of new dispersed phases in the hyaloplasm, the dissolution or
disappearance, transformation, and fusion of existing dispersed phases
such as plastids and mitochondria.
The ultimate aim in fixing protoplasm is to make invisible or barely
visible structures easily observable. The increase of optical contrast
between structures is generally considered to be more important than the
preservation of the substances forming these structures. A marked
optical contrast results from the great difference
in the physicochemical
characteristics among the various systems making up proto-
colloidal
plasm, and another results from the distinct phase boundaries of these
colloidal systems. Now, when the final irreversible change in the col-
loidal system occurs as a result of fixation, what has taken place is
actually destruction. The changes in optical differentiation just men-
tioned are the results of the destruction of the original colloidal systems.
In the original condition (t.e., in the living condition), there is little or no
optical differentiation; in the final condition there is usually great optical
contrast, provided a suitable fixing fluid has been chosen. The final
condition depends both on the nature and on the concentration of the
chemical reagents composing the fixing and on the particular nature
fluid,

of the protoplasm. The be interpreted as meaning

latter expression is to
the difference in the nature of the protoplasm between, say, a red alga and
an angiosperrn root tip.
According to most authorities, the hydrogen-ion concentration (pH)
of the fixative influences the destructive action rather than the preserva-
tive action. For example, the higher the concentration of hydrogen
ions, the less perfect is the prevServation of purely cytoplasmic structures.
Protoplasm, regarded as similar to a protein solution, coagulates most
easily at the isoelectric pointand absorbs the minimum quantity of the
various ions. Karyoplasm, with respect to the isoelectric point, is known
to be more acidic than cytoplasm. Since ordinary fixatives are strongly
acid, the lower the pH of a fixative, the less is its coagulating power and
the more is its dissolving power; this explains why strongly acidic fluids
give such poor fixation of vacuoles, for example. The dissolving action
is considerable in those fluids, such as Gilson\s or Petrunkevitsch’s modi-
fication thereof, whose pH is less than 1.

To put it briefly, fixation is a contradictory process, consisting as it

does of both the preservation of some structures and the destruction of
 KILLING AND FIXATION 31

others. The more impor-

latter factor is to the microscopist actually the
tant of the two since it is by
means that optical contrast is obtained.
this
Since staining of tissues is but another method of enhancing optical
differences, it in turn is dependent upon the nature of the fixed proto-
plasm. Therefore, if the staining is not clear and sharp, the more or less
illogical conclusion is that the protoplasm was imperfectly fixed. It is
really possible to get perfect preservation of structures but to have
practically no optical or stain contrast, as all experienced technicians are
fully aware.
The failure to get sharp staining following “pcrfecd/^ fixation is to a
considerable extent also bound up with the chemical properties of the
various dyes and the circumstances under which they combine with the
different types of fixed substances. This obviously is strictly a chemical
problem, and extremely little is known about il
at present The presence .

of (excessive amounts of phlobaphene (tannin) compounds can cause com-

plications during both fixation and staining, and, as another example, the
[)ractice in some quarters mixing potassium bichromate and chromic
of
acid (which really gives an excessive amount of chromic acid) gives poor
fixation and difficult staining. In any event, it is agreed that whatever
the mechanism of fixation and staining, all other conditions being equal,
the type of fixation employed greatly the ultimate staining of
influen(^6\s

the tissues (Naylor 1926; Sheinin and Davenport 1931; Tolstoouhov

1927, 1928).
One effect of fixation is the hardening of the tissues, but hardening is

ordinarily brought about by the dehydrating fluids. The latter differ

considerably in the degree of their hardening effects, but as a rule any
tissue that becomes hardened by one reagent is liable to become more or
less hardened by all other reagents. The tissue hardening that occurs
after fixation is presumed to be caused by precipitation, but swelling or
shrinkage may also influence it (Sheinin and Davenport 1931). Then'
is apparently no correlation between tissue hardening and tissue preser^ a-

tion. While good tissue hardening does not necessarily result in good
tissue preservation, the latter must be preceded by the former.
Certain powerful fixatives, such as Carnoy-LeBrun\s fluid, bring about
their effectsby desiccating the tissues. Some plasmolysis, therefore,
must be expected.
It has recently been demonstrated that much of the criticism leveled
against certain killing and fixing fluids was not merited sinc^e the trouble
was actually caused by inadequate and, to a certain extent, improper
dehydration methods. It should be borne in mind that killing and fixing
are only two phases of the entire process of transforming living materials
into permanent preparations and th^it each phase must be examined both
32 GENERAL METHODS

independently and in conjunction with each other phase. Violent

dehydration methods can easily give a false fixation picture, and it is
illogical to fasten the blame on the fixing fluid.

PRINCIPLES INVOLVED IN COMBINING REAGENTS IN KILLING

AND FIXING FLUIDS
Each reagent used for purposes of killing and fixing has both its

advantages and its disadvantages.

Some reagents used for killing purposes penetrate rapidly, and others
penetrate slowly. Both classifications in turn iiudude reagents which
simply do what is expected of them and have no further effect upon the
tissues, whereas other reagents will damage the tissues if their destructive
effects are not df^terred by the addition of other n^agents, or by washing
them out. Osmium tetroxide may be taken as an illustration: it is the
fumes that actually do the killing, which occurs within a few minutes; if
the substance is allowed to react for longer than hour or is used in too
large concentrations, the tissues become first brown, then blacker and
blacker, and will eventually become reduced to a (diarred mass.
The reagents used for fixation may be classified similarly, but the
definition is far sharper among this class of reagents than with those used
primarily for killing.
The characteristics of the more important reagents used for killing
and fixing are noted below. The general principle involved in the com-
bining of these and other substances to form a killing and fixing fluid is to
secure a balance between all the properties of the reagents involved. A
substance which tends to shrink cytoplasm, for instance, should be com-
bined with one which tends to swell cytoplasm. In other words, a
disadvantage inherent in one reagent can be counterbalanced by that
peculiar to some other reagent if the two disadvantages are of a directly
opposite nature. Two reagents which have the identical disadvantages
should never be combined by themselves.
Moreover, substances which are easily oxidized should not, as a rule,
be combined with reagents that are powerful reducers.
Practically all suggested formulae are flexible insofar as the quantities
or proportions of each reagent are concerned. In fact, most proposed
formulae are merely variations of some one basic formula. No one fluid
serves equally well f or all orga nisms because of the^4mmepse''diversity in
"
Mructiife, chemical and physical organization, etc., ai^ng plants. "Some”
^elasticity is necessary, and the proportions, of each reagen^in any given
formula require adj u e tment aMofding^ totire~8pec|e8, with relation to iU
ptructurp and composition, Jf the standard proportions do not give
satisfactory fixation, it is ordinarily possible to determine which reagent
needs to be increased or decreased, ae the case may be, or some other
 KILLING AND FIXATION 33

combination tnight be experimented with. A formula intended primarily

for cytological purposes docs not always give good fixation for anatomical
purposes, and vice versa.
apparent from the foregoing discussion that the choice
It is therefore
of the killingand fixing fluid is an important matter. The nature of the
—
material being worked upon should first be determined whether it is soft
and easily damaged or tough, resistant, and not readily penetrated by
reagents in solution. Next the purpose for which the material is being
prepared should be considered. A fluid which might give good preserva-
tion of root tips does not necessarily render the chromosomes of the
mitotic figures in the tips in a fit (condition for (critical study, c;onsequently
there will be numerous occasions when it becomes necessary to sacrifice
some desirable qualities for the sake of others.

HANDLING OF MATERIAL
The greatest care should be exercis(jd in handling tissues or organisms
preparatory to placing them in the killing fluid. Very small organisms,
such as filamentous, colonial, and unicellular algae and filamentous fungi,
Euglena, small Bryophyta, may be placed entire in the fluid. With
larger organisms, more or less dissection or reduction to smaller portions
is required. Avoid pressure of any sort, and work quickly so that a
minimum time will elapse between removal from the plant, from the
aquatic habitat, etc., and placing in the killing fluid.
^^he smaller the piece of tissue and the larger the surface exposed to
the direct action of the killing fluid, the better the killing and fixation.
The weaker fixatives penetrate with difficulty. Occasionally it will be
found that the outer layers of cells are overfixed, while the innermost
portions are badly underfixed. This is caused by poor penetration of the
fixative portion of the fluid. The remedy, of course, is to use one of
stronger penetrating power. Acetic-alcohol and picro-aceto -formalin
combinations penetrate rapidly aiicl' easily, while mixtures containing
chromic and osmic acids penetrate poorly. Buds which are covered with
dense hairs should be treated first with a strong alcoholic; solution for a few
minutes and then placed in another fluid, preferably an aqueous one.
For example, buds of nearly all the Asteracieae are beautifully fixed if
they are placed in Carnoy’s fluid at the time of collecting and after not
more than 10 minutes transferred to Navashin's fluid. All superfluous
tissue should be carefully removed. In general, organs or pieces of tissue,
for the optimum results, should never exceed 5 mm. in any one direction.

FIXATION IMAGES
Fixation images are of two types, and the characteristics of each are so
pronounced that the technician should learn to recognize them promptly.
 34 GENERAL METHODS

1. Acid Fixation Image. —Chromatin, nucleoli, and spindle fibers

preserved; cytoplasm fixed as stringy spongioplasm ;
nucleoplasm and
mitochondria dissolved.
2. Basic Fixation Image .
—chromatin and spindle fibers dis-
^^Resting
solved; nucleoli, nucleoplasm, and mitochondria preserved; cytoplasm
fixed as hyaloplasm, and vacuoles more or less preserved.
Nearly all the fixing fluids in common use give the acid fixation image.
Very few fluids giving the basic image have been compounded, and the
most useful ones have been developed only during the past few years
(Zirkle 1927 et seq.). However, there are variations in each image accord-
ing to the material for instance, mitochondria are occasionally found after
;

distinctly acid fluids were used. Investigations on the effect of fixing

fluids have generally failed to take into account the changes produced by
the dehydrating reagents. The correct procedure for describing the
final effect should include both the killing and fixing fluid, as well as the
dehydrating reagent and the infiltrating medium. The picture given
by Bouin\s fluid with absolute ethyl alcohol and xylol as the dehydrating
fluids is quite different when tertiary butyl alcohol is used for
dehydrating.
With certain reagents there is some overlapping of the t.wo images.
Copper bichromate, for example, if used in a solution at pH 4.8, preserves
both chromatin and mitochondria (Zirkle 1928).
\y If the fixing fluid contains two or more substances, the resulting fixa-
tion image is determined primarily by the component which penetrates
more rapidly (Zirkle 19336). The various reagents, it will be recalled,
penetrate at different rates, and also according to their respective
concentrations.
For most purposes a fluid giving an acid image will be the most satis-
factory, particularly since the majority of the preparations that the
technician has become accustomed to observing have been from such
fluids. The employed mainly for studies on cytoplasmic
basic fluids are
structure and for cytoplasmic iindusions such as mitochondria.

KILLING AND FIXING FLUID REAGENTS

Under this heading will be described the substances most commonly

used in killing and fixing fluids, and the formulae for the various fluids will
be given in a succeeding section. The student should familiarize himself
with these substances and be able to recognize all of them instantly by
means of appearance, odor, and other characteristics.
A.|isolute Ethyl Alcohol. —
This is a fair killing and fixing fluid if
immediate results are desired. The time should be very short; in any
event, not over 1 hour should elapse before the fluid is poured off, fresh
 KILLING AND FIXATION 35

absolute ethyl alcohol is added to ensure complete dehydration and the

infiltrationthen begun.
—
96% Ethyl Alcohol. This is a fair general preservative but has noth-
ing whatever to recommend it for even passable work. Alcohol is a
reducing agent and is easily oxidized to acetaldehyde and the latter in
turn to acetic acid. Chromic acid, potassium bichromate, and osmium
tetroxide should therefore not be mixed with this alcohol. It is much
more useful and efficient when in combination with formalin and glacial
acetic or propionic acid. Unless some glycerin be added, tissues left in
95% alcohol become too brittle for further use. Material which has been
killed and fixed in most of the standard fluids may, after the original fluid
has been thoroughly washed out, be kept indefinitely in 70% ethyl
alcohol. Proteins are precipitated by alcohol; the precipitates are
insoluble. Nucleic acid is precipitated, but not insolubly. Fats and
phospholipides are dissolved.
Alcoholic fluids should not be used at low temperatures.
—
Chloroform. Chloroform is never used alone. It is a constituent of
Carnoy’s fluids.
—
Formalin. It is best to use only the chemically pure solution, which
costs but a trifle more than the ordinary brands. Used alone, fair fixation
sometimes occurs, the results are generally unpredictable because
some types of mateoiil ai:g.«^runken and overfixed, while others may
become swollen and vacuolized. Formalin as a rule penetrates slowly
(Underhill 1932), and when used al^)»e gives the basic fixation image.
Formalin is one of the best hardening agents. It does not precipitate
proteins or render them insoluble in water but merely prevents ethyl
alcohol from hardening them excessively, by exerting a different harden-
ing effect of its Formalin neither preserves nor destroys fats but
own.
more or less preserves phospholipides? til using formalin in combination

with other reagents, such as chromic acid, it should be kept in mind that
formaldehyde is a powerful reducing agent; it becomes oxidized to formic
acid. The fumes of formalin are extremely irritating to mucous
membranes.
Formalin may be neutralized by adding about 5% pyridine.
Most marine algae are successfully fixed in sea water to which has been
added from 6 to 10% formalin.
Glacial Acetic Acid. —
This acid is very commonly employed as a
constituent of fixing fluids, on the assumption that its presumed tendency
to swell cytoplasm counteracts the shrinking effects of certain other
reagents, such as chromic acid and formalin. Actually, however, acetic
acid shrinks tissues (Zirkle 19286). Its usefulness lies in the fact that it is a
fat-soluble acid which penetrates rapidly and produces the acid fixation
image in the tissues, The acid otherwise is more of a preservative than a
36 GENERAL METHODS

fixative,as may be recalled from its use in the pickling industry; it

preserves both ordinary proteins and fats, but they can subsequently be
dissolved in water. It is primarily a chromatin preservative.
Fixation images identical with that of acetic acid are produced by
ferric, cupric, chromic, and mercuric acetates. Propionic, butyric, and
valeric acids may, under most circumstances, afford better fixation than
acetic acid.
Nitric Acid. — ^Little use is made of this acid in botanical microtech-
nique, but has been included in certain fixatives.
it

Picric Acid. —
This acid, which comes in the form of a yellow crystalline
solid, sometimes slightly moistened as a precaution against explosions,
is invariably used in the form of a saturated solution. Jt shrinks strongly
and penetrates tissues readily. Proteins, nucleoproteins, and nucleic acid
are all precipitated. In whatever solution or fluid it is employed, it

should alwaj^s be washed out \\dth 70% ethyl alcohol, never with water,
unless the fluid contains some other reagent which indissolubly precipi-
tates chromatin. The washing may be facilitated by warming the wash
alcohols to as high as 40°C. always necessary to get rid of all the
It is not
yellow color imparted to the tissues; if this is found necessary, lithium

carbonate may be added to the 70% wash alcohol until the color has
disappeared from the tissues.
Anthraquinone is superior to picric acid as a killing reagent and may
be substituted; it is used in exactly the same way.
—
Chromic Acid. Microtechnicians, and particularly those engaged in
cytological work, can scarcely get along without chromic acid. It is the
aqueous solution of chromic anhydride, which comes in the form of
reddish-brown, extremely deliquescent crystals. Chromic anhydride
should not be dissolved in alcohol as it will quickly become reduced to
chr^ou^,tudde or sesquioxide, neither of which is of any value as a fixa-
tive. Chromi? acid is the basis of a long series of killing and fixing fluids,
which commonly also contain acetic acid.
Chromic acid tends to shrink tissues; to counteract this tendency,
other substances are usually mixed with it. It does not penetrate very
well into many types of tissues. Some technicians contend that at low
concentrations the acid exerts a considerable solvent action upon tissues,
but this apparently is more true of animal than of plant tissues. The
one great defect of chromic acid resides in the fact that, because it is a
poj^erful hardening agent, it tends to cause tissues to be come brittle.
Ine acid is one of the most powerful precipitants of proteins, nucleo-
proteins, and nucleic acid, none of which can later be dissolved. Fats
are not affected nor are lipoids.
Chromic acid stock solutions are best kept in strengths of 2 and 10%.
Practically all formulae in general use are based upon dilutions of these
 KILLING AND FIXATION 37

stock solutions. Large volumes of any killing fluid containing chromic

acid should be used. Such fluids should always be washed out thoroughly
with either running water or frequent changes of large volumes of water.
Fixation with chromic acid and other chromic compounds is essentially
a tanning process, and tissues consequently become more or less browmish
in color. The tanning is most pronounced after the use of those fluids
which contain both chromic acid and potassium bichromate {e.g., La
Courts fluids). If the sections appear to have too much chromate fixa-
tion, which is invariably the case after such fluids, bleaching may be
effected by dipping the slides into 1 %
aqueous potassium permanganate
for about 1 minute, rinsing very briefly in water, then dipping in 5%
aqueous oxalic acid for 1 minute, and finally washing thoroughly with
water. Or differential acidification (page 169) may be attempted.
As a rule, a cytologic.al fixative weak in chromic acid will cause
chromosomes to become attenuated and to reveal constrictions that a
fixative strong in the acid will not show up.
Osmic Acid. —
Osmic acid is the name commonly given to osmium
tetroxide, although this compound is not strictly an acid. It is an
extremely expensive substance and usually comes in the solid form in
0.5- or 1-g. sealed tubes. The weight cited is not always accurate but
because of the extreme difficulty of checking up on this point, any prob-
able discrepancies may for most practical purposes be ignored. Solutions
of osmic acid are very readily reduced by the presence of the least bit of
organic matter. Exposure of plain aqueous solutions to the light does
not nec^essarily bring about reduction, as is commonly believed, provided
that the water is absolutely pure and quite free from dust. Such a
solution should be kept in a thoroughly cleaned dropping bottle with
grooved stopper. Before breaking the tube in which the acid comes,
clean it carefully, removing every evidence of the label, and finally rinse
in several changes of pure water. It would be a far more satisfactory
procedure to purchase a 1 %
solution in 1 %
chromic acid direct from the
manufacturer.
Reduction of solutions in distilled water may as a rule be prevented by
the addition of (1) a small amount of sodium iodate, (2) about 10 drops
of a 5% aqueous solution of mercuric chloride to each 100 cc. of 1%
osmic acid solution, or (3) enough potassium permanganate to give a
barely perceptible rosy tinge to the solution, adding more of the per-
manganate whenever the solution becomes colorless.

Osmium tetroxide penetrates plant tissues poorly. The fumes or

vapor kill just as efficiently as does the solution, especially spores, zoo-
spores, small unicellular algae, and similar organisms. The solution, how-
ever, should never be used in a strength exceeding 2%. Many workers
consider 0.1 to 0.5% to be strong enough. Unacidified osmium tetroxide
38 GENERAL METHODS

precipitates proteins instantly and coarsely; acid solutions do so slowly

and more evenly. On the
other hand, each of the two solutions works in
the opposite way on nucleoproteins.^^.^-^mium is the only reagent which
preserves both fats and conjugated lipides. Objects should always be as
small as possifiTeV whether-'ftJf killing by the fumes or the solution.
Fixation is complete as soon as the material has become brown through-
out. Killing fluids containing osmium tetroxide must always be thor-
oughly washed out before dehydration isbegun. If the material has been
left in the killing fluid too long, it will become blackened, with the result

that it is necessary to bleach the sections before a proper stain reaction

can be secured.
Under certain circumstances the use of osmic acid is contraindicated.
For example, it should not be used on the Rhodophyta because it aggra-

vates the natural tendency of this group of plants to break to pieces

during almost any stage of the subsequent processes; nor, except for
special purposes, should it be used on tissues rich in oily substances.
—
Mercuric Chloride. Corrosive sublimate is a synonym for this
extremely poisonous substance. Mercuric chloride is never used alone.
It is a rapid fixer and a powerful precipitant of proteins and nucleic acid,
but it tends to shrink tissues. Transparent tissues are rendered opaque
as soon as fixed;™ Aqueous solutions should always be based upon
distilled water. The sublimate must be very carefully washed out after
fixation. Aqueous solutions may be washed out with water, although it
would be preferable to use an alcohol of a strength below 70%. Alcoholic
solutions should be washed with alcohol of the same percentage as that in
the killing fluid. In the case of material which is to be embedded, the
deposits left by the mercuric salt are more easily removed from the sec-
tions. Transfer the slides from 70% alcohol to 50% alcohol to which are
added about 1 g. of iodine and 2 g. of potassium iodide per 100 cc. After
a few minutes, transfer to a 0.2% solution of sodium thiosulphate (ordi-
nary photographers’ hypo is satisfactory) to remove the iodine, then
proceed to the staining after a thorough washing in water. The hypo
solution must be kept up to, but not beyond, its original strength by
the addition of a tiny crystal from time to time.
Material killed in solutions containing mercuric chloride should be
embedded as quickly as possible.
The use of mercuric chloride as the main or a minor component of a
killing fluid to be used on material intended for cytological study is not
to be recommended.
Iodine. —A weak aqueous solution of iodine and potassium iodide
is excellent for fixing microscopic plant forms; glacial acetic acid and
formalin may advantageously be added. Penetration is rapid. Wash
out thoroughly with water.
 KILLING AND FIXATION 39

Potassium Bichromate. —This not often mentioned in

substance is

the literature on botanical microtechnique, but it is a decidedly useful

substance. It is the principal ingredient of many fluids devised for

the study of mitochondria. It is a superb hardening agent but has the
serious fault of penetrating so slowly and poorly that some plasmolysis
must be expected in large pie(;es of tissue. It has no effect on fats,
hut its effect on mitochondria will be noted in the following discussion.
The bichromate reacts in two entirely different ways, depending
upon whether it is used alone or in combination with an acid. If the
solution containing the bichromate is acidified to make itmore acid
than pH 4.6, the fixation image is that of chromic acid. The chromo-
somes are well fixed, cytoplasm and chromatin are precipitated as
networks, and mito(‘hondria are dissolved. When the solution is less
acid or more alkaline (f.c., with a pH of over 5.2), the chromosomes are
dissolved, a chromatin network is not apparent, the cytoplasm is homo-
geneously preserved, and the mitochondria are well fixed.
It appears clear from the pre(*eding discussion that mixtures including
potassium bichromate with chromic acid, and sometimes also acetic
acid, arc illogical since the* only eff(‘ct is that of an excessive proportion
of clvromic acid.
Other Reagents. — Dozens, if not hundreds, of other substances have
])een f)ropos(;d as ingnMlients of all sorts of mixtures.No good reason
(‘xists for the use of some but others, whi(4i might after all be
of them,
found to have ^^aluable properties, were employed at a time when too
little was known concerning the tjffects both of these substances and of

the other reagents with wdiich they were mixed, and they were conse-
quently discarded as of no value.

KILLING AND FIXING FLUIDS

Killing and fixing fluids, in the present text at least, are classified
according to whether they produce a basic or an acidic fixation image.
Those which give a basic image are too few in number to require
subclassification, whereas thereis practically no logical method of

differentiating the numerous concoctions that give acid images. All

classifications which have previously been proposed are wholly arbitrary
and need not be considered further.

Mixtures Giving Acid Fixation Images

In the following discussion the various mixtures are grouped accord-
ing to whether the principal ingredient is formalin, glacial acetic acid,
chromic acid, or various combinations of these three substances, either
alone or with the addition of other reagents.
40 GENERAL METHODS

The optimum time required for fixation to be effected and the method
of washing out the fluid are given for each formula.

Acetic Acid- Alcohol Mixtures

y —
Carnoy’s Fluids. Two different fluids bear Carnoy^s name. In
the literature there has beien hardly any mention as to which was
used, which may provide an explanation for the inequalities in published
reports. The second fluid is the one more commonly employed. The
formula for Farmer's fluid is identical with the first one noted below.

(1) 100% ethyl alcohol 15 cc.

Glacial acetic acid 5 cc.
(2) 100% ethyl alcohol 30 cc.
Glacial acetic acid 5 cc.
Chloroform 15 cc.

Action of both fluids is very rapid, because of the great penetrating

power of each. For root tips, 15 minutes is long enough; for anthers,
1 hour should suffice. The practice of certain botanists of leaving
material in such violent fluids for over 48 hours is beyond all reason and
merely leads to overfixation and the production of artifacts such as
cytomixis. These fluids alone are not to be recommended for studies
on plant cytology, but for such difficult subje(;ts as the ova of Ascaris
and most insects the second formula is about the best yet devised. Wash
in two changes of 95% ethyl alcohol and proceed to paraflSn as quickly
as possible.
Cfiurnoy-LeBrun’s Fluid. —
This mixture is probably the most rapid in
action of all. not especially recommended for routine fixation but
It is
might be tried where rapid action and strong penetration are required.
Wash in 95% ethyl alcohol.

100% ethyl alcohol 10 cc.

Glacial acetic acid 10 cc.
Chloroform 10 cc.
Mercuric chloride To saturation.

Gilson’s Fluid. —This is a rather unusual combination that has been

recommended by some workers for the fleshy fungi, particularly for the
softer, gelatinous forms such as Tremella. The mixture does not keep
for more than a day. Allow to react for 18 to 20 hours. Wash out
thoroughly with 50% ethyl alcohol, and remove the mercuric deposits
from the sections.
60% ethyl alcohol 50.0 cc.
Distilled water 440 0 . cc.
Glacial acetic acid 2.0 cc.
Nitric acid, 46® strength, about an 80% solution. . . 7.6 cq.
Mercuric chloride 10.0 g.
 KILLING AND FIXATION 41

Petrunkevitsch’s Fluid. —This is a variation of Gilson^s fluid, which

may be employed when must be penetrated in
large masses of tissue
order to reach deeply embedded portions. It has given good preserva-
tion of megagametophytes in angiosperms. Care must be taken not to
Dverfix. Most ovaries require 12 to 20 hours. Wash out as directed
For Gilson^s fluid, and embed as quickly as possible.

40% ethyl aleohol 125.0 ec.

Glacial acetic acid 27.5 cc.
Concentrated nitric acid 2.5 cc.
Mercuric chlorid(‘ To saiiiration.

Formnlin-A cciic Acid- A Icohol Mixtures

—
Formalin -Aceto-Alcohol. This fluid, more familiarly known as FAA,
^ might almost be called the *^standard preservative’^ of botanical micro-
technique, since it is used mor(‘ (^xttmsively than any other. The varia-
tions that have Ixhui proposed are almost endless, and some have even
been given the names of the technicians who first mentioned them (Lang-
don, Rawlins, Lavdowsky, among others). The standard proportions
are:

50% (or 70%) et hyl alcohol 90 cc.

Glacial acet ic acid 5 cc.

Formalin 5 cc.

Some technicians liabitually use 50% alcohol, others use 70%; the
lower percentage should be employed with the more delicate materials,
especially the thalloid Bryophyta. The proportions of both the acetic
acid and the formalin —
may indeed, they sometimes must be varied —
according to the nature of the material, as determined by experience.
For hard woody materials, for instance, it would be advisable to decrease
the amount of acetic acid and to increase the formalin, since the latter
penetrates more slowly than the former.
This reagent may be used with almost any plant material intended
for anatomical or morphological study. It is unsuitable for chromosome
studies. Material may be left in it almost indefinitely without appreci-
able damage; this property of nearly perfect preservation makes formalin-
aceto-alcohol the ideal fluid to take on long collecting trips. The
minimum time of fixation is 18 hours. If the tertiary butyl alcohol
dehydration method is employed, it is unnecessary to wash out the
killing fluid; one may go directly to the 50% stage of that method. If

other methods are used, washing in two changes of 50% ethyl alcohol
is all that is necessary. Woody materials should be washed for two days
in running water and softened for three to six weeks in a 50% aqueous
solution of hydrofluoric acid, if the use of the desilicifying ^cid appears
necessary.
42 GENERAL METHODS

Fonnalin-Propiono-Alcohol. —Since propionic acid appears to give

more adequate fixation than acetic acid, it may be substituted for the
latter, in the same proportions, in the formula given above. The time
of fixation and manner of washing are identical.

Chromic Acid and Acetic Acid Mixtures

Chromic acid is rarely used alone, but combinations of chromic and

acetic acids in various proportions, to which formalin is sometimes added
(see the following section), are extensively employed. Such combina-
tions are almost endless, consequently the formulae for only a few will b('
cited here. By studying those given, the student can easily adjust the
joroportions of the two acids to fit any particular circumstance. A large
\’olum(i of fluid containing these two acids should be used, and it cannot
be used more than once. The time required is rarely less than 24 hours;
a few days^ immersion will usually do no appreciable harm. After
fixation, all fluids which contain chromic acid as an ingnulient should be
washed out thoroughly with water, otherwise staining of the sections will
be obscured and differentiation rather poor.
The chromic acid should be made up as a sto(;k solution of 1 %, 2%, or
even 10% in distilled water. The tendency of the crystals of chromic
anhydride, from which chromic acid is prepared, to deliquesce so readily
makes it a disagreeable task to weigh out small quantities at a time. The
aqueous solution is perfectly stable. Practically all other formulae are
based upon one of these percentage solutions.
—
Stock Chrom -Acetic Fluid. A stock solution used by some workers
as a general purpose fluid consists of 1 g. chromic acid, 1 cc. glacial acetic^
acid and 100 cc. water and is commonly known as ^‘1% chrom-acetic.^'
It however, not a very precise fixative.
is,

Weak Chrom-Acetic. —
This is a more precise formula and may be used
for filamentous algae and fungi, Bryophyta, prothallia of the Pterido-
phyta, moss capsules, and similar subjects that are easily penetrated.

10% aqueous chromic acid 2 5 cc.

.

10% aqueous acetic acid 5.0 cc.

Distilled water To 100 0. cc.

Medium Chrom -Acetic. —This

an excellent mixture for root tips
is

and small ovaries or megagametophytes. About

isolated ovules with
2% of maltose or urea or 0.3 to 0.5% saponin should be added to facilitate
penetration.

10% aqueous chromic acid 7 cc.

10% aqueous acetic acid 10 cc.
Distilled water , To 100 cc.
 KILLING AND FIXATION 43

Strong Chrom-Acetic. —^For

woody materials, tough leaves, and
similar objects. As indicated for the preceding mixture, the addition
of maltose, urea, or saponin will be found beneficial.

10% aqueous chromic acid 1 cc.

10% aqueous acetic acid 10 cc.

Distilled water To 100 cc.

Chromic Acetic and Osmic Acid Mixtures

y y

These fluids constitute the so-called Flemming fluids, named after

their originator. They represent the mixtures which will, for the various
types of material for which each is best adapted, afford by far the most
truthful representations obtainable by present methods. The following
solutions should be made up only just before being used.
Weak Chrom-Osmo-Acetic. —For more delicate tissues.

10% aqueous chromic acid 1.5 cc.

10% aqueous acetic acid 1.0 cc.

2% osmic acid in 2% aqueous chromic acid. ... 5.0 cc.

Distilled water 96.5 w..

Strong Chrom-Osmo-Acetic. —For more resistant tissues.

10% aqueous chromic acid 3.1 cc.

10% aqueous acetic acid 30.0 cc.

2% osmic acid in 2% aqueous chromic acid. ... 12.0 cc.

Distilled water 1 1 . 9 (^c.

Taylor’s Chrom-Osmo-Acetic for Smears. —This is a very satis-

factory killing fluid for smear preparations and gives fairly faithful
preservation of chromosomal structures. It is offered as a substitute
forNavashin/s fluid, commonly used for similar purposes. The mixture
should be made up with a 10-cc. pipette graduated to hundredths. The
amount of maltose to be added should be determined by experiment on
each species. The purpose of the maltose is to preserve the identity of
trabants (satellites) and to avoid the obliteration of constrictions.

10% aqueous chromic acid 0.20 cc.

10% aqueous acetic acid 2.00 cc.

2% osmic acid in 2% aqueous chromic acid. ... 1 .50 cc.

Distilled water 8.30 cc.
Maltose (approximately) 0.15 s-

Taylor’s Modified Benda’s Fluid. — Benda's was one of the earliest

fluidsused in investigations on chromatin and is still preferred by some
workers. It is valuable in the study of prophase stages of meiosis in
microsporocytes.
 44 GENERAL METHODS

10% aqueous chromic acid 3. 1 cc.

Glacial acetic acid 8 drops
2% osmic acid in 2% aqueous chromic acid. . . 12.0 cc.
Distilled water 41.9 cc.

—
Chamberlain’s Chrom-Osmo-Acetic. This combination is suitable
^ only for fresh-water algae, filamentous fungi, and similar organisms and
should not be used for root tips, stem tips, or other morphological
material.

Chromic acid 1 g.

Glacial acetic acid 3 cc.

1% aqueous osmic acid 1 cc.

Distilled water 100 cc.

Chromic Acid, Acetic Acid, and Foi'malin Mixtures

Mixtures of these three reagents constitute the so-called Navashin
fluids, of which a considerable number of modifications have been
proposed, some bearing the names of the originators. All are designed
more for cytological than for morphological fixation purposes. They
are excellent for smears of microsporocytes, for anthers, buds and root
tips. Many cytologists fix buds first in Carnoy’s fluid for from 5 to
10 minutes, then pour off the fluid and replace with a Navashin mixture.
This procedure .is more satisfactory on the Astcraceae than on species
from other families and is better for buds covered with dense hairs or
which are difiicult for the aqueous solution to penetrate. Microtoming
may be difficult with some materials, and the sections are liable to become
loosened from the slides.
If only a small quantity is needed for immediate use, the following
modification may be used, Belling^s variation, which is always made
up in two parts and mixed just before being used, is to be recommended
under other circumstances. The great majority of other proposed
combinations differ merely in the proportion of formalin to be added.
This does not constitute sufficient reason for giving them new names.
Taylor’s Modified Karpechenko Fluid.

10% aqueous chromic acid 1.5 cc.

10% aqueous acetic acid 10.0 cc.

Formalin 0 83
. cc.
Distilled water 23.67 cc.

—
Modified Navashin Fluid. Make up the two solutions
Belling’s
^ separately, mixing equal volumes of each just before using. For meta-
phase smear preparations, Solution B may be composed of 100 cc.
formalin and 275 cc. distilled water. Three hours appears to be long
enough for most smears, but immersion for as long as 12 hours does no
injury. After fixation, transfer smear preparations to a stender of
 KILLING AND FIXATION 45

0.5% aqueous chromic acid for not longer than 10 minutes, to remove
the formalin, then proceed with the staining.
Buds, root tips, and similar materials may be left in the fluid almost
indefinitely. Shortly after being mixed, the solution will turn a brownish-
green to greenish color, which indicates the reduction of the chromic acid.
Wash thoroughly in several changes of water when ready to commence
the dehydration.

Solution A; Chromic acid crystals 5 g.

Glacial acetic acid 50 cc.
Distilled water 320 cc.
Solution B: Formalin 200 cc.
Distilled water 175 cc.
Saponin 3 g.

"
Randolph’s Modified Navashin Fluid. —The following fluid, to which
the abbreviation Craf has been applied, is claimed to be superior to
other Navashin-type mixtures (Randolph 1935). Fix for 12 to 24 hours.
Transfer the material, without w^ashing, directly to 70% ethyl alcohol,
changing the alcohol three or four times at 15-mimite intervals.

Solution A: Chromic acid 1 g.

Glacial acetic acid 7 cc.
Distilled water 92 cc.
Solution B: Neutral formalin 30 cc.
Distilled water 70 cc.

Mix equal portions of A and B just before using.

Potassium Bichromate Mixtures

As has been noted in the general discussion above, the mixing of
potassium bichromate with chromic acid is illogical and gives unsatis-
factory results; therefore fluids of this type are scarcely to be recpin-
mended. LaCour’s fluids, which have come into favor in some English
institutions but have not met with the same re(;eption in the United
States, are in this category. Tissues become so heavily tanned that
they are hard to bleach suffi(aently and staining has always been poor.
In fact the use of these fluids has been claimed by some observers to be
the direct cause of the erroneous interpretations of certain cytological
phenomena by one school of thought in that field.
The first fluid mentioned below has considerable to recommend it

for certain special purposes, but the others are included merely as a
matter of record.
Tellyesnicky’s Fluid. —Inmixture potassium bichromate is
this
substituted for chromic acid. has been used on the Bryophyta and
It
also on leaves with excellent results. It might be tried if a similar
46 GENERAL METHODS

fluid containing chromic acid fails to work. Fix 24 to 48 hours, prefer-

ably changing once, and wash out in running water for 12 hours.

Potassium bichromate 3 g.
Glacial acetic acid 6 cc.
Distilled water 100 cc.

LaCour’s 2BE Fluid.

1 % aqueous chromic acid 90 cc.

Potassium bichromate 1 g.
Saponin 0 05 g,
.

6% aqueous acetic acid 10 cc.

2% aqueous osmic acid 16 cc.

Distilled water 46 cc.

LaCour’s 2BD Fluid.

1 %
aqueous chromic acid 100 cc.

1 %
aqueous potassium bichromate 100 cc.
Saponin 0.1 g.
2% aqueous osmic acid 30 cc.
5 % aqueous acetic acid 30 cc.

Picric Acid Mixtures

The such mixture to be proposed was the familiar Bouin^s fluid,

first

so widely used by zoological technicians. The original mixture is hardly

suitable for plant material since the tissues are apt to become brittle and
difficulty is often experienced in microtoming the material. The original
formula is as follows:

Saturated aqueous picric acid 76 cc.

Formalin 25 cc.
Glacial acetic acid 6 cc.

Anthraquinone may
be substituted for the picric acid and should
give more Fix tissues for 24 hours, rinse quickly
satisfactory results.
with water, then wash thoroughly with 50% alcohol.
Allen’s Modified Bouin’s Fluid (Allen’s B-15). —
This fluid has
proved to be very satisfactory with the buds of many species which
give poor results with alcoholic and chrom-acetic fixatives. Immediately
before using, heat 100 cc. Bouin^s fluid to 37°C. and add 1.5 g. chromic
acid. Stir thoroughly, then add 2 g. urea. Keep the fluid at between
37 and 39°C. while the material is being placed in it; when finished,
allow it to cool gradually. The fluid will become greenish within hour
and rapidly loses its efficiency. Four hours’ time should give thorough
fixation, but the material may be allowed to remain in the fluid overnight.
Wash with frequent changes of 70% ethyl alcohol over two days or until
no more yellow color is extracted.
 KILLING AND FIXATION 47

The morphology of metaphase and anaphase chromosomes is well

brought out by this mixture, but it is not suitable for other stages in
either mitosis or meiosis. Cleland adds 1 g. of chromic acid to freshly
prepared Bouin^s fluid and substitutes 1 g. of maltose or lactose for the
urea. With these modifications he has secured satisfactory results
with the very difficult Onagraceae. Staining is usually brilliant with
iron hematoxylin.
—
Sass’ Modified Bouin’s Fluid. Beautiful results are obtained witli
buds and anthers of the Liliaceae by means of this mixture. Dehydrate,
without washing out the mixture, in grades of acetone (5, 10, 15%, etc.),
and clear in five grades of acetone-xylol or acetone-tertiary butyl alcohol.

1% aqueous chromic acid 50 cc.

Saturated aqueous picric acid 35 cc.
Formalin 10 cc.

Glacial acetic acid 5 cc.

Mercuric Chloride Mixtures

—
Schaudinn’s Fluid. This mixture is widely employed on the Protozoa
and related organisms. It may be used to fix on the slide plant sper-
matozoids and zoospores and certain of the flagellated unicellular algae.
For this purpose it is used at a temperature of 70°C. It was originally
made by adding 10 cc. of absolute ethyl alcohol to 20 cc. of a saturated
aqueous solution of mercuric chloride. A later version is to add 10 cc.
of absolute ethyl alcohol to 40 cc. (or twice the original quantity) of the
chloride. Most protozoologists add from 1 to 5 parts of glacial acetic
acid just before using, but seems better to dilute the fluid one-half
it

with water and to add 2% glacial acetic acid immediately before using.
Fix for several hours, and wash out with a medium strength alcohol,
to which is added a little iodine solution to remove any mercury deposits.
—
Worcester’s Fluid. Worcester's fluid has been found to give excellent
results with plant tissues whose cells are congested with various sub-
stances. The enzyme-secreting cells of seedlings are well fixed. Allow
to react about 20 hours, and wash thoroughly with 70% alcohol to which
is added about 1 %
potassium iodide.

Saturated aqueous mercuric chloride 96 cc.

Formalin 4 c^,
10% aqueous glacial acetic acid cot >

Mixtures Giving Basic Fixati^^^ages

^^..^kle-Erliki Fluid. —For mitochondria. "Ae^^agd is cbmplete^y,

basic, and all chromatin is dissolved (Zirkle Fix for 48 hours;

wash with water.
48 GENERAL METHODS
Potassium bichromate 1 25.
g.

Ammoniuin bichromate 1.25 g.

Cupric sulphate 1 .00 g.
Distilled water 200.0 cc.

—
Reduced Chromic Fluid. For mitochondria and vacuoles
Zirkle’s
(Zirkle 1932). Formalin when added to solutions containing unreduced
chromium compounds immediately reduces the latter, (jonsequently
such mixtures are unsatisfactory. This disadvantage is obviated by
using a reduced chromium salt, such as chromic sulphate.

Chromium sulphate 5 g.
Cupric oxide Slight excess
Formalin 10-50 cc.
Distilled water 90-50 cc.
(Total amount of fluids should be 100 cc.)

Fix 48 hours; wash with water. The purpose of the copper is to bring
the pH The concentration of the formalin depends upon the
to 4.6.
material being fixed and must be determined by experiment. If used

in too great a concentration, there will be some plasmolysis and swelling

of the vacuoles; if in too low a concentration, the mixture may be so
diluted by the liciuid contents of the tissue that an erratii* fixation irnagc'
results. Even when this fixative seems to be at its best, perfect preserva-
tion of the vacuoles and their tonoplasts does not always result.
 CHAPTER VI

STAINS

The subject of dyes or stains and methods of using them has become
one of vast j)roportions. The number of dyes available is enormous,
and ways of (uri{)loying stains are almost as iiumerous as the workers
using a particular dye or combination of stains. Standardization in the
manufacture and certifi(^ation of dyes of American manufacture has been
attained, but the majority of technicians are still individualistic in the
utilization of these stains. These facts, however, need deter no one
because the most convincing and satisfactory results have been, and still
are being, obtained with a few w(‘ll-known dyes. For c'xample, plant
morphologists use safranin and fast green more extensivc^ly than any
other combination of stains; cytologists consider that crystal violet
has no rival as a stain for dividing chrpniatin; the bacterioTiigist would
be almost helpless without methylene blue; and Delafield’s or Harris’
hematoxylin with a counb^rstaiii of eosiii is in universaruse^'Gy zoological
and climcal technicians. The preparation and use of stains has all
but become a scicmce in itself, and there exists a most valuable journal,
Stain Technology^ devoted to these and reflated subjects. Most of the
abstracting journals, American and foreign, devote special sections to
stains and staining methods. One should make it a habit to read these
abstracts in order to keep abreast of current trends. Experimenting
with new dyes and new staining schedules is a most fascinating occupa-
tion, and there is a very great deal yet to be learned about stains and
their utilization.
It is not proposed to burden the present text with accounts of the
history of stains, their preparation, chemical constitution, and similar
purely technical details. For such information, the student cannot do
better than consult the very valuable manual by Dr. H. J. Conn, “Biologi-
cal Stains” (Biological Stain Commission, Geneva, N. Y., 3d ed., 1936).
The greater part of the information contained in the remainder of this
chapter has been derived from the publications of the Stain Commission
and other reliable sources.
The older botanical technicians placed much of their reliance upon
natural or textile dyes. Very few of the natural, and practically none
of the technical, dyes are still in use. The latter were always of doubtful

purity since manufacturing methods could not always be so standardized

49
50 GENERAL METHODS

as to give an identical product each time a batch was prepared, and they
were too often insoluble in the technician's customary solvents. Nearly
all the dyes now used in biological technique are synthetic chemical

compounds made from the substances found in coal tar. The two types
of dyes, natural and coal tar, are being discussed separately.

THE NATURAL DYES

Only three natural dyes are still used by botanical technicians.
These dyes have not yet been manufactured synthetically. All are
important cytological stains. One, cochineal and its derivatives, is of
insect origin the others arc from plants belonging to the Caesalpiniaceae.
;

Brazilin. —
This dye is obtained from different trees known collectively
as brazilwood,^’ but principally from Caesalpinia crista or C. echinata.
It is closely related chemically to hematoxylin but is neither so active
nor so strong a stain. It has recently come into extensive use as a stain
employment for the purpose having originated with Belling,
for smears, its
but refinements in the method have since been made (Capinpin 1930).
Brazilin is ordinarily employed as a 0.5% solution in 70% alcohol,
which is allowed to ripen for about a week. Keep containers well
stoppered, away from light and air. Brazilin by itself is not a dye; it
reacts only after mordanting with ferric ammonium sulphate.
—
Hematoxylin. Hematoxylin is a chromogen derived from logwood.
Hematoxylin campechianum L., and is one of the most important of all
stains. It is a homologue of brazilin, possessing one more hydroxyl
group in its chemical constitution. The dye solution itself has little?
or no affinity for tissues, unless iron or aluminum is present in the latter,
consequently mordanting in some form is necessary. The stain is made
up in combination or in conjunction with various metallic salts, prin-
cipally those of iron (always in the ferric form), aluminum, and copper.
Some of the schedules are progressive; others are regressive. The color
effects of hematoxylin vary with the character of the medium in which
it is dissolved and according to the after treatment. In the presence of
acids the color is red; in the presence of alkaline solutions it is blue. By
Heidenhain’s iron-alum schedule, structures such as chromosomes and
pyrenoids are stained black, while the cytoplasm of sporogenous cells, for
example, is stained gray. However, by exposing the sections to the
action of ammonia fumes, the color is turned to blue.
The solution of the dye as used in Heidenhain’s iron hematoxylin
technique is simply a 0.5% solution in divstilled water. It will be found
to be more satisfactory to prepare a 10% solution in absolute ethyl
alcohol and to dilute a portion with distilled water to the 0.5% strength
when required. Formerly it was necessary to permit the solutions to
stand for some time to ripen (into hematein), but the certified dyes will
 STAINS 51

often be sufficiently ripe after a few days. The process may be hastened
by placing the solution in a very wide and shallow evaporating dish and
exposing, at a distance of approximately 2 feet, to any rather powerful
quartz mercury- vapor arc for about 45 minutes. Stir the solution
frequently during exposure.
At high temperatures hydrolysis of hematoxylin solutions occurs; a
metallic scum or film forms on the surface. Such solutions must be
discarded. Spoilage is also indicated when the solution starts to turn
brown. A relatively stable hematoxylin solution may be prepared by
adding 5 cc. of a 10% absolute alcohol solution to 100 cc. methyl cello-
solve, 50 cc. distilled water, and 50 cc. tap water that contains calcium
compounds in solution. If the solution on shaking does not acquire a
rich wine-red color, add a pinch of sodium bicarbonate and shake vigor-
ously. Ripening usually occurs immediately, and the solution can be
used at once. This solution does not spoil at high temperatures; it also
retains its staining capacity for far longer periods than do simple aqueous
solutions.
Most of the difficulties with hematoxylin arise during the destaining
and differentiation. The same solution should not be used for both
mordanting and destaining. Solutions of reagents other than ferric
ammonium sulphate frequently give better results: ugly precipitates
are avoided, thicker sections may be clearly stained, and disagreeable
tan or brownish colors do not result (Hutner 1934).
Tuan^s use of picric acid (Tuan 1930) has been extensively followed
by numerous technicians. If the picric acid solutions are warmed to
about 50°C., the destaining will be greatly accelerated. It is not
necessary that the color effects at the end of the destaining process be in
the nature of sharp black and white (or colorless) contrasts. This some-
times indicates that one has merely painted various structures black and
has not really stained them in order to reveal details clearly. Sections
differentiated in picric acid may be blued by washing out the acid thor-
oughly and by adding 1 or 2 drops of ammonium hydroxide to the 70%
dehydrating alcohol.
Ferric ammonium sulphate is both an acid and an oxidizer. A fairly
close approximation to the effect obtained with this reagent can be gotten
with the following mixture:

0.26% solution of hydrochloric acid in 96% alcohol. . 2 parts

Merck's Superoxol 1 part

The solution should be freshly prepared. Destaining completed, wash

the slides with 70% alcohol, and blue with ammonium
hydroxide. Take
care that the higher dehydrating alcohols do not become acidified by the
hydrochloric acid.
52 GENERAL METHODS

Ferric chloride may be substituted for ferric ammonium sulphate,

both as mordant and as differentiator. Use a 2 to 5% solution for the
mordanting, for about 30 minutes. Rinse, then stain. Differentiate
in a 1 to 3% solution. The stain is more blue than black, and the
differentiation is more precise (Haggquist 1933).
Delafield’s hematoxylin is still being made according to the original
formula, in which dye and mordant are combined. To 400 cc. of a
saturated aqueous solution of ammonium aluminum sulphate, add drop
by drop a solution of 4 g. hematoxylin crystals in 25 cc. of 95% ethyl
alcohol. Expose to light and air for four days. Then add 10 cc. c.p.
glycerin and 100 cc. methyl alcohol. Allow to stand for at least two
months, exposed to the air, until the color is sufficiently dark. The
process may, however, be accelerated by exposure to the quartz mercury
lamp, as described above for plain hematoxylin solutions, the time of
exposure being about 2 hours. It is rather dangerous and unsatisfactory
to follow the recommendation of some microsc^opists to ripen the solution
with hydrogen pcToxide. 'The ripened undiluted solution is a powerful
stain; a much more can be obtained by diluting the
prt‘cise differentiation
staining solution with as much as twice its bulk of distilled water. The
stain is differentiated in either water or 70% ethyl alcohol acidified with
hydrochloric acid; the procedures arc outlined in the following chapter.
Harris^ hematoxylin resembles Delafield^s in its color effects but is
made up in an entirely different manner. It is a superb in toio stain.
The formula is as follows:
Hematoxylin crystals 5 g.
Aluminum ammonium sulphate 3 g.
50% tahyl alcohol 1000 cc.

Dissolve the dye and the alum with the aid of heat, then add 6 g.
mercuric oxide (use only the red powder) and boil for 30 minutes. Filter,
then bring up again to the original volume with 50% alcohol. Acidify
in the proportion of 1 drop hydrochloric acid to each 100 cc. of solution.
When one is in doubt as to the proper stain to use, he may safely
use either Harris^ or Delafield^s hematoxylin. Even after they become
thoroughly experienced in the use of other stains and combinations of
stains, skilled technicians often encounter difficulties. Many, if not
most, of them resort to either of these two hematoxylins and generally
obtain the desired effects. Delafield’s will sometimes stain chromatin
well when the regular chromatin stains fail; it also brings out cellulose
walls more sharply than most other stains. If proper care in differentia-
tion is taken, sporogenous cells are unusually well defined by both
hematoxylins.
Mayer’s haemalum has been recommended for. the nuclei of filamen-
tous algae and fungi, having
little or no effect upon cell walls or plasticU*
 —

STAINS 53

The improved formula is as follows: dissolve 1 g. hematoxylin crystals

in 1 liter water. Then add 2 g. sodium iodate and 50 g. aluminum
potassium sulphate. The solution acts much like Delafield^s hematoxylin
but does not keep well; add a crystal of thymol to prevent mold growth.
Excellent results are secured by diluting the stain about ten times with
distilledwater and allowing it to act overnight. Follow any balsam
method with algae or fungi; transfer to the stain from water,
infiltration
and wash out thoroughly with water before proceeding to the next step.
Ehrlich^s hematoxylin has been extensively used with safraniii on
woody tissues:

Distilled water 50 ee.

Absolute ethyl alcohol 50 cc.
Glycerin, c.p 50 cc.
Glacial acetic acid 5 cc.
Hematoxylin crystals 1 fz;.

Alurniniitn potassium sulphate To excess

Keep in a dark place until the color becomes a deep red, or it may be
ripened in 3 or 4 hours by exposure to a quartz mertairy lam[) as directed
in the discussion of Heidenhain's heinatoxyliu above. Transfer sc^ctioiis
to the stain from 35% alcohol or water, allow to remain for 5 to 30 min-
utes, wash off excess stain with water or 35% alcohol, and proceed with
the dehydration. Orange G or erythrosin may be used as a counterstain.
If safranin used with Ehrlicdi’s hematoxylin, stain first in th(> safranin.
is

—
Hematein. Many workers consider hematein superior to hema-
toxylin (Kornhauser 1930) oji the ground that ^Mt is easy to prepare, easy
to us(', saves time, and gives good rtisults.^^ A MacAndrews and Forbes
product should bt' us('d.
To prepare the stain, grind 0.5 hematein in a glass mortar with
g.
10 cc. of 95% ethyl alcohol, and add 5% aqueous aluminum
to 500 cc. of
potassium sulphate. Transfer the slides to the stain from water. The
stain is progressiv(^; consequently the slides should be inspected fre-
quently for 5 minutes or so, which is th(‘ average time recpiin^d. Next
rinse 1 to 3 seconds in tap water. One may counterstain 1 to 3 seconds
with eosin bluish (1 part stock solution —0.5% solution in 20% alcohol
to 2 parts distilled water). Wash in several changes of tap water,
dehydrate, and mount.
—
Cochineal and Derivatives. Cochineal is a yellowish-red powder,
obtained by grinding the dried bodies of the female cochineal insect and
extracting the coloring matter. Cochineal and its derivatives are some
of the most important bulk stains yet available. They are all progressive
stains but may be used retrogressively. Cochineal itself is not used by
botanists to any great extent.
54 GENERAL METHODS

Carmin is a bright red in color and is the lake obtained by adding

alum to cochineal. It is a complicated mixture of which the essential
coloring agent is carminic acid. Carmin and carminic acid are so easy
to use and give such astonishingly beautiful effects that it is amazing
to what a slight extent they are used by botanical technicians. Cytolo-
gists, however, have a considerable appreciation of carmin, at least, as
indicated by its extensive use in the iron-acetocarmin smear technique.
Carmin is only slightly solul)le in water at a neutral reaction; hence
solutions must be either acid, as is the customary cas(^, or alkaline.
Carminic- acid is the active dye principle of cocihiiuial and carmin.
It is a fairly strong dibasic acid. It is best known to botanists as th(^
principal ingredient of Mayer^s carmalum.
Indigocarmin. — Indigocarmin is only indirectly a natural dye, but
it is included among the natural dyes because it is based upon one. It

is the sodium salt of indigodivsulphonic acid, and conseciuently has acid

properties. In color it is bluish; Init when mixed with picric* a(*id to
make the picro-indigoc^armin stain irsed in stainitig algae, the color
becomes green. The solubility of indigocarmin at 25°Cy. is 1.68% in

water and 0.01% in 95% alcohol.

THE COAL-TAR DYES

The older classifications of coal-tar dyes are largely artificial. Tlu'
classification at present used is bavsed upon similarity in chemicail struc-
ture. Six groups have been recognized (( 'Onn 1986):
1 . The nitro dyes.
Example: Picric acid:
2. The azo dyes.
Exam,ples: Orange G, Bismarck brown, Sudan IV. ^
3. The anthraquinone dyes.
Example: Alizarin.
4. The quinone-imide group of dyes:
a. Indainins: none in common use.
b. Thiazins.
^ Examples: Methylene blue, thionin.
c. Oxazins: none in common use.
d. Azins; subdivided into
(1) Exanfple: Neutral red.
Arnido-azins.
(2) Examples: Safranin 0, inagdala
Safranins. red.
(3) Indulins. Example: Nigrosin.
5. The phenyl-methane group of dyes:
a. Diphenyl-rn ethanes: none commonly used.
< 6. Diamino-triphenyl methanes.
Examples: Fast gree6, light green, malachite green.
c. Triamino-triphenyl methanes.
Examples: Acid fuchsin, crystal violet, anilin^lue.
d. Hydroxy-triphenyl methanes: none generally used.
 STAINS 55

6. The xarithene group of dyes:

a. Pyronins: none used in botanical technique.
b. Rhodainines: none used.
c. Fluorane derivatives.
Examples: Eosin, erythrosin.

The nomenelature of dyes is in great confusion because practically

no system was used in naming them. Unscrupulous dealers have
given new names to old dyes, actuated solely in adding to their profits
by the introduction of a supposedly new product. The syn-
onymy of some dyes has reached amazing proportions. The confusion
in nomenclature of histological dyes has fn^pumtly misled users. ()n(‘

would do well to follow the recommendations of the Commission on

Biological Stains as to acceptable names. This is the procedure followed
in the pnisent text.
On lal^els and in certain published papers one will frequently meet
with numbers preceded by the initials C.I., e.g., C.I. No. 841 ;
or by the
name Schultz, c.g., Schultz No. 679. The first refers to a publication
by the Society Dyers and Colourists of England, known as the Colour
of
Index. It is a very complete manual listing textile dyes, their synonymy,
chemical formulae, methods of preparation, and distinctive cliaracter-
istics. Schultz refers to the German equivalent, rnucli less compre-
hensive, of the Colour Index, viz,, Schultz’s Farbstofftab(‘lln. The
numlx^rs arc those given in the respective index; the two cit(‘d above
refer to Safranin 0 as given by each. Numbers are not being cited
in the present text as they are of little practical value to the average
technician.
In the following list the various vstains are arranged alphabetically,
rather than according to the chemical classification given above, in order
to give greater convenience in reference. First comes the accepted
name with occasional citation of synonyms whenevc'r
of the dye, together
such are in common use. Next comes an indication as to whether the
dye is acidic or basic, then its chemical classification. Following this
is given the solubility of the dye at 26°C. in water and in 95% ethyl
alcohol, as expressed in percentages. This indicates roughly how much
of any st^in is needed to obtain a saturated solution in each solvent,
but allowances must always be made for the presence of impurities in the
dye sample, particularly if it is uncertified or of foreign manufacture.
Solubility statistics are not given for dyes which are mixtures of two or
more dyes. Finally, information is given concerning the customary
solvents for each dye, together with occasional brief mention of treatment
subsequent to staining. Detailed stain schedules are reserved for the
following chapter.
 .

56 GENERAL METHODS

Acid Fuchsin. —Acid; triamino triphenyl methane (rosanilins).

A(;id fuchsins are acomplex mixture of sulfonated derivatives of basic
fuchsin, and any given sample is probably a mixture of several different
acid fuehsiris. In plant technique it is used to stain the cortex, pith
parenchyma, and cellulose walls and is excellent for differentiating
zoosporangia and f)araphyses in the Laminariales. It has some use as a
mitochondrial stain.
A 1 % solution in 70% alcohol is preferable, but a 0.5 to 1% solution
in distilled water sometimes works equally well. The stain reacts very
rapidly and is easily extracted by the higher alcohols. In staining
morphological material, the stain should be allowed to react for 2 hours.
If the solution in 70% alcohol is used, one may differentiate in a saturated
solution of picric ficid in 70% al(;ohol for about 1 minute and then rinse
in 70% alcohol until a bright reddish-magenta replaces the yellow of the
acid. Complete dehydration quickly, and mount at once.
Acid fuchsin must not be confused with basic fuchsin; when fuchsin
alone is mentioned by careless writers, without specifying the acid or
basic form, it is usually the basic form which is meant.
Acid Green Sec Light Green, SF.
:

Alizarin Red S. —
Acid; oxyqujnone group. Solubility: 7.69% in
water; 0.15% in alcohol. Used for differentiating chromatin when
crystal violet has been used for staining mitochondria. It has recently
been employed for staining chromosomes (Backman 1935).
Anilin Blue (Syn. WS
cotton blue, water blue, China blue).—
:

Strongly acid; tri amino-triphenyl methane group. During manufacture,

the exact composition of this dye cannot be controlled, hence it is rather
unsatisfactory for precise staining procedures. The dye is a mixture,
and no two lots will react alike. Nevertheless, it is very valuable as a
counterstain, especially to safranin when used on plant tissues. It will
stain cellulose walls and the achromatic figure, and (in conjunction with
erythrosin or phloxine) it is said to be excellent for the filamentous
Chlorophyta.
The dye is supposed to be soluble in water only, but an aqueous
solution is practically useless. A 1% solution in 90% ethyl alcohol
serves well; after a brief differentiation in 95% alcohol, the anilin blue
should be fixed and intensified in 95% alcohol slightly acidified with
hydrochloric acid. The most satisfactory to dissolve as
writer finds it

much stain as possible in methyl cellosolve, and then to add clove oil
and a little absolute alcohol as diluents. Use such a solution from a
dropping bottle. Clear in clove oil or synthetic oil of wintergreen
(methyl salicylate), wash in xylol, and mount in balsam.
—
Aurantia. Acid; nitro group. Solubility: nil in water; 0.33% in
alcohol. Used for the dernonstration of mitochondria.
 STAINS 57

Basic Fuchsin: See Pararosanilin.

—
Bismarck Brown Y. Basic; azo group. Solubility: 1.36% in water;
1.08% in alcohol. This stain w'as formerly much favored but has
fallen into disuse. A 1% solution in 70% alcohol gives the optimum
results. Itworks poorly on material fixed in reagents containing chromic,
acid. It rarely overstains and is quite permanent. Mucin and cellulose
walls are brilliantly colored. Solutions of the dye should never be heated,
otherwise the composition of the dy(i will be changed.
Bordeaux Red (Syn. : fast red B or P). — Acid; azo grouj). Solu-
bility: 3.83% in water; 0.19% in alcohol. A cyto])lasmic stain aftei*

iron hematoxylin. cemtrosomes more brilliant. Use a 1%

It renders
aqueous solution, and stain for 12 to 24 hours.
—
Congo Red. A(?id; azo group. Solubility: nil in water; 0.19% in
alcohol. In plant technique it is used as a cytoplasmic stain in contrast
to iron hematoxylin and other nu(‘l(‘ar dy(‘S. It also stains mucin and
cellulose. As a cytoplasmic stain, a solution in weak alcohol may be
used. Congo red should be us(‘d last, whcai employed in combination
with any oth(*r stain, and d(‘hydration should be as rapid as possi]>le.
In the presence* of free acid, solutions of this dye b(*come bliu*.
yy'Crystal Violet (Syiu.''. gentian violet).- -Basic; triamino-triphenyl
methane group. Solubility 1 .68 % in wat(*r 1 3.87 % in alcohol. Crystal
:
;

violet is the most completely methylat(‘d of the various violets. It is

one of the most useful stains and is becoming increasingly important,
especially in cytology. In many cases it affords results not obtainable
from safranin, iron hematoxylin, or other nuclear stains. In nearly
all procedures it is now used in place of the mixture of dyes known as

gentian violet, but even if the latter dye is specified, crystal (or
methyl) violet may nevertlu'less be safely substituted. In the Flemming
trijde combinations ‘^gentian violet'^ is consid(*red l)y some workers to
react better than either crystal or methyl violet in staining the achro-
matic figure, but such worker’s have apparently never bothen^d to asc.er-
tain whether other violets would not work equally well. Various lots
of crystal violet will be found to behave somewhat diffcu'ently under a
given set of circumstances.
All the violets may be used in 1% distilled water solutions. These
solutions keep for some time, but in most of the critical staining proce-
dures it is ordinarily the better part of discretion to use a freshly prepared
solution. All the violets wash out so quickly in the dehydrating alcohols
that one should either mordant the stain, use one of the special methods,
or dissolve the stain in clove oil. The clove oil solution may be kept in a
dropping bottle. another method is to make a saturated solution
Still
in clove oil and to add a few drops of this solution to a staining dish full of
xylol. This mixture is very unstable, and when in use needs to be
 58 GENERAL METHODS

brought up to strength by the addition of more stain from time to

time.
The violets frequently overstain the cytoplasm of buds and root
tips, thus making chromosome counts difficult. To avoid this, the
sectionsmay be transferred from water to 2% aqueous acetic acid for
about 30 minutes, then washed for twice the length of time in running
water.
Eosin, Bluish. —Acid; fluorane derivative. Solubility: 39.11% in
water; 0.75% in alcohol. Sometimes used as a counterstain but of little
value on plant materials. Erythrosin is far more satisfactory.
Eosin, Yellowish. — Acid; fluorane derivative. Solubility: 44.20%
in water; 2.18% in alcohol. When
mentioned, this is the type
eosin is

meant. Little used by botanists, but a most valuable cytoplasmic stain

for animal tissues, especially after a hematoxylin stain. The eosin
should be used last, and preferably in a strong alcoholic solution.
For paraffin se(^tions a 1% solution in 95% alcohol may be used;
stain for but a few seconds. For matcTial to be mounted whole, cither
in glycerin jelly orby any balsam infiltration method, us(» a 1 aqueous %
solution, and stain several hours or overnight. It. lias been recom-

mended that material l^e transferred directly from the stain to a 2%)
aqueous solution of acetic acid for 5 to 10 minutes, changing the acid
several times, then transferring to 10% glycerin, leaving about 1 cc,.
of the acid to keep the whole solution slightly acid. When the glycerin
is sufficiently concentrated, mount in glycerin jelly. By the balsam
method wash out the glycerin with 95% alcohol slightly acidified with
acetic acid, and do not drain off the last alcohol too completely l)efore
transferring to the diluted balsam. Eosin seems to keep better when the
mounting media are slightly acid.
Erythrosin, Bluish. —Acid; xanthene group (fluorane derivative).
Solubility varies according to the salt: 0.15 to 11.10% in water; 0.04 to
1;87% in al(;ohol. Used generally by botanists instead of eosin. An
excellent counterstain, and fine for staining the gelatinous sheaths of
algae (e.g.j Nosioc), Erythrosin is much like eosin in all respects but is

an iodine rather than a bromine derivative. Use a 1% solution in

95% alcohol or in clove oil; stain for not longer than 3 minutes, although
the clove oil solution stains sufficiently in about 10 seconds. Erythrosin
may be substituted for magdala red or phloxine in all schedules calling
for these two dyes.
Erythrosin a tetraiodo compound corresponding to the tetrabrom
is

compound (Conn 1936). It is the presence of the iodine

of typical eosin
in erythrosin which apparently makes it such an excellent counterstain
to crystal violet in certain cytological staining methods (Johansen
1932).
 STAINS 69

^ Fast Green FCF. —Acid; diamirj^-triphenyl methane group. Solu-

bility: 16^.04% in water, 0.33% in alcohol. This dye is far superior to
light green SF and fades praotuSly not at all. It stains more intensely
in a shorter time. Solutions should always be made up several days in
advance of using and should be weak; not over 1% aqueous or 0.1%
alcoholic. The recommended as
clove oil-absolute alcohol solution is

the one giving the optimum add enough dry dye to a mixture of
results:
equal parts of methyl cellosblve, absolute alcohol, and clove oil to give
a dark greenish solution (about 0.5%), and try it on a sample slide. If
too strong, dilute with clove oil until satisfactory. Fast green solutions
can be used for differentiating safranin, but the results in the writer’s
experience have always been rather messy and a really sharp differentia-
tion cannot be obtained in this fashion. Fast green turns blue in alkaline
solutions. On the stems and leaves of aquatic plants and with most
gymnosperm material it is generally blue to bluish-green, rarely a bright
green.
Gentian Violet. — crystal violet and nu^thyl
Sec' violet.
Iodine Green. — Basic; triamino-triphenyl nu*thane group. Closely
related to nu^thyl green. A selective chromatin stain; also excellent for
lignifi(Kl cell walls. Ordinarily used in contrast to the fuchsins. Stain
for 1 hour, or, if tin* stain washes out too rapidly, for 24 hours, in a
1 % solution in 70% alcohol.
Janus Green B. — Basic; r(‘lated to both
the safranin and azo groups.
Solubility: 5.18% 1.12% in alcohol. Used in various high
in water;
dilutions as a vital stain for fungi and for the flagella of the protozoa.
The vital stain is prepared by dissolving from 0.001 to 0.41 g. in 100 cc.
of physiological saline solution.
Light Green SF.— Acid; diamino-triphenyl methane group. Solubil-
ity: 20.35% in water; 0.82% in alcohol. An excellent cytoplasmic
stain, and very extcnsiv(dy employed for staining cellulose walls. Aft('r
certain killing fluids it will show up the achromatu* figure beautifully.
The stain, Tuifortunately, fades within a short time. Fast gro'cn FCF
is an (^xc(dlent substitute and far more permam'nt. Howc'ver, on some
filamentous algae, light green should be used rather than fast green as th(‘

latter tends to overstain badly even when irsed in very high dilutions.
The solution, in whatever medium, should not be stronger than 0.5%;
a 0.2% solution is strong enough. Staining is rapid. The solution in
95% alcohol is commonly used, but many prefer to dissolve the dye in
absolute alcohol and dilute with clove oil, keeping the solution in a
dropping bottle. The stain will reduce other coal-tar dyes, especially
safranin, consequently it should not be allowed to react too long.
If a saturated solution of light green in cither water or alcohol is

acidulated with hydrochloric or acetic, acid, a differential stain for

60 GENERAL METHODS

lignified tissues is obtained.Simple washing with water will remove the

stain from save lignified cell walls. By combining light green with
all

alcoholic Sudan IV, suberized and cutinized tissues are differentiated

from lignified tissues.
Care should be taken not to confuse light green with malachite green,
acid green, fast green, or methyl green.
—
Magdala Red. Basic; safranin group. There is much doubt as to
just what was meant by magdala red^^ in many schedules calling for
this dye. In plant technique, schedules calling for this dye give very
different results with various lots of the dye. It would be better to
substitute phloxine, which gives substantially identical results.
Malachite Green (Syn.: emerald green, light green E). Weakly —
basic; diamino-triphenyl methane group. Now largely replaced by
methylene (not methyl) green.
Malachite green may used as a 0.5% or somewhat stronger solu-
V)e

tion in either 95% or clove oil.

alcohol, distilled water, It may be
applied for 1 minute when used alone or for 20 seconds following 20 min-
utes in safranin. The safranin should be 1% aqueous and is not differ-
entiated before applying the green. When used alone, the green should
reveal clearly all such histological elements as the cell walls, endodermis,
bast, cytoplasm, nuclei, and chloroplasts. When used following safranin
on plant pathological material, the host nuclei, xylem, and cutinized
walls, as w(^ll as the nu(;lei of the infecting fungus, should appear red; the
cytoplasm and cellulose walls of the host should appear green.
—
Martius Yellow. Acid; nitro group. Solubility: sodium salt, 4.57%
in water; 0.16% in alcohol —
calcium salt, 0.05% in water; 1.90% in
alcohol. Used with malachite green and acid fuchsin for staining
infected plant tissiu^s, and used alone for pollen tubes in styles.
—
Methyl Green (Syn.: light green). Basic; triamino-triphenyl
methane group. This is generally an impure dye, as it usually contains
some methyl violet. The color thus is sometimes purple and not green,
as the name might indicate. A chromatin and nuclear stain, also for
lignified plant tissues. Very useful for staining chromatin in protozoa,
and for the gonococcus. The dye does not usually give a good stain after
fixation in a fluid that does not contain acetic acid. It does not ordinarily
keep well, according to many technicians. AJ% aqueous solution is
recommended.
—
Methyl Violet (Syn.: gentian violet). Basic; triamino-triphenyl
methane group. Solubility: 2.93% in water; 15.21% in alcohol. A
mixture of dyes (methyl pararosanilins). There are many shades,
designated by letters: R indicating the reddish shades and B the bluish.
The bluish colors are preferable as the reddish ones usually leave a
disagreeable transparency in chromosomes and other tissues. Use as
indicated for crystal violet.
 STAINS 61

Many cytologists prefer methyl violet 2B to all other types of violet

dye.
—
Methylene Blue. Basic; thiazin group. Solubility: 3.55% in water;
1.48% in alcohol. The most valuable bacteriological stain. Used to a
slight extent by zoological technicians, but rarely by botanists. It is a
good stain for yeasts, but tends to overstain.
—
Methylene Green. Basic; thiazin group. Solubility: 1.46% in
water; 0.12% in alcohol. Derived from methylene blue. Sometimes
substituted for methyl green for lignified tissues and chromatin, but
otherwise is little used.
—
Neutral Red. Weakly basic; azin series. Solubility: 5.64% in
water; 2.45% in alcohol. Used in vital staining, especially with the
protozoa, and to indicate tlui reactions of the (contents of living plant
cells.

Nigrosin (Syn.: indulin black). — Basie; composition ])oorly under-

stood: a variable mixture. A 2% may l)e made up
acpi(‘ous solution
as a stock solutionand a few drops addend to th<‘ waUu* containing (diloro-
phyta or Cyanophyta to be mounted in glycx'rin. Stop acdlon when the
stain s(*ems to be right; it should look somewhat like iron luanatoxylin.
The stain (;annot be satisfactorily used on plant material embedded in
paraffin.
Orange G. — Acid; azo series. Solubility: 10.86% in water; 0.22% in
alcohol. One of th(‘ most us(*ful cytoplasmic* counterstains and specified
in innumerabh' s(*hedul(‘s. A gemu'al ratlua* than a sc'lectivc* stain. The
medium in whi(‘h the dye* is dissolved variens according to the sptH'ific;

teehniciue. The dye usually does not completely dissolve, but 1 g. of

the dyc' to 100 cc. of solvcuit may be used. Actionextremely rapid.
is

The methyl cellosolve solution is most satisfactory, and this may be

diluted with other solvents.
Pararosanilin (Syn.: basic; fiichsin). — Basic; triamino-triphenyl
methane group. Solubility: 0.26% in water; 5.93% in alcohol, l^ara-
rosanilin is the chief c*onstituent of the majority of samples of basic;
fuchsin submitted for certification as biological stains (Conn 1936); it is
therefore; more desirable to use the name of the spcicific dye rather than a
generic; description. Basic fuchsin has never enjoyed much of a reputa-
tion in general botanic;al technique, but since it is the stain involvc^d in
the Feulgen nucleal reaction, an interesting future may be predicted. It
is a powerful nuclear dye and also stains mucin and bacteria. For
ordinary purposes a saturated aciueous solution will be found to be
satisfactory.
—
Phloxine B. Acid; xanthene group. Solubility varies according to
the salt: 3.75 to 50.90% in water; 0.45 to 29.10% in alcohol. Phloxine
has been claimed to be a good algal stain, but the results in the writer's
hands have been disappointing.
62 GENERAL METHODS
/

v/picric Acid. —Strongly

acid; nitro group. Solubility: 1.18% in
water; 8.96% Sometimes employed as a general cytoplasmic
in alcohol.
stain. In alcoholic solutions, picric acid is coming into extensive use as a
differentiator for the hematoxylins, safranin, and the violets. It is
almost invariably used as a nearly saturated solution.
—
Resorcin Blue. Basic; oxazin series. Used as a microchemical
reagent for callose, and, combined with martins yellow under the name
^^lacmoid,’^ for pollen tubes.
Safranin O. —Basic; azin (safranin) group. Solubility: 5.45% in
water; 3.41% in alcohol. This is perhaps the most important stain
known to botanists and is used in both morphology and cytology. It
stains lignified, cutinized, suberized, and cliitinized structures as well as
chromosomes, nucleoli, and centrosomes.
Safranin ordinarily dissolves better in strong alcohol than in water,
despite the contrary indications given by the solubility data mentioned
above. A stock solution may be prepared by dissolving 2.25 g. of a
certified sample in 225 cc. of 95% ethyl alcohol, and a part of this stock

solution may be diluted with an equal volume of distilled water when

needed for use. should prove to be too strong, it may be
If this solution
further diluted wnth 50%
Various other formulae for making
alcohol.
safranin solutions have been publish(‘d, and certain schedules also call
for safranin to be dissolved in particular ways; it is best to follow such
directions explicitly in order to secure the expected results.
The by dissolving 4 g. of the
waiter, for example, prepares safranin
dye in methyl cellosolve; when solution is complete, 100 cc. each
200 cc.

of 95% alcohol and distilled water are added, followed by 4 g. sodium

acetate and 8 cc. formalin. The purpose of the acetate is to intensify
the stain; that of the formalin is in the nature of a mordant. Slides are
left in the solution for 24 to 48 hours; differentiation is exceptionally

easy, giving sharp and brilliant contrasts.

Safranin invariably overstains indiscriminately and therefore requires
differentiation. This, in the past, was done in 50% alcohol slightly
acidulated with hydrochloric or acetic acid, but a much sharper differenti-
ation can be obtained by adding picric acid to the 95% dehydrating
alcohol. After tissues have been stained with safranin, the excess stain
should always be washed away with water, otherwise there is great
danger that ugly irremovable precipitates may be deposited in the
tissues.
Sudan III. —Weakly acid; azo group. Now replaced by Sudan IV.
Sudan IV Scharlach R, fat ponceau R). Weakly acid; azo
(Syn. :
—
group. Solubility: nil in water; 0.09% in alcohol. About the only
specific fat stain. Use a saturated alcoholic solution for about 10 min-
utes, and wash rapidly in alcohol. Mount in glycerin jelly. As alcohol
 STAINS 63

is a fat solvent, avoid leaving in the alcohols any longer than necessary.
Tissues should be fixed in a solution which does not extract or dissolve
fats. Lecithin, resins, latex, wax, and cuticles arc also stained by
Sudan IV chloroplasts
;
arc^ stained a dull red.
Thionin. — Basic; thiazin group. both water and
Solubility: 0.25% in
alcohol. This dye is little^ but because
called for in botanical technique,
of its metachromatic propc^rties (i.e.y the ability to impart different
colors to different c(‘ll structures) it is us(‘ful with animal tissues. To
stain chromosomes, allow a saturat'd solution' in water or rather w(‘ak
alcohol to act for about 5 minutes. Following fixation in a fluid contain-
ing mc^rcairic chloride and usc^d dilut(‘d for about 5 minutes, it is a specific
1

stain for mucin; the mucin is red, and everything else is blue.
Vital Red (Syn.: brilliant Congo red). —
Acid; azo group. An impor-
tant vital stain.

OTHER SUBSTANCES ACTING AS STAINS

Stains, frequently of a sp(‘cific type, are imparted by certain reagents
which act partly as color indicators of a microchemical nature.
Iodine. — lodirn^ is well known as a si)ecific color indicator for starch,
when made up in combination with potassium iodide. The color reac-
tions of iodine on sections of fresh material are as follows:

Blue Bkown Yellow

Starch Cellulose Pectin
Saponin Proteins Cutln
Inulin deposits Callosc
Alkaloids Cork

Osmic Acid (Syn. : osmium tetroxide). — Osmic acid has the peculiar
property of blackening certain cell inclusions. This j)roperty has been
taken to be specific proof of tln^ identity of such inclusions, but it has
recently been severely questioned. probable that osmic acid
It is highly
is reduced to an insoluble black precipitate by so many different sub-

stances that it is rather dangerous to draw inferences from its ability to

blacken various cell constituents. By using special fixatives and staining
with iron hematoxylin, more precise and reliable results may be obtained.

TABLES OF STAINS FOR SPECIFIC STRUCTURES

In the following tabulated summary the structures or substances and
stains are arranged alphabetically. No discrimination is being made
between the different stains under any particular heading since
it is

clearly recognized that under varying circumstances and on different

materials the same stain will not react equally well.
64 GENERAL METHODS
Achromatic Figure Carmin
Anilin blue Carminic acid
Erythrosin Hematoxylin
Fast green FCF Iodine green
Methyl or crystal violet Methyl green
Light green Safranin
Bulk Stains Fats
Bismarck brown Y Sudan III or IV (specific)
Carmin Lignified Cell Walls
Carminic acid Crystal violet
Harris' hematoxylin Iodine green
Callose Methyl green
'Anilin blue Methylene green
Resorcin blue (specific) Safranin
Cellulose Cell Walls Middle Lamellae
Acid fuchsin Iron hematoxylin
Anilin blue Ruthenium red (specific)
Bismarck brown Y Mitochondria
Congo red Acid fuchsin
Dclafield’s hematoxylin Aurantia
Fast green F(^F Crystal violet
Light green Iron hematoxylin
Chitin Janus green B (vital)
Safranin Nuclear (General)
CuTiNiZED Cell Walls Carmin
Acid fuchsin Crystal and methyl violets
Crystal or methyl violet Hematoxylin
Erythrosin Iodine green
Methyl green Methyl green
Methylene blue Methylene blue
Safranin (specific) Pararosanilin
Cytoplasm Safranin
Acid fuchsin Thionin
Anilin blue Plant Mucin
FiOsin Y Bismarck brown Y
Erythrosin B
Congo red
Fast green FCP"
Pararosanilin
Indigocarmin
Plastids
Light green
Crystal or methyl violet
Malachite green
Methyl orange Iron hematoxylin

Nigrosin Proteins
Orange (I Safranin
Phloxino Suberized Cell Walls
Dividing Chromatin (Chromosomes) Safranin
Brazilin Sudan III or IV (specific)
 CHAPTER VII

STAINING PROCEDURES
In this chapter t he met hods oi‘ using dyes will be dealt with. As was
intimated in th(‘ preceding chapter, the technician has at his service
some 250 diffenait dy(*s. No one (‘ould possibly h^arn how to use correctly
all these dyes, singly or in various coml)inations, whether according to
more or less rigid sp(M;ifications or empirically. This, fortunately, is a
matt('r whicdi need give us no concern. One would do well to follow the
example set by competent t(‘chnicians by selecting a very few combina-
tions and ])racticing with them on ajl sorts of tissm^s until the pro(;edure of
using ea(^h of tht\se dy(^s has been thoroughly mastered. The three
stain combinations which are undoubtedly used by a larger number
of botanical teclmic'ians than any othca* groups of dyes include (1) iron
h(‘matoxylin with or without a suitable (a)unterstain; (2) safranin and
fast green, and (3) a variant, of Flemming’s triple combination. While
all scIkkIuIcs will be outlined in sufficient detail, the foregoing three
methods will b(^ (‘lalK)rated at considerable length.
The nature of the dyeing or staining reactions which occur when
tissu(‘s are imnu'rsed in a solution of a dye are still imperfectly under-

stood. Th(H)ri(‘s to account- for the r(‘actions have ))een based almost
ex(*lusively upon eitlaa* cln'micail or physi(‘.al phenomena. A long dis-
cussion of thes(' phenomena could be }:)rt‘sent,ed, but as it is extremely
doubtful whetlun* such an a(*count would !)(» of practical value it is being
omitted. An ('xception, however, is being made in the case ofhematoxy-
lin in order to explain the necessity for the use of a mordant since the
substance by itself do('s not stain tissues.
The qu(‘stion of using buffer solutions for the control of the hydrogen-
ion (‘oncentration in staining procedures has recaaved some attention
(French 1930), but such solutions have been chiefly used with compli-
cated schedules involving compound stains on other than plant tissues.
Botanical technicians have generally ignored the emtire subject.
The staining schedules which are given below have been divided
two groups, according to whether the primary stain is
artificially into
produced by a natural dye or by a coal-tar dye. The artificiality occurs
because the two are occasionally used together; in such instances the
procedure is listed in the second group of schedules.
Solvents. —The nature of the most appropriate solvent for each dye
has been cited under that dye in the preceding chapter. Summarizing,
66
66 GimERAL METUODSi

it may be stated that the basic coal-tar dyes, as used in b,otanical tech-
nique, are dissolved in either water or 50% ethyl alcohol; the acid dyes
employed as cytoplasmic counterstains are usually dissolved in an alcohol
of high percentage' or in clove oil, because^ they ordinarily wash out very
easily;hematoxylin may be dissolved in either water or absolute alcohol
but never used without mordanting; thc^ carrnins are always in aqueous
is

solution with the eaistomary addition of various salts or acids.

Under certain cir(nimstanc(‘s it may b(' desirable to employ a solvent
not ordinarily used with a ])articular dy(\ Whether such solvents can
be employed dei^ends entirt'ly on whc'ther tlu' dy(^ or dyes will dissolve in
them. This can be ascertained only by trial unless one has a record of
such a solvent having been used by others. There is, of course, no reason
why solvents other tlian those specified cannot be employed, but th('
effects of the solvent upon the tissues themselv(is and upon other stains
(if any are used) should be can^fully watched, and the necessjiry adjust-
ments madc^.
The basic coal-tar dyes are often dissolved in acetin (glycc^rin mono-
acetate), the acid dyes in an alc.ohol-etlu'r such as methyl (H'llosolve or in
beechwood creosote. Lactic acid or leonlinic a(dd (dissolve the colorless
crystals in either wat('r or alcohol) is a good solvent of the indulins
(^^alcohol-soluble’’ indulin and nigrosin).
—
Strength of Solutions. In the preceding (*hapter the extent to which
each dye will dissolve in water and alcohol lias been indicated wherever
known. These figures hold only for certified dyes of known dye
content.
On all dyes c.ertified by the Stain Commission is printed
the labels of
the totalamount of soluble dye contained in each batch tested. If the
dye content amounts to 80% or more, one may consider it sufficiently
high for most practical purposes. It is neither necessary nor desirable
to split hairs in su(;h matters because it is the general rule to overstain
tissues and then to differentiate the stain until the desired intensity or
depth of staining has been acquired. This means that a dye certified as
having 80% or higher dye content may be used in the proportion of 1 g.
to 100 cc. of solvent to give a 1 %
solution. However, if the dye content
is below 80%, appropriate adjustments in the weight of the dye used to

make a solution of specific percentage must be made. For example, if

the dye content is given as 50%, 2 g. of such dye to 100 cc. of solvent
would be required to make a 1% solution. On the other hand, if a
saturated solution is indicated, the problem is practically devoid of
complications.
Dyes may now be purchased put up in capsules which when dissolved
in the appropriate solvent will give a solution of a specified strength or
percentage.
 STAINING PROCEDURES 67

The solubility of certain dyes is accelerated by the application of heat,

but some dye users have exceeded the bounds of common sense in this
respect. Despite contrary recommendations, the violets gentian, —
crystal and methyl —
should never be boiled because the heated solutions,
whether filtered or not, .generally leave unsightly irremovable precipitates
in the tissues.
Many dyes remain in good (condition for only very short periods, no
matter what the solvent. It is as a rule very risky to use spoilt solutions.
—
Mordants. The earmins and hematoxylins are always used after or
concurrently with various mordants, the salts of different metals serving
this purpose.
The coal-tar dyes do not as a rule require mordants but many t(‘ch-
nicians liave little' strategems designed to improve the selectivity or
intensity of certain of tluise dyes, tiosin and ('rythrosin, for instance,
are supposed to be improved by the addition of a trace of a(*(‘tic acid or
to be differemtiated in some solve'.it acidified with this acid. Barium
chloride (2 to 4% solution) has bec'U us(‘d as a mordant for the' acid dyes;
silicotiingstic acid (the' technical quality in a 4% aqueous solution) for the^
basic* dyes. Other mordants include potassium permanganate (a 1%
solution), ammonium (ihromate (to 4%) or ammonium dichromate (to

3%), aluminum hydroxide* or aluminum potassium sulphate (‘‘potash

alum”) (to 3 or 4%). All tliese* are bc'st used for 5 to 10 minutes on the
slides afte'i* they have l)een l)rought down to watc'r. Exea'ss mordant
should always be' was he ‘el off with water be'fore the slide\s are' plac'ed in the
staining solution. The' additie)n of a- traca^ of lithium carbonate', is claimed
to improve the ae*tion of the basic dyes.
Iodine and picrie* ae*id are can ployed as mordants for the various violet
dyes after the stains have be'e'ii allowe*d to react on the tissues.
The pH of the fluid use'd for washing out and differe'.ntiating the coal-
tar dyes determines the sc'lectivc' retention of the stain to a far greater
extent than has laeen generally realized. Insufficient experimental work
has been done upon this important problem. Basic dyc'S should as a rule
be washed out with solutions which are definitely acid in reaction, while
most acid dyes (which are all used as counterstains) should be differ-
entiated in solutions which are slightly alkaline. It would be far better
if the solution containing the acid dye were buffered to a specified pH,

determined by trial, as affording a satisfactory stain which required no

differentiating. It seems possible that the excellent results occasionally
obtained with counterstains were due to one^s having unintentionally
gotten the optimum pH.
Effects of Fixatives on Stains. —
It is not so much the chemicals of the
fixatives themselves that affect the later staining of various plant mate-
rials as the chemical nature of the various tissues plus the compounds
68 GENERAL METHODS

resulting from chemical reactions involving these tissues and the reagents
in the killing and fixing fluids which to a great extent determine the
results that are obtained with different dyes.
Let a few examples be taken. (1) Safranin has a strong affinity for
chromatin. Fluids containing chromic acid and formalin, perhaps by a
sort of mordanting action, leave chromatin-containing structures such as
nuclei and chromosomes in a nearly perfect condition for staining with
safranin. If, however, the chromic acid reacts with substances in the

cells to leave them colored dark brown, the safranin is correspondingly

dulled, the dullness and brownness increasing with the intensity of the
reaction between the acid and the cell contents. A point will be reached
where there no longer any differentiation between cytoplasm and other
is

cell contents (as when the tissues are saturated with tannin compounds),

and bleaching as a prelude to staining becomes necessary. (2) Some

killing fluids preserve the plastin of mitotic figures perfectly; of’ these
fluids some mordant the plastin so that it stains, but others fail to mordant
it. This explains why certain staining procedures which are supposed
to reveal the so-called “spindle fibers’^ occasionally fail: the fault is not
because the staining was not carried out properly but because the struc-
tures involved were inadequately mordanted by the preservative.
Navashin^s fluid, for example, preserves plastin but rarely mordants it.
(3) The triple combinations work as they should only on material origi-
nally fixed in a fluid that contained osmic acid or on sections that were
mordanted in a weak solution of osmic acid in chromic acid. Otherwise,
the chromosomes tend to appear purple rather than red, i.e., they have an
affinity for the violet rather than for safranin.
Bleaching. — Sections cut from materials which were excessively
darkened by the fixing fluid require to be bleached before a proper stain
can be obtained. One should not, however, bleach sections without
being fairly certain that it should be done because bleaching frequently
is more or less damaging to the tissues. Many types of material which an^
naturally dark (such as the mechanical tissue in fern rhizomes) do not
actually require bleaching and can be beautifully stained if a proper
selection of dyes is made.
Bleaching fluids have been described elsewhere (see page 21) but a few
additional methods are being described below.
For the bleaching of sections to be stained in a triple combination, see
under StockwelFs variation of this combination (page 85). This proce-
dure is also excellent for removing excess phlobaphene precipitates.
If the sections appear to have suffered from too much chromate
fixation, immerse the sections for 1 minute in a 1% aqueous solution of
potassium permanganate, rinse, remove the permanganate in a 1%
aqueous solution of oxalic acid, wash again, and proceed to the staining.
 STAINING PROCEDURES 69

Chlorine gas fumes bleach excellently. Place enough crystals of

potassium chlorate to approximate the size of a grain of wheat in a coplin
and pour on a little dilute hydrochloric acid. As soon as the greenish-
yellow fumes become apparent, fill the jar quickly with 50% alcohol.
About 20 minutes^ minimum immersion of the vsections is required to effect
bleaching. Sections, however, cannot be stained if commercial bleaching
fluids containing chlorine are used.
Boric acid solutions have been used for bleaching as has a 2% aqueous
solution of ammonium persulphate. The latter should be used with
caution as it is a pow(n’ful oxidizer.
The blackening caused l)y osmic acid may be removed by dissolved
chlorine gas or by immersing the sections in hydrogen peroxide diluted
one-half with water. The process may be hastened by pla(‘ing the con-
tainer in the sunlight.
To reston^ the staining capacity of tissues, bleach if necessary, then
soak for 15 minutes in a 10% solution of benzoyl peroxide in acetone;
wash out in a solution of 2 parts xylol to 3 of acetone, followed by absolute
alcohol.
Differential Acidification. —In order to intensify and localize stains in
tissues whose elements do not present sufficient contrast in their relative
degree of acidic or basic reactions, the hydrolysis phase of Fculgen’s reac-
tion may be employed to render nuclei more acid and thus to give them a
greater affinity for nuclear stains. This procedure is especially adaptable'
to more or less thick materials which are to be mounted — such as
entire
fern prothallia, Ectocarpus, root tips, Elodea leaves, and similar objects.
The slides or fixed materials are brought down to water, placed in cold
1/N hydroe^hloric acid for exactly 1 hour, rinsed with one change of dis-

tilled water, and then placed in the nuclear stain. The acid must not be
heated, as is done in the regular Feulgen technique, otherwise the material
is very likely to become dissociated.

—
Progressive and Regressive Staining. By observing the progress of
the staining under the microscope from time to time, any desired intensity
may be attained. This is known as ^^progressive staining.^^ A sharp
differentiation usually cannot be obtained by this method. Conse-
quently, the general practice is to overstain considerably and then to
destain or otherwise to differentiate until a satisfactory optimum has been
reached. This is known as regressive staining.^^ Practically all
the schedules given in the present chapter concern regressive staining
procedures.
—
General and Specific Stains. A general stain is one that stains every-
thing indiscriminately. There is little or no selective differentiation, but
such may to a slight extent be obtained by regressive destaining. iota

staining of some classes of plant material represents this type of stain;

70 GENERAL METHODS

what dififerentiation occurs is usually produced by the dehydrating fluids.

Botanists find it more desirable to use specific or selective stains because

they need to distinguish the structural elements of tissues, between
lignified and nonlignified cell walls. The after treatment of the stain
determines almost wholly the degree of selectivity that is attained.
Some dyes are highly selective. Sudan IV, for example, stains only
fats or fat-containing elements such as suberin, Congo red is almost
specific for plant mucins, and alkanet gives a bright red color to oils.
In plant microtechnique, dyes have occasionally been classified
according to the structures for which they have the most marked affinity.
Some dyes may be found in more than one category, safranin being a
prominent example. Of course, the selectivity of a dye may by appro-
priate chemical reactions be made to reverse its usual affinities, but such
procedures may be considered as being unnatural. At the end of the
preceding chapter a list of dyes recommended for particular structures is
given, and reference may be made to this list whenever one is uncertain
what stain or combination of stains to use.
THE NATURAL DYES
Cochineal, Carmin and Carminic Acid
Mayer’s Alum Cochineal. — Dissolve
12 g. aluminum potassium
sulphate in 160 cc. distilled Bring to a boil, add 12 g. powdered
water.
cochineal, and continue the boiling for 20 minutes. Pour off the clear
supernatant liquid after th6 mixture has cooled, add more water to the
cochineal, and boil again. Decant as before, adding this liquid to the
first, filter, and evaporate down to 160 cc. To prevent the growth of
molds, add a small crushed crystal of thymol.
This solution is suitable for in toio staining. The time required is
from 24 to 48 hours. Wash in water for 20 to 60 minutes to remove the
alum. No differentiation is required, but to prevent the stain from
becoming extracted, run up through graded alcohols to 70% alcohol.
The material, if of a suitable type, may be mounted whole in balsam if
first carried through hygrobutol; or the material may be embedded and

the sections counterstained with Bismarck brown (in 70% alcohol) or

other suitable stain.
—As used on smear preparations,
Iron-Acetocannin. this stain is fully
described in the chapter onSmear Methods.
Grenacher’s Alum Carmin. —Boil powdered carmin
1 g. for 10 to 20
minutes in a 5% aqueous solution of aluminum ammonium sulphate.
Filter when cool. This is one of the easiest carmin stains to use since it
rarely overstains. After the killing fluid has been washed out, transfer
bulli material to the dye, and leave until it appears to be properly stained;
this may occasionally be a matter of several days. Dehydrate (preferably
 STAINING PROCEDURES 71

with hygrobutol, acetone, or dioxan), embed, and section. After the

sections have dried to the slides, remove the paraffin with xylol, and
mount in balsam.
With many materials some differentiation should be carried out.
After the material has been in the stain for a day or longer, transfer to
acid alcohol (2 drops hydrochloric acid to each 100 cc. of 70% alcohol)
until the color changes to a clear red. The time required varies from 1
hour to 2 or 3 days. Wash in a few changes of 70% alcohol to get rid
of the acid, then complete dehydration, and proceed to the embedding.
Alcoholic Borax Carmin. —
Dissolve 2 g. carrnin in 100 cc. of a 4%
aqueous solution of borax by boiling for 30 minutes. Set aside for at
least three days, then dilute with an equal volume of 70% alcohol, “and
filter. Leave the material in the stain until it appears to be thoroughly
stained. Differentiate in acid alcohol (5 drops hydrochloric acid to
each 100 cc. of 70% alcohol) until the material becomes clear and trans-
parent. Wash
out the acid with a change or two of 70% alcohol, then
j)roceed with the dehydration. This is perhaps the best stain for bulk
staining of plant materials^.
Mayer’s Carmalum.- Dissolve 1 g. carminic acid and 10 g. aluminum
ammonium sulphate? in 200 cc. distilled water, using heat if necessary.
Filter, and add 0.2 g. salicylic acid or a small crushed crystal of thymol to
avoid mold growth. This is an excellent m toto stain as one may control
the differentiation satisfactorily. If it is desired that the cytoplasm
remain stained, wash out the excess stain with distilled water; if a purely
nuclear stain is wanted, use a 5 to 10% aqueous solution of aluminum
ammonium sulphate for differentiating.
Some types of materials may be given -a double stain if 1 volume of a
saturated aqueous solution of picric acid be added to 10 volumes of the
carmalum solution; or 1 volume of a 0.2% aqueous solution of indigo-
carmin to 4 to 6 volumes of carmalum. One might also experiment
with other counterstains, which should be used in high dilutions.
—
L3mch’s Borax-Carmin. This is one of the most superb in toto stains
for small animals and th6 protozoa, and there is no reason why it cannot
succeed equally well on various types of plant material which are suitable
for staining with carmin. The following variation has worked well on
the Volvocales:
1. After fixation, wash out the killing fluid and run up to 50% ethyl
alcohol.
2. Stain in Grenacher’s alcoholic borax-carmin, made up with
Griibler’s ‘^rubrum opticum ” carmin (other samples of carmin dye have
not worked successfully), and diluted in the proportion of 12 cc. stain to
100 cc. of 70% alcohol and 1 cc. hydrochloric acid, for 2 hours or as much
longer as might be required to penetrate the objects thoroughly.
72 GENERAL METHODS

3. Agitating the container constantly, drop in hydrochloric acid with

a pipette until a barely perceptible brick-red precipitate is produced.

Avoid excess acid. Stir well, then let remain overnight.
4. Decant the stain,and transfer material to slightly acidulated 70%
alcohol. Differentiate by adding 5% or stronger hydrochloric acid, and
wash with several changes of 80% alcohol when the differentiation appears
to be satisfactory.
5. If a counterstain is desired, a saturated solution of indulin (Griibler)
in 80% ethyl alcohol may
be added to the 80% alcohol containing the
material until appears a light blue. Watch the process as the indulin
it

is difficult to remove if allowed to overstain. A few minutes to 1 hour

may be required. Stop action of the stain by pouring off the 80% alcohol
and adding 95% alcohol.
6. Complete dehydration with absolute alcohol, pass through absolute
alcohol-xylol, pure xylol, and mount in balsam.

Brazilin, Hematoxylin, and Hematein

Brazilin. —This dye is scarcely used in general botanical technique

but is valuable on smear preparations (page 163).
—
Heidenhain’s Iron Hematoxylin. This is by far the most important
stain in the technician's repertoire and is the one which above all others
he should strive to master completely as soon as possible. For this
reason the method of using this stain is being described in considerable
detail. All technicians use fundamentally the same solutions, but no
two follow exactly the same schedule. Som(^ even protest that the
procedure cannot be written down in precise terms since so much depends
upon the individual using it. It is almost wholly a matter of personal
idiosyncrasy: one should study the outline described below, striving to
understand fully the reason for each step, experimenting with all sorts
of properly prepared material, and noting the differences which ensue
upon varying the periods in the mordant and in the staining solution
itself. After that, one will soon find that he has practically formulated
a schedule of his own, which can readily be adapte^d to fit the necessities
of particular cases as they arise during the course of his work.
1. Bring sections down to water (i.c., remove the paraffin with xylol,

and pass through a mixture of equal parts of xylol and absolute alcohol,
allowing 10 minutes in each; then pass into a mixture of equal parts of
absolute alcohol and ether plus 1% celloidin for 3 minutes, remove slides,
and keep in air until the sections become opaque but are not completely
dried out; plunge into 70% alcohol for 5 minutes, and finally pass through
35% alcohol into water).
2, Wash thoroughly with water, and finally rinse in distilled water.
 STAINING PROCEDURES 73

3.Mordanting is now carried out. Hematoxylin by itself does not

stain,even after it has been ripened by oxidation into hematein.
(However, if the tissues contain traces of iron, copper, or aluminum, the
dye will stain such tissues without mordanting.) The chemical most
commonly used for mordanting is ferric ammonium sulphate, colloquially
known may also be used. The
as “iron alum/^ although other substances
crystalsmust be a clear violet color; any that have turned yellow should
be discarded. The strength of the solution is usually 3% some tech- —
nicians use a 2%, others a 4% solution. Weaker strengths should be
used on more delicate objects, such as filamentous algae. Solutions must
be freshly prepared and used only once; or a quantity of the following
mixture, which keeps perfectly, may be made up: to 500 cc. distilled
water add 5 cc. glacial acetic acid, 0.6 cc. c.p. sulphuric acid, and 15 g. of
the alum crystals. The alum may be pulverized in a mortar to facilitate
solution.
The time required for mordanting should not exceed 2 hours. An
hour is a longer time for diluted solutions or
sufficient with thin sections,
thicker sections. Sections over 12m thick cannot be satisfactorily stained
with iron hematoxylin.
4. Wash thoroughly in running water for 5 minutes.

5. Rinse in a change of distilled water. (This step is necessary if

the tap water is hard. In any event, any water containing a high per-
centage of dissolved salts will min the staining solution.)
6. Stain in 0.5% aqueous hematoxylin, prepared as described in the
preceding chapter (page 50). As a rule, the slides should be left in th(^
stairi at least as long as they remained in the mordant, but in most cases
24 hours is the optimum time.
7. Wash off excess stain with water.
8. Destain in 2% ferric ammonium sulphate (or use ferric chloride),
using a large volume of the The
tim(‘ which will be required for
fluid.

proper destaining varies with the material, the thickness of the sections,
and other variable factors. The slides may be arranged around inside a
stender, taken out in order, one by one, and examined on a glass plate
placed on the stage of the microscope. If a glass plate is not used, the
microscope will be ruined by the iron compound. Put the slide on the
plate (section side up) while dripping wet, and examine quickly to judge
whether differentiation is proceeding properly. Watcli chromatin-
containing bodies only, rather than judge by other structures which may
have been colored by reagents other than the hematoxylin. As soon as a
slide seems just right, place in a flat staining dish filled with water.
Remember a considerable difference between tlie refractive
that there is

indexes of water and balsam; the stain when just right looks grayisli-
black while in the water. When destaining bulk material, one will learn
 74 GENERAL METHODS

to look for a grayish fluorescence emanating from the material: when it

appears, it is a fairly safe indication that differentiation has progressed far

enough.
9. Wash in running water for at least 30 minutes, but preferably for 1

hour. The washing should be as thorough as possible; if traces of the

destaining solution remain in the tissues, their continued action will cause
the preparation to fade completely.
10. Dehydrate in 50, 70, and 95% alcohols, allowing at least 5 min-
utes in each.
(11. Counterstains are sometimes undesirable, but if one seems
necessary or desirable, orange G, gold orange, or fast green may be used.
These are best used in methyl cellosolve-absolute alcohol-clove oil

solution. Remove a slide from the 95% alcohol; with a clean cloth
quickly wipe dry the underside, hold the slide level, and apply with a
pipette just enough staining solution to cover the sections completely.
Let remain a few moments, then pour the stain back into the bottle.
Wash off with waste clove oil diluted considerably with equal parts of
absolute alcohol and xylol; then clear in a mixture of equal parts of
clove oil, absolute ethyl alcohol, and xylol; wash with xylol plus a trace
of absolute alcohol to take care of any moisture that might be brought
over, then proceed to step 13.)
Absolute alcohol and xylol, equal proportions, 5 minutes.
12.
13. Xylol,two changes, 5 minutes in each.
14. Mount in balsam.
The periods noted for steps 3 and 6 are merely suggestive and need
more or less to be determined for each type of material. Bryophytes,
for instance, differentiate with extreme rapidity, but most root tips
differentiate comparatively slowly. When the slides are taken from the
stain, they should have the appearance of being badly overstained every-
where. The excess stain will come out in clouds at first, but differentia-
tion of chromatin will proceed slowly. After a little experience in
judging appearances under the microscope, it can be controlled to a
nicety, and any desired intensity of stain may be obtained., A sharp
black-and-white contrast is undesirable; there should be variations in
color from light gray through gray to black.
Iron Hematoxylin and Bismarck Brown. —
This particular combina-
tion was designed for use on the phloem tissues of woody plants and is a
very useful combination for these structures (Harrar 1928).
1. If sections were cut freehand or on the sliding microtome, place

in a 2% aqueous solution of ferric ammonium sulphate for 20 minutes.

If materials have been killed and fixed, the time may be reduced by half.
2. Drain mordant, and wa^b in M
le^t five changee of distilled water
to insure removal of mordant,
 STAINING PROCEDURES 75

3. Flood with distilled water, and add 2 or 3 drops of 1% aqueous

hematoxylin. Watch progress of the stain under the microscope, and
stop at the desired point by quickly changing to water.
4. Change the water two or three times, then cover the sections

with a 1 %aqueous solution of Bismarck brown for 3 to 4 hours, depending

on the thickness of the sections.
5. Wash out excess stain with water.
6. Dehydrate gradually, and use at least two changes of absolute
alcohol to remove all traces of water.
7. Clear with xylol, and mount in balsam.
Stone hard woods stain a cherry red, bast fibers a brilliant
cells of

orange, the ray cells and other parenchymatous tissues a chestnut brown,
and the middle lamellae a dark blue. The bast fibers, parenchymatous
tissues, and middle lamellae of Coniferophyta stain as indicated, but the
stone cells turn a vivid burnt orange.
Iron Hematoxylin and Safranin.— As originally intended, this com-
bination is best used after a killing fluid containing picric acid as one of its
constituents, but it has been successfully used after chromic a(;id fixatives.
The completed preparation, which should be examined with an immersion
lens, is frequently of great beauty. The particular value of the combina-
tion is to enable one to differentiate between nucleoli, which take a bright
red color, and other nuclear constituents in plants with large nuclei. The
chromosomes during metaphase and anaphase are a bright red and stand
out sharply against the dark background during prophase and telophase
;

the chromosomes are much darker. Trabants are easy to identify, they
often being a bright red and connected to the chromosome by a black
thread.
1. Bring theslides down to water (see p. 72).
2. Mordant in 3 % aqueous ferric ammonium sulphate for 2 to 3 hours.
3. Wash running water for 5 minutes, then rinse in distilled water.
in
4. Hematoxylin solution. (Make a 10% solution of the crystals in
absolute alcohol. For use, dilute 10 cc. of this solution with 90 cc. of
distilled water.) Leave the sections in the stain as long as they were left
in the mordant.
5. Differentiate in 3% aqueous ferric ammonium sulphate with great
care. Pay especial attention to the decolorization of the nucleoli. When
they are almost colorless, stop action by transferring the slide to water.
6. Wash with running water for at least 1 hour.

7. Safranin solution. (Originally equal quantities of saturated solu-

tions in 95% alcohol and anilin water, respectively, were specified. The
older the anilin water solution, the better the action of the stain. How-
ever, any standard solution will serve.) Leave in the stain for 12 to 15
hours.
76 GENERAL METHODS

8. Differentiate either in 70% alcohol acidified with a few drops of

hydrochloric acid or use 95 %
picro-alcohol for not longer than 10 seconds.
9. Proceed as usual to balsam.
Alcoholic Iron Hematoxylin. —
Sometimes the alcoholic solution of
hematoxylin affords better results than does the aqueous solution. In
other cases, as with many of the marine algae which are with the utmost
difficulty retained on the slides and would wash off if brought down to
water, only the alcoholic hematoxylin can be used. The following
schedule has consistently given a beautiful and sharp stain.
1. Bring sections down to 70% alcohol.

2. Mordant for 5 hours or longer in a solution composed of 10 parts

of 50% alcohol and 1 part of a 4% aqueous solution of ferric ammonium

sulphate.
3. Rinse briefly in 70% alcohol.
4. Stain for 12 to 24 hours in a hematoxylin solution made up of 10
parts of 70% alcohol and 1 part of the following hematoxylin solution:

Hematoxylin crystals 1 g.

Absolute ethyl alcohol 10 cc.

Distilled water 90 cc.
Thymol Small crystal

Dissolve the hematoxylin in the alcohol, then add the water.

5. Differentiate in the same solution that was used for mordanting,

which should finally be discarded. Watch the progress of the destainirig

under the microscope; it will probably take a long time.
6. Wash thoroughly in several changes of 70% alcohol to remove the

mordant.
7. Dehydrate in 95% and absolute alcohol.

8. If a counterstain is desired, a solution of orange G in clove oil is

preferable. Use the methyl cellosolve-absolute alcohol-clove oil solution.

9. Xylol, then moutit in balsam.

Harris’ Hematoxylin. —
This stain was originally devised as a sub-
stitute for Delafield\s hematoxylin in zoological technique but has proved
to be more satisfactory than the latter stain with most plant materials,
such as fern prothallia, the Rhodophyta, many fungi and similar subjects
which are to be mounted whole, and for cytological staining.
1. Bring slides down to water, or wash the killing fluid out of the

material with water.

2. Stain for about 20 minutes in the hematoxylin solution.
3. Rinse in distilled water until all the excess stain has been washed
away.
4. Destain sections in acid water (about 5 drops or less of hydrochloric
acid to each 100 cc. of water). As a rule, the time is about 5 seconds, but
 ,;

STAINING PROCEDURES 77

one can easily stop the action of the acid, rinse the material with tap water
(which will ^^blue^^ the stain), and examine under the microscope. Bulk
material will require a considerably longer period.
5. Wash for a few moments in tap water. If the water is not suffi-

ciently alkaline to ‘^blue^! the material, add 1 or 2 drops of ammonia to

the water. If it is desired to clear the cytoplasm, place the slides or
material in a weak solution of lithium carbonate in water. About 10
minutes suffices.
6. Rinse in water again, then proceed with the dehydration as usual.

If a counterstain is desired, erythrosin is very satisfactory for mariin^

algae and also for many fresh-water forms (such as Batrachospermum)
fungi, and other cryptogams, while fast green may be used on fern pro-
thallia and bryophytes. Both dyes should be in fairly strong solution,
and the sections or material should be left in the stain until compk^tely
and evenly stained.
Delafield’s Hematoxylin. —
thoroughly rip('ned solution should
always be used.
1. Transfer to th(^ stain from eithcu* water or 50% alcohol. The
length of time the sections may be left in the stain depends partly on th('
character of the mat('rial, partly on the nature of the killing fluid, but
mainly on the thoroughness of the washing out of the latter. Som(‘
materials are well stained in 3 minutes, while others require as long as
30 minutes. As a trial, a few slides may be left in the stain for 10 minutes
if the time seems too short or too long, the duration of staining may
accordingly be regulated for future batches of the same material.
2. Wash running tap water a few minutes to remove completely
in
all excess stain.The washing should be as thorough as possible to avoid
the formation of troublesome precipitates.
3. Treat with acidulated water: add about 2 drops of hydrochloric
acid to each 100 cc. of water. The sections, which should preferably
be slightly overstained, are treated for a few minutes until they turn a
pale pinkish-purple, but care must be taken not to extract too much of
the stain. Then transfer quickly to water, and wash in running tap
water until the sections acquire a rich purple color. If the tap water
is not sufficiently alkaline, immerse the slides for a few moments in

water to which a little sodium or lithium carbonate has been added,

then complete the washing.
4. Pass through 50, 70, and 95% alcohols. A few minutes in each
strength is sufficient.

5. If desired, mount directly from 95% alcohol in euparal or diaphane.

These mounting media possess the property of intensifying hematoxylin
stains. Otherwise pass through absolute alcohol, equal parts of absolute
alcohol and xylol, then xylol, and mount in balsam.
78 GENERAL METHODS
A counterstain is ordinarily of not much value on plant tissues,
but most animal tissues are improved by counterstaining with eosin Y.
From step 4 in the above schedule, proceed to the counterstain dissolved
in 95% alcohol; allow to react for about 3 to 5 minutes, then wash excess
stain in a change of 95% alcohol. Mounting may be in euparal or
diaphanc, or dehydration may be completed in absolute alcohol and the
slides passed through xylol to balsam.
Orange G or erythrosin B in clove oil solution may in some instances
prove satisfactory counterstains. Stain on the slide after dehydrating
in absolute alcohol, clear in clove oil, wash in xylol, then mount.
The clearing fluids and balsam are by most technicians required to
be perfectly neutral in reaction, otherwise the rich purple color will
turn red and finally disappear if these reagents are in the least acid in
reaction.
Sass^ Modified Mayer’s —
Haemalum. Primarily a histological stain.
Bring slides down to tap water.
1.

2. Leave in the following staining solution until the dc^sired depth

has been reached (in about 15 to 30 minutes) Dissolve 50 g. aluminum

:

amrnonium s\ilphatc in 1 liter boiling water. Remove from the heat,

and add 1 g. hematoxylin crystals. When dissolved, add 0.2 g. sodium
iodate, cool, and filter. Later filter whenever a metallic scum appears.
The solutiou is best used fresh but will keep for a few months.
3. Wash in distilled water, then in tap water (or in J/foo sodium or

lithium carbonate) and again in distilled water.

4. Dehydrate, and clear as usual.

Any desired counterstain may be used. Nuclear selectivity may be

increased by the addition of acid: the alum can be increavsed to saturation
and 2 to 5% of glacial acetic acid added. The results vary with the
nature of the fixative used.
Komhauser’s Modified Mayer’s Haemalum. The customary pro- —
cedure of adding sodium iodate as an ingredient of Mayer's haemalum
has been criticized on the ground that the life of the solution is shortened
so that it turns brown and fails to stain (Kornhauser 1930). The
revised method is as follows:
1. Run the sections down to water.
2. Stain an average of 5 minutes in the hematein solution prepared
thus: grind 0.5 g. hematein (of MacAndrews and Forbes manufacture)
in a glass mortar with 10 cc. of 95% alcohol, and add the mixture to
500 cc. 5 %
aqueous aluminum potassium sulphate. The solution is ready
for immediate us(\
3. Rinse for a few seconc^s in tap water. It is claimed that, if the
rinsing is quick, a little of the alum solution is carried over and serves
to fix the counterstain in the tissues.
 :

STAINING PROCEDURES 79

4. Counterstain with any suitable acid counterstain dissolved in

distilled water. The method was originally devised for animal tissues,
and eosin was therefore specified. This dye, of course^, is of little value
on plant tissues, consequently erythrosin B is the first substitute to be
chosen. If, on washing with tap water, the counterstain (;omes out,

dehydrate as far as 95% ethyl alcohol, and apply the desired counterstain
from the usual alcoholic clove oil solution.
5. Complete dehydration, pass through xylol, and mount.

Ehrlich’s Hematoxylin. —
An excellent in iota stain for algae, fungi,
small bryophyt(\s, and similar subjects.
1. Bring slides down to 50% alcohol (or freehand sections or bulk
material up to 35% alcohol).
2. Stain 5 to 30 minutes in the following solution

Distilled water 100 ee.

Absolute alcohol 100 cc.
Glycerin 100 ce.
Glacial acetic acid 10 cc.
Hematoxylin crystals 2 g.
Aluminum ammonium s\ilphate In excess

Ripen in the light until the solution acquires a dark red color. If kept
well stoppered, the solution keeps for years. Hemat(‘in may be sub-
stituted for th(‘ hematoxylin; 0.4 g. is the correct amount.
3. Wash out ex(!ess stain with 50% alcohol.
4. For woody tissues counterstain with safranin, oi* proceed with the
dehydration and counterstain with erythrosin B or orange G in clove oil.
5. Clear, and mount in balsam.

Ide-Roza Variation of Weigert’s Hematoxylin. mordant is The

mixed with the stain; and an excellent general stain is produced:
Weigert’s hematoxylin (dissolve 1 g. hematoxyliu in 10 cc. of
absolute ethyl alcohol and dilute to 100 cc. with distilled water) 15 cc.

Double distilled glycerin 5 cc.

Mordant (5 g. ferric ammonium sulphate and 6 g. aluminum

chloride in 100 cc. distilled water) 5 cc.

Stain for 12 to 24 hours, and differentiate with 2 to 2.5% aqueous ferric

ammonium sulphate. The staining appeals much more uniform through-
out a section in spite of accidental variations in thickness, a feature not
obtainable with other hematoxylin schedules.

THE COAL-TAR DYES

In employing coal-tar dyes, it is important to use the basic dye first,
if two stains are being used in combination. The basic dyes are generally
the non-cytoplasmic stains; they stain such prominent structures as
nuclei, chromosomes, and lignified cell walls. The basic dyes are usually
80 GENERAL METHODS

dissolved in alcohol of around 70%, some in water, a few in anilin water,

one or two in clove oil, and rarely in other solvents. Of course, one may
use a single dye, but the final result is invariably improved by the

employment of a contrasting stain. Sometimes two basic dyes may be

used together (as safranin and crystal violet), but the rule is for an acid
cytoplasmic stain to follow a basic stain.
Most of the coal-tar dyes are used as regressive stains. A few
may be employed, in high dilutions and with continual scrutiny of the
sections, as progressive stains; this type of staining has most frequently
been applied to counters taining, particularly of filamentous algae foi-
whole mounts.

Safranin and Combinations

Safranin is the most important and valuable of the coal-tar dyes to

the plant technician, just as the violets are indispensable to the cytologist.
It iseasy to put a slide into safranin: the problem is to obtain an accurate
differentiation. The custom formerly was to use a solution of hydro-
chloric acid in alcohol, but many of the more discriminating technicians
prefer to use picric acid,which seems both to differentiate and to mordant.
Advantage taken of the fact that fast green not only provides an
is

excellent counterstain to safranin, but serves to remove the red com-

pletely from structural elements where its presence is undesirable. In
fact, a fast green solution alone may be used for differentiating safranin,
but both colors finally appear too dull for this procedure to deserve
recommendation.
When a safranin stain is specified, the dye known as safranin O is
always meant; if a safranin of another type is required, it is indicated
by the letter Y or B, as the case may be. Both safranin Y (yellowish)
and safranin B (bluish) are on the market; both are worthless in botanical
technique as they have never given results at all comparable in brilliancy
and accuracy of differentiation with safranin O. There are two types of
safranin 0 on the market, one which acts slowly and the other rapidly,
but they can be recognized as such only by trial.
In using""safranin, one should always remember the fundamental rule
of washing out the excess stain with water before proceeding with the
differentiation. If this precaution is not observed, an irremovable pre-
cipitate will be produced in the sections; this is particularly liable to
happen if the picro-alcohol method is used for differentiating.
—
Safranin and Fast Green. Long experience with this schedule leads
the writer to prefer it above all others. It has proved to be wholly
dependable on sections of almost every type of plant material except the
algae.
 STAINING PROCEDURES 81

1. Bring slides down to 70% alcohol (or freehand sections up to

^5% and give any special treatment which the nature of the
alcohol),
killing fluid might render necessary.
2. Stain in a 1% solution in methyl cellosolve~50% alcohol (see

page 62 for formula) for 2 to 24 or even 48 hours. Gymnosperm

material should have the minimum period.
3. Wash excess stain with running water for a few moments.
4. Simultaneously differentiate and dehydrate with 95% alcohol, to
which 0.5% (i.e.j almost to saturation) picric acid crystals are added (if
the acid comes in the moist form, make allowances for the water). A
stock solution should be made considerably in advance as the acid dis-
solves slowly. The used solutions should be kept in separate bottles
and may be used until they become somewhat saturated with safranin.
About 10 seconds in the 95% alcohol suffices, unless the slides have been
left in the stain too long, which naturally would call for a longer washing

in the picro-alcohol.
5. Stop actionof the acid by immensing the slides in 95% alcohol
to which added about 4 to 5 drops of ammonia to each 100 cc. Avoid
is

excess ammonia. The sections should appear to be a trifle overstained

at this juncture. Do not leave the slides in the alcohol-ammonia solution
longer than 2 minutes, as the alcohol during this period is extracting
some of the stain.
6. Complete dehydration with absolute alcohol for about 10 se(;onds.

(The writer goes only as far as 95% alcohol with purely morphological
material, but absolute alcohol should be used on cytological objects.)
The slides may remain in this alcohol as long as 10 minutes if a large
number are being brouglit up and are being counterstained individually.
7. Counterstain with fast green, prepared by making a nearly satu-

rated solution in equal parts of methyl cellosolve and absolute alcohol

and adding enough of this solution to a mixture of 25 parts absolute
alcohol and 75 parts clove oil to give the desired intensity. Keep th('

stain in a dropping bottle; it may be used over and over again for hun-
dreds of slides. Sometimes one may prefer a weak, sometimes a deep,
stain, according to the nature of the material. The fast green is such a
powerful dye that its action should not be allowed to proceed for more
than 15 seconds.
8. Pour the counterstain back into the dropping bottle and rinse off
excess stain with used clove oil. (As a matter of economy, the clove
oil clearing solution specified in the following step, after it has become

too saturated with dye, poured into a bottle, diluted one-half with
is

equal parts of absolute alcohol and xylol, and used for washing off excess
counterstain into a waste jar. See Fig. 15 for arrangement of apparatus
for counterstaining.)
 — .

82 GENERAL METHODS

9. Clear in a mixture of 50 parts clove oil (U.S.P. brand serves as

well as the more expensive c.p.), 25 parts absolute alcohol, and 25 parts
xylol for a few moments.
10. Removeclearer by washing slide a few seconds in a xylol wash
(add 3 to 4 drops absolute alcohol to take care of any moisture acci-
dentally brought over the slides should not show any cloudiness)
;

11. Two changes of pure xylol, then mount in balsam.

The safranin should appear a brilliant red in nuclei, chromosomes,
and in lignified and cutinized cell walls; while the fast green should be
equally brilliant in the cytoplasm and on cellulose cell walls. In some
cell walls at certain developmental or formative stages, portions will be

more or less sharply stained by the safranin, other portions weakly by

the green. Safranin and fast green are both durable and should show no
signs of fading after six years.
—
Safranin and Anilin Blue. Anilin blue may be substituted for fast
green in the preceding schedule. As the blue does not affect the safranin
to any appreciable extent, the latter must be somewhat more accurately
differentiated than is necessary when fast green is used as the counter-
stain. The anilin blue solution is made l)y preparing a saturated solution
in equal parts of methyl cellosolve and absolute alcohol and later diluting
with an equal volume of clove oil. The stain should be allowed to react
for about 1 minute; it scarcely ever overstains and requires no differenti-
ating. To avoid extracting too much of the blue, clearing is usually
carried out in pure methyl salicylate (synthetic oil of wintergreen)
instead of in clove oil.

Anilin blue is a little more precise than fast green on many types of
plant materials. If the effects given by the fast green are too strong or
too gaudy, substitute anilin blue. The combination is superb on gymno-
sperm ovules, archegonia, and embryos and on angiosperm stems and
roots. The anilin blue is occasionally a little fugitive, especially in acid
balsam.
Safranin and Picro-anilin Blue. —
The combination is intended for
use on freehand sections of woody tissues. First stain the sections in
safranin as usual, the optimum time being 2 hours. Wash out the excess
stain with water and destaih with either 95% alcohol saturated with
picric acid or with a mere trace of hydrochloric acid in 50% alcohol.
Stain for 2 hours in a picro-anilin blue stain (make up saturated solutions
of picric acid and anilin blue in 95% alcohol, and when ready to use, mix
in the ratio of 78% picric acid to 22% of anilin blue). Wash for 10 sec-
onds in absolute alcohol, clear in clove oil, and mount in balsam after
going through xylol.
Safranin and Crystal Violet. A rapid schedule involving these two
«?tains has given very good results on various gymnpsperm tissues, It
 STAINING PROCEDURES 83

has b^en successfully used on various stages in the development of

the ovule and associated structures of Finns, Cedrus, Ephedra, and
Ginkgo.
1. Stain in safranin as usual and differentiate with 95% alcohol

saturated with picric acid, then complete dehydration with a change of

absolute alcohol.
2. Dip into a solution of equal parts of absolute alcohol and xylol
for several seconds, then into another composed of 25 parts of absolutci
alcohol and 75 parts of xylol.
3. made by putting 6 to 8 drops of a
Stain in a crystal violet solution
saturated solution of the dye in equal parts of absolute alcohol and clove
oil into a coplin of xylol. This staining solution is very unstable and
should be freshly made up for each batch of slides. The strength of the
violet needs to be renewed after every 30 or so slides by the addition of
another drop of stock solution. The stock solution keeps indefinitely.
Leave the slide in the stain while a second one is being taken through the
picro-alcohol, and differentiate while the second is staining.
4. Differentiate the (*rystal violet in a mixture of equal parts of clove

oil and xylol. A few seconds generally suffice.

5. Wash thoroughly in xylol, pass through a change of pure xylol, and

mount in balsam.
Safranin and Harris’ (or Delafield’s) Hematoxylin. —The following
schedule maybe used either for freehand sections or for paraffin sections.
It is especially serviceable for semi woody tissues.

1. Bring freehand sections up to 35% alcohol, or paraffin sections

down to 70% alcohol.

2. Stain in safranin (use the 1% methyl cellosolve-50% alcohol
solution) for 18 hours.
3. Wash out excess stain with water.
4. Differentiate carefully in 50%
alcohol slightly acidulated with
hydrochloric acid. When xylem
appears bright red and cellulose walls
are a deep pinkish color, the stain is about right. Avoid too much
destaining since more of the safranin will be removed during subsequent
steps.
Wash thoroughly in water for 5 minutes.
5.

Transfer to the hematoxylin for about 15 or 20 minutes. After

6.

removing from the stain, the color of the sections should be a deep purple
all over.
Treat with water slightly acidulated with hydrochloric acid for a
7.
very few seconds. (Slides should be handled individually.) As soon
as the sections appear reddish, transfer to tap water, and wash thor-
oughly. It is vitally important to remove every trace of acid, hence the
washing process should be continued for at least 20 minutes. If the
 j —

84 GENERAL METHODS

hematoxylin not sufficiently blued by the tap water, dip momentarily

is

in a stender of water plus a few drops of ammonia.

8. In the case of paraffin sections, run up through 50 and 70%

alcohols to 95%, allowing 3 to 5 minutes in each. Mounting may be in

euparal or diaphane, or one may proceed to absolute alcohol, and pass
through xylol to balsam. With freehand sections dehydrate by gradually
substituting hygrobutol for the water, finally infiltrate with balsam,
and mount.
Safranin, Crystal Violet, and Orange G (Flemming^s triple stain).
Botanists, and biologists in general as well, seem to be divided on the
merits of this combination. Nevertheless, it has its uses and its value,
but by some it is underrated, by others overrated, while still others have
flagrantly abused it. By this it is meant that it was used when other
and more appropriate stain combinations should have bcnni chosen.
In cytological work, Flemming\s triple stain may be routinely
(employed to supplement and corroborate the results obtained with
iron hematoxylin. The contrasting colors are at times a pronounced
advantage. In a properly stained cytological preparation, the chromatin
of the metaphase and anaphase chromosomes should show tlu^ safranin
predominating, while the chromatin during prophase stages should retain
the violet. In the chromosomes the condensed chromatin should be red,
while the parts in which little or no chromatin is present should be violet.
In cytological terminology, in other words, the chromonemata should be
red and the chromosome matrix purplish, but it is exlTomely rare to
obtain such an accurate diflferentiation. The nuch'oli should be red,
the spindle fibers and plastids violet, and the cytoplasm a buff-gray color.
The orange G is used not to provide a third (cytoplasmic) stain it is —
in this respect thatmost technicians are in error —but to differentiate
between the other two dyes.
In general only tissues fixed in a mixture containing chromic and osmic
acids can be, successfully stained by a Flemming combination. Material
killed in other fluids should first be run down to water and mordanted
for a day or so in a 1% solution of osmic acid in 2% chromic acid, or as
directed under StockwelFs variation described below.
A. A typical schedule which many prefer, is as follows:
1. Slides in 70% alcohol (or, if they have been mordanted, first

place in 35% alcohol for 5 minutes).

2. Stain in a standard safranin solution. The time varies according
to the material; in any case, it should not be lessthan 2 hours. For
most plant materials 6 hours is sufficient, but there is no harm leaving
in the stain for 24 hours.
3. Rinse thoroughly in water.
 .

STAINING PROCEDURES 85

Stain in a 1% aqueous solution of crystal violet.

4. The time here
must be determined experimentally. Fifteen minutes to 1 hour or more
are required.
5. Rinse in water to remove surplus stain.
6. Dip twice in 95% alcohol and three or four times in absolute
alcohol.
7. Remove slide, wipe off underside, and put on just enough orange G
(saturated solution in clove oil) to cover the sections. The time should
not exceed 10 seconds.
Pour the orange back into the bottle, wash off the excess stain
8.

with used diluted cloveoil, and drop slide into a stender of pure clove

oil. In a few moments put the slide on a glass plate, and examine under
the microscope. When the violet is satisfactory, wash in xylol to stop
action of the clove oil.

9. Two (changes of xylol, then mount in balsam.

B. A variation of the preceding schedule followed by some technicians
may be outlined, but the result is frequently sloppy in appearance.
1. Proceed to the end of step 4 in the preceding schedule.

2. Remove slide from staining jar, wipe off the underside, and flood

with a saturated aqueous solution of orange G. About 30 seconds

suffice.

3. Drop 95% alcohol on the slide until clouds of color no longer

arise from the sections,
4. Wash with used clove oil, then differentiate as before with pure
(^love oil.
C. StockwelVs Variation , — If the sections are too dark, overchromated
or contain too much phlobaphene, they should first be run down to water
and bleached overnight in the following solution, which prepares the
sections for very sharp staining:

Water 90 cc.
Potassiuiri bichromate 1 g.

Glacial acetic acid 10 cc.

Chromic acid 1 g.

The theory underlying the use of this fluid, taking as examples

tissues such as the root tips of Quercus, buds of Dudleya and other
Crassulaceae containing much phlobaphene, is that the chromic acid
renders the precipitated tannins soluble, the acetic acid then removes
them, and the dichromate thereupon catalyzes the tissues.
If the material was not fixed in a solution containing sufficient
chromic acid to mordant it, the slides should first be mordanted in a
1% aqueous chromic solution for at least 1 hour, but preferably overnight
Wash out the acid thoroughly before proceeding to the stain.
 86 GENERAL METHODS

Stain 1 to 24 hours in the following solution:

1 %
aqueous solution of gentian (crystal) violet 1 part
I %
aqueous solution of safranin 2 parts
Distilled water 1-4 parts

For most plant tissues an hour is sufficiently long, but a dilute stain
acting over a period of 24 hours presumably gives superior results.
Wash with tap water, place for 30 seconds in 1
off excess stain potas- %
sium iodide plus 1% 70% iodine in
alcohol, then pass through the follow-
ing fluids, allowing but a few seconds in each: 50 and 70% alcohols,
95% alcohol plus picric acid (about 1 g. per 100 cc.), 95% alcohol plus
ammonia (8 to 10 drops per 100 cc.), pure 95% alcohol, absolute alcohol,
clove oil plus orange G (0.2 g. per 100 cc.), pure clove oil, and finally
through three jars of xylol to make certain that all traces of clove oil are
removed before mounting in balsam.
The slides may
be examined in the first xylol after the staining has
been completed. seems to be too much safranin, return the
If there
slide to absolute alcohol, then back to xylol. Excess violet may l>e
reduced by returning the slide to clove oil, then back to xylol.
Chromosomes are stained varying shades of light to dark purple;
the spindle fibers are purplish, the nucleoli red, and the cytoplasm orange.
With morphological and anatomical material, the final effect is the same
as with the usual versions of the triple combination.
D. An abbreviated form of the preceding schedule, for use on non-
cytological materials, is as follows:
1. Bring slides down to water, then stain for 1 hour in the staining
solution.
2. Rinse thoroughly in water.
3. Rinse in 95% alcohol for a few minutes. This serves to remove
most of the surplus safranin.
4. Rinse in absolute alcohol.
5. Differentiate the violet in the clove oil-orange G solution for a
few seconds.
6. Wash in xylol, and examine under the microvscope to determine
if the violet is satisfactory. If not, go back to a plain clove oil, and
differentiate until satisfactory.
7. Wash thoroughly in xylol, then mount in balsam.
E. Craigie^s Modification,
1. Bring slides down and mordant in 0.5% aqueous osmic
to water
acid for 30 minutes to an hour. This step is omitted for material which
has been fixed in a fluid containing osmic acid.
2. Wash thoroughly in water and stain 1 to 3 hours (or longer if
necessary) in 1% aqueous safranin.
 STAINING PROCEDURES 87

Rinse in water and differentiate the safranin in 0.025/N hydro-

3.

chloric acid. Allow the acidulated water to react until the outline of
the nucleus is definite and chromatin granules are discernible. Do not
I’emove too much stain at this point. After differentiation, rinse thor-
oughly in water.
4. Stain in a 0.27 to 0.3% solution of ciystal violet in 7% alcohol
for about 30 minutes.
5. Rinse in water and differentiate in a 0.025/N solution of hydro-

chloric acid. Differentiation is far more important than the time of

staining; it should be stoj)ped when the nuclei take a definite outline.
6. Rinse in water and put in Gram\s solution (1 g. iodine and 3 g.

potassium iodide to 300 cc. wat('r) for 1 to 3 minutes, or until the slides
have turned a de(^p black.
7. Wash away excess iodine solution thoroughly and immers(' slides

in a 1% solution of mercuric chloride for 1 to 3 minutes, ov until the

sections have turned a bright blu(».
8. Wash in water and blot exccNSs cautiously, not letting tlu' sections

become comph^tely dry.

9. Immerse in 95% alcohol for 4 to 6 seconds and transf(‘r to carbol-
xylol (25 parts phenol to 75 parts xylol) w^hilc the violet is still coming
out in clouds.
10. Leave in carbol-xylol from 15 seconds to several minutes or until
proper differentiation has occurred, which will be when prophas(‘ stages
are deep blue and chromosomes and nucleoli are definitely red.
11. Wash in xylol, and stain in a 1% solution of orange G in clove oil
for not longer than a minute.
12. Clear briefly in pure clove oil, wash thoroughlj'^ in xylol, and
mount in balsam.
If it is desired that the violet be intensified in the spindles, sections
should first be mordanted vdth potassium permanganate.

Quadruple Combinations

Safranin, crystal violet, fast green, and orange G may be combined

for use on tissues in which there is either a variety of cell types or con-
siderable chemical difference in the structure of the cells. The combina-
tion is not suitable for tissues with little differentiation, as in meristematic;
regions.
Conant’s Quadruple Stain. —^An excellent combination, giving clear
results.
1. Bring slides down to 70% alcohol.
2. Stain 2 to 24 hours in 1% safranin in 50% alcohol.
3. Rinse thoroughly in water.
 88 GENERAL METHODS

4. Stain in a saturated aqueous solution of crystal violet for about a

minute.
5. Rinse in water.

6. Dehydrate through two changes of absolute alcohol.

7. Dip slides rapidly, for 5 to 10 dips, in 1% fast green in absolute

alcohol, then transfer quickly to a saturated solution of gold orange

(orange G may be substituted) in clove oil, agitating the slide until the
adhering alcohol is completely diffused through the clove oil.
8. Transfer through tJbree more jars of orange-clove oil solution,
allowing several minutes in each, for further differentiation and clearing
of the background.
9. Rinse thoroughly in xylol, then mount in balsam.
—
Johansen’s Quadruple Stain. The preceding method is not intended
to correlate stain affinity with specific structures. To achieve as closely
as possible this effect, the following procedure has been devised, employ-
ing the newer stain solvents. It is a simple procedure, even if the mix-
tures are rather complicated, inasmuch as differentiation is automatic
and little need be left to personal judgment.
1. Bring slides down to 70% alcohol.

2. Stain in the methyl cellosolve-50% alcohol safranin solution

(page 62) for 24 to 48 hours. Overstaining is not possible.

3. Rinse in tap water.
4. Stain in 1% aqueous methyl violet 2B for 10 to 15 minutes.
Rinse in tap water.
5.

6. Rinse for 15 seconds in a mixture of equal parts 95% alcohol,

methyl cellosolve, and tertiary butyl alcohol.

7. Immerse for 10 to 15 minutes in a fast green FCF solution prepared

thus: take 1 part of a saturated solution of the dye in equal parts of

clove oil and methyl cellosolve, 3 parts 95% alcohol, 3 parts tertiary
butyl alcohol and 1 % glacial acetic acid. The time may require some
experimental determination according to the tissues and the fixing fluid.
(The time cited is for formalin-aceto- (or propiono-) alcohol; tissues fixed
in chrom-acetic fluids may require more time.)
Rinse briefly in a mixture of equal parts of 95% alcohol and tertiary
8.

butyl alcohol plus about 0.5% glacial acetic acid.

9. Immerse for about 3 minutes in an orange G solution prepared

as follows: 1 part each of a saturated solution of the dye in methyl

cellosolve, pure methyl cellosolve, and 95% alcohol.
10. Rinse briefly in a wash composed of 1 part each of clove oil,

methyl cellosolve, and 95% alcohol.

11. Rinse in a wash made up of equal parts of clove oil, absolute
alcohol, and xylol.
12. Rinse in two changes of xylol, then mount in balsam.
 J^AINING PROCEDURES 89

The staining solutions can accommodate a large number of slides

before replacement isrequired, but the washing solutions in steps 6, 8,
and 10 will need replacement frequently. As soon as one of them
becomes saturated with dye, it should be renewed, otherwise the sharp-
ness of the stains will be diminished.
The staining effects should be as follows: dividing chromatin red,
resting chromatin purplish, mndeoli red (occasionally violet), nucleoplasm
colorless or greenish, lignified cell walls bright red, cutinized cell walls
reddish-purple, suberized walls red, cellulose cell walls greenish-orange,
cytoplasm bright orange, middle lamellae green, starch grains purple
with green or orange halos (the (*olor of the halos soon becomes replaced
by the purple in some typ(\s of material), plastids purplish to greenish,
invading fungal mycelium green, the callose portion of the guard cells of
stomata bright red and the nunainder purple, and Casparian strips red
and the remainder of the cc^ll wall of the endodermis yellow. In sections
of roots for origin of tlie lateral r(>ots, the cytoplasm of the latter should
be stained green, with purplish nuclei, while the cyt()])lasm f^lsewhere
should be orange with red nuclei. The combination is exceptionally
good for sections of lichens, as the algae are well differentiated, and also
for Puccinia grarninis telia and uredinia.

Otheu Coal-tar Dyes

—
v^rystal Violet and Erythrosin. This method is especially valuable
where a sharp differentiation between weakly lignified and nonlignified
tissues is desired and is also particularly useful for general differentiation
as vascular structures are rendered readily distinguishable from neighbor-
ing tissues (tlackson 1926). The schedule must be adapted for different
types and lots of material and often also according to the nature of the
fixing fluid; in some materials the xylem elements stain satisfactorily
in a few minutes, but in other types 30 minutes or longer may be required.
The action of the erythrosin must be closely watched as it tends to replace
the violet in lignified cell walls.

1. Bring slides down to water.

2. Stain in 1% aqueous crystal violet, 15 minutes as a trial.
3. Rinse quickly in tap water.
4. Dehydrate quickly but thoroughly in 95% and absolute alcohol.
5. Immerse in a saturated solution of erythrosin B in clove oil for
1 to 5 minutes.
6.Absolute alcohol and xylol, equal portions, 2 minutes or less.
7.Wash thoroughly in xylol, then mount in balsam.
—
Cooper’s Triple Combination. Primarily a cytological stain. Stain
sections in 1% aqueous methyl green (use a strictly fresh dye for making
90 GENERAL METHOm
up the solution) for 1 hour, counterstain in 1% aqueous acid fuchsin
for minute, then stain in 1% aqueous erythrosin B for 2 to 3 seconds.
1

Chromatin granules and nucleoli in early prophase stages of micro-

sporogenesis are stained green, and the linin threads are stained red. In
later stages the chromosomes are stained a brilliant green (Cooper 1931).
—
Smith’s Picric Acid-Gram Stain. This is likewise primarily a cyto-
logical stain (F. S. Smith 1934).
1. Bring sections down to 95% alcohol, or smears up to 70%.

2. Mordant 10 to 20 minutes in a solution composed of 1 g. each of

iodine and potassium iodide to each 100 cc. of 80% alcohol.

3. Rinse in water, then stain in 1 % aqueous crystal violet for about
15 minutes.
Rinse in water again, then place into a second iodine solution, of
4.
the same proportions as that noted in step 2, for a few minutes.
5. Rinse in 95% alcohol.

6. Flood slide quickly with a saturated solution of picric acid in

absolute alcohol, then wash immediately with plain absolute alcohol

for only a few seconds. The purpose of the picric acid is to fix the
violet in chromatic materials,
7. Differentiate in clove oil. The violet will be extracted from the
cytoplasm but not from the chromosomes.
8. Wash in xylol, then pass through two changes of pure xylol,

allowing the slides to remain in one of them for about 1 hour before
finally mounting in balsam.
—
Johansen’s Methyl Violet-Erythrosin Stain. This is a beautiful com-
bination for mitose^s in root tips (Johansen 1932).
1. Bring slides down to water.

2. Stain 15 to 30 minutes in a 1% solution of methyl violet 2B in

distilled water.
3. Rinse off excess stain with water, then simultaneously differentiate
and dehydrate with a saturated solution of picric acid in 95% alcohol.
About 10 to 15 seconds suffice.
4. Stop action of the acid by placing the slide for 15 seconds in 95%
alcohol plus 2 to 3 drops of ammonia per 100 cc.
5. Wash
in pure 95% alcohol, for about 15 seconds.
Counterstain with a nearly saturated solution of erythrosin in
6.

equal parts of absolute alcohol and clove oil for 5 to 10 seconds.

7. Clear in clove oil for about 30 seconds, then wash thoroughly in

two changes of xylol, and mount in balsam.

Resting and dividing chromatin is stained a brilliant purple; plastin
dark r^ed, cell walls red, and the cytoplasm pinkish.
—
Nfewton’s Gentian Violet-Iodine Method. For this method, see the
chapter on Smear Methods (page 156).
 STAINING PROCEDURES 91

Modified Cajal Basic Fuchsin-Indigocarmin Stain.^ —A cytologi(;al

stain valuable on root tips (Hruby 1933).
1. Bring slides down to water.
2. Stain 5 to 20 minutes in a saturated aqueous solution of para-
rosanilin (basic fuchsin). (Do not use a dye labeled with the synonym
‘^diamond fuchsin” or with the German Diamantfuchsin.” Neither of
these dyes (;an be differentiated properly.)
3. Rinse in water until free stain no longer comes out.

4. Stain 5 to 15 minutes in a mixture of equal portions of saturated

aqueous solutions of picric acid and indigocarmin.

5. Pass rapidly through 70% alcohol, in which the sections wdll

appear red, then through 95% and absolute alcohol until the sections
appear greenish.
6. Clear in xylol, and mount in balsam.

If acid instead of basic fuchsin is used, the procedure varies slightly:

1.. Proceed to the end of step 4 in the above schedule.

2. Wash in water to which a trac^e of acetic acid has been add(‘d.

3. Wash rapidly in 70, 95%, and absolute alcohol, as indicated in

step 5 above, then clear in xylol, and mount in balsam.
With onion root tips as an example, the coloration should be as
chromosomes and
follows: late prophase stages various shades of bright
or dark red, early prophases bluish-red, nucleoli clear blue, spindle fibers
and cell walls a dark blue against a light blue cytoplasm.
Foster’s Tannic Acid-Iron Chloride Method. — This method was
primarily designed for the cell walls in meristematic tissues but is capable
of wider application, as noted in the Northen methods cited below.
1. Bring slides down to water.
2. Mordant in 1 % aqueous solution of c.p. tannic acid for 10 minutes.
(Add 1 g. sodium salicylate to each 100 cc. of solution to prevent growth
of molds.)
3. Wash
thoroughly in water.
4. Bring out stain by placing slides in 3% aqueous c.p. ferric chloride
for several minutes. Then examine under the micTos(*ope if the cell :

walls of the meristem appear black or dark blue and the nuclei and cyto-
plasm gray, the stain is correct. If the stain seems to be too weak, wash
the slide thoroughly in water, and return to the tannic acid solution.
Alternate between steps 2 and 4 until differentiation is satisfactory, tak-
ing care to wash the slide thoroughly in water each time a transfer is

made.
6. Immerse in 50% alcohol for a few minutes.
6. Stain for 48 hours in a 1 % solution of safranin in 50% alcohol.
7. Rinse in water, and destain cautioasly in 70% alcohol very weakly
acidulated with hydrochloric acid.
92 GENERAL METHODS

8. Pass through 70% and higher alcohols to xylol, then mount in

balsam.
Cell walls should be coloredan intense black, the cytoplasm violet
or pink, plastin blue, and the and chromosomes red. Other
nucleoli
cytoplasmic or nuclear stains may also be used.
—
Northen’s Variations of Foster’s Method. These procedures add a
third stain to those given by the preceding method (Northen 1936) and
are especially adapted to the staining of stems and of roots showing
stages in the development of lateral roots.
A. If the safratlin is retained by the tissues, the safranin may be
applied first:

1. Stain 24 hours in 1% safranin in 50% alcohol.

Rinse for 10 seconds in 50% alcohol.
2.

3. Immerse for 15 to 30 seconds in 0.5% tannic acid in 50% alcohol

(use c.p. Merck or Mallinckrodt tannic acid).

4. Pass through two coplins of 70% alcohol, allowing 10 to 15 seconds

in each.
5. Immerse for 10 to 20 seconds in 1% ferric chloride in 70% alcohol.
6. Place in 80% alcohol for 10 seconds, in 95% alcohol for 20 seconds,
and in absolute alcohol for 30 to 40 seconds.
7. Stain for 30 to 60 seconds in 0.5% crystal violet in clove oil.

8.Rinse in xylol (plus a trace of absolute alcohol), pass through purer

xylol, then mount in balsam.
When properly applied, the procedure should stain parenchyma and
collenchyma cell walls purple, fiber walls red or purple, lignified cell
walls red, walls of the cambial cells black, nucleoli red, and cytoplasm
purple. If too much safranin has been extracted, reduce the time in the
alcohols; if the safranin has been insufficiently differentiated, allow more
time in the alcohols or add a drop of hydrochloric acid to one of- the 70%
wash alcohols.
B. If the safranin is easily extracted, the tannic acid-ferric chloride
part should be applied first.
1. Bring slides down to 70% alcohol, then leave for 2 to 3 minutes
in 1% tannic acid in 50% alcohol.
Rinse for 15 to 20 seconds in each of two coplins of 50% alcohol.
2.

3. in 3% ferric chloride in 50% alcohol for 30 to 60 seconds.

Immerse
The slides will become blackened, but this is of no moment.
4. Rinse in 50% alcohol.

5. Stain for 24 hours in 1% safranin in 50% alcohol.

6. Rinse in 50% alcohol,

7. Destain partially in 70% alcohol; since more safranin will be

removed during the following steps^ the differentiation should not be

carried too far.
 STAINING PROCEDURES 93

8. Dehydrate in 80, 96%, and absolute alcohol for 10 to 30 seconds

in each.
9. Stain for 1 to 3 minutes in 0.5% light green (or substitute fast
green) in clove oil plus 7 cc. absolute alcohol to each 100 cc. clove oil.

10. Rinse in absolute alcohol, then in xylol, and mount in balsam.

By this procedure, cambial cell walls and parenchyma cell walls
are black, lignified walls are red, collenchyma walls are a blackish-red,
nuclei are red, and the cytoplasm a bluish-green.
—
Acid Fuchsin and Fast Green. This histological combination is
suggested for the marine Phaeophyta as a quick and easy method giving
good differentiation.
1. Bring slides down to 95% alcohol.

2. Stain 20 minutes in 1% acid fuchsin in 70% alcohol.

3. Rinse off excess stain by slowly plunging slide 2 to 3 times into a

large dish of tap water.
4. Rinse in 95% alcohol for a few seconds.

5. Wipe off underside of slide, and cover sections with the usual

solution of fast green in methyl cellosolve-absolute alcohol-clove oil for a

few seconds.
6. Clear in absolute alcohol-clove oil-xylol mixture.
7. Wash in xylol, and mount in balsam.
The sections should, as with Phaeophyta, have been covered with
all

removing the paraffin with xylol. When

celloidin, either before or after
used on sections of the fruiting frond of any of the Laminariales, for
example, the zoospores are stained a brilliant magenta, the nuclei and
pigment bodies various shades of red, the cell walls light green, and the
layers of the slime cap are differentiated by the green. This is the only
known combination which will reveal clearly the internal structure of
the paraphyses.
Iodine Green or Methyl Green and Acid Fuchsin. — If one wishes to
stain lignified tissues with a green dye, iodine or methyl green (not
methylene green) works admirably. These two dyes stain similarly to
safranin. Methyl green is probably easier to differentiate than iodine
green, but is extracted more rapidly.
1. Bring slides down to water (or have freehand sections in water).

2. Stain 12 hours or longer in 1% aqueous iodine or methyl green.

3. Wash in water until the stain is almost but not entirely removed
from nonlignified elements.
4. Counterstain about 3 to 8 minutes in 1% aqueous acid fuchsin.

The stain should not be allowed to react long enough to extract the
green from the lignified tissues. The point may be determined by
removing a slide from the staining solution and examining under the
microscope.
94 GENERAL METHODS

5. Rinse rapidly in 95% and absolute alcohol.

6. Clear in clove oil solution, pass through xylol, and mount in balsam.
Both stains may be used as 1% solutions in 70% alcohol if desired;
washing may be in 70% alcohol. This method may be better than using
wash naturally
the stains in aqueous solutions, but the use of an alcoholic
makes somewhat more expensive. The combination may be used as a
it

cytological stain, but the washings must be made very quickly. Methyl
green is have a trace
sensitive to alkalies, so that all solutions should
of acetic acid added to them. If there was no acetic acid in the killing
fluid, a chromatin stain is unobtainable. The resistance of the stain
to alcohol may be increased by mordanting the sections for 5 minutes
previous to staining with 1 %
aqueous iodine solution. The green stains,
unfortunately, do not keep any too well. Chromosomes and nuclei are
stained green, plastin and cytoplasm light red.
Feulgen’s Nucleal Reaction. —This is not so much a staining pro-
cedure as a chemical reaction for chromatin; consequently it is being
described elsewhere (page 95).
 CHAPTER VIII

SPECIAL METHODS
Feulgen Nucleal Reaction. —This procedure was originally devised
as a microchemical test to distinguish the particular type of nucleic
acid found in chromatin from similar types. The essential feature of
the method is the production of a specific purple (or occasionally magenta)
color when a reduced, or colorless, form of pararosanilin (basic fuchsin) ,

is brought into contact with an aldehyde in the presence of the nucleic*

acid peculiar to chromatin.
The original version (Feulgen and Rossenbeck 1924) has undergoru^
innumerable variations. The majority of the proposed changes have
since been demonstrated to be of little or no value (de Tomasi 1936).
Botanical technicians who have experimented with the technique have
demonstrated beyond all doubt its gn^at utility on all types of material
(c.^., Margolena 1932a, Westbrook 1930), and it has been of especial

service where it is necessary to differentiate between true chromatin

and other substances whicli give a staining reaction similar to that of
chromatin with ordinary coal-tar dyes.
The staining solution is prepared as follows (de Tomasi 1936): Dis-
solve 0.5 g. basic fuchsin [use a Stain Commission type especially certified
for use in the Feulgen technique (Scaiilan and Melin 1937)], by pouring
over it 100 cc. and shaking thoroughly. Cool
boiling distilled water
to 50°C., and filter. To the filtrate add 10 cc. 1/N hydrochloric acid,
then 0.5 g. potassium metabisulphite. Shake thoroughly, stopper
tightly, and place in the dark for about 18 hours.
All substances should be of the highest purity: the water should
be glass-distilled and the chemicals of reagent quality. If a satisfactory
certified basic fuchsin sample is not available, Grubler\s Diamantfuchsin,
as specified by the originators, almost always works well. The 1/N
hydrochloric acid may be prepared with sufficiently close accuracy by
adding 82.5 cc. reagent hydrochloric acid to 1 liter of glass-distilled
water.
The staining is carried out as follows: Bring the slides down to dis-
tilled water, and rinse in cold 1/N hydrochloric acid; place in fresh 1/N
hydrochloric acid, and heat the solution quickly to 60°C. (but not above
that temperature), allowing the slides to remain for 4 to 5 minutes.
Next rinse in cold 1 /N hydrochloric acid, then in distilled water. Trans-
95
 96 GENERAL METHODS

fer the slides to the staining solution, which should be either colorless
or of a light straw color. The optimum time for animal tissues is 2 hours,
but most plant tissues require from 3 to 5 hours. Remove a slide from
the stain, touch the lower edge to absorbent paper, then pass quickly to
the first of three closed coplins, each containing the following differentiat-
ing solution: 1/N hydrochloric acid, 5 cc.; 10% aqueous potassium
metabisulphite, 5 cc.; distilled water, 100 cc. Wash the slides for 10
minutes in each of the three jars. Then rinse in distilled water and
dehydrate. Plant materials may be counterstained momentarily with
0.05% fast green in 95% alcohol. Mount preferably in dammar balsam,
although strictly neutral Canada balsam serves equally well.
Solutions should be made up only as required, since none of them
keeps well. In time the staining solution will become colored and should
then be discarded.
Various methods have been devised to secure satisfactory samples
of the basic fuchsin dye or to purify such samples (Scanlan and Meliii
1937), but such treatments are either unnecessarily cumbersome or call
for more apparatus and chemical knowledge than the average technician
possesses. Given a certified dye sample, the staining solution can be
rendered practically colorless by adding a decolorizing carbon to the
solution after it has been allowed to bleach for 24 hours (Coleman 1938).
To each 200 cc. of the basic fuchsin solution prepared as directed above,
add 2 g. potassium metabisulphite and 10 cc. 1/N hydrochloric acid.
After bleaching for 24 hours, add 0.5 g. of a powdered decolorizing carbon
or charcoal (the brand known as Supra-neutral Norit, obtainable from
L. A. Salomon, 216 Pearl Street, New York, N. Y., has been recom-
mended). Shake, for a few minutes, then filter rapidly through coarse
filter paper.
A procedure has recently been devised (Semmens and Bhaduri
1939) for the differential staining of the nucleoli following the application
of the Feulgen technique. The originators claim that the procedure
/"does not affect the chromatin stain, but experience has shown that the
fuchsin is often completely removed. The method may be briefly
outlined:
1. Mordant for 1 hour in filtered 5% aqueous sodium carbonate.
2. Wash for 30 minutes in distilled water.
3. Stain for 10 minutes or longer in a 0.5% solution of light green SF
yellowish in 100 cc. of 90% alcohol plus a few drops of anilin oil.

4. Rinse with 70% alcohol saturated with sodium carbonate. This

step has been the most troublesome one. The amount of carbonate
that dissolves in the alcohol is highly variable: if too little dissolves,

differentiation is poor, wher^as^ if the proportion is too great, complete

discoloration results,
 —

SPECIAL METHODS 97

5. Transfer to 96% alcohol, and leave until the green dye has been
removed from the cytoplasm. At least 10 minutes will be required.
6. Immerse in absolute alcohol for 10 minutes or longer.
7. Place in absolute alcohol and xylol, equal parts of each, for 10

minutes.
8. Clear through xylol, and mount.
Osmium Impregnation Methods. —
methods of osmic acid impreg-
All
nation are in general tedious, variable, and frequently unsuccessful
(Bowen 1929). The following rules have been laid down:
1. Material should be absolutely fresh. Do not use plants which
have lingered about the laboratory or greenhouse under unfavorable
(londitions. Use pieces of material cut into as small portions as possible.
Large root tips and other massive parts will probably not react success-
fully unless subdivided.
Use osmic solutions, both for fixing fluids and for the osmication
2.

process, —
which are positively fresh not over one or two weeks old
and which have been kept properly clean and preserved from the action
1.
of light.
3. Keep the ingredients of fixing fluids in separate stock solutions,
and mix them just before using.
4. All fixation, and especially all osmication, steps must positively
be conducted in glass-stoppered bottles. If such bottles are not available,
it is not worth wasting time and supplies attempting osmication.

5. After the material is finally washed out in running water after

osmication, do not spend more than five or six days at the most in the
alcohols before embedding in paraffin. For these post-osmication
processes, the use of corked shell vials is permissible.
The Kolatchev Method (Bowen^s Schedule).
Fix for 24 hours in Champy^s fluid:

1% aqueous chromic acid 7 cc.

3% aqueous potassium bichromate 7 cc.

2% aqueous osmic acid 4 cc.

Do not attempt to work up too much material in one bottle. Gently

agitate the bottle occasionally.
2. Wash in running water for 24 hours. It is very important to
remove all traces of the chromium.
3. Place the material in new glass-stoppered bottles and commence
the osmication process. This may be carried out by one or all of several
methods:
a. Place the material in 1 % osmic acid solution,
and then
(1) Put the bottles in an incubator at 35°C. for periods of
4“9 days.
98 GENERAL METHODS

(2) Put the bottles in an incubator at 40®C. for 8 hours. Then

transfer the bottles to an incubator at 35®C. without
changing the osmic solution. Leave in this second
incubator for periods such that the total time in the acid
is from 4 to 9 days.
h. Place the material in 2% osmic acid solution, and then proceed
(1) As in method a(l).
(2) As in method a (2).
It is generally advisable, to obtain a greater chance of success, to
divide each lot of material into four portions and to try all four methods
simultaneously. Otherwise, method most likely to afford suc-
6(2) is
cessful impregnation. In all methods, a volume of osmic acid solution
at least equal to twice the bulk of the material should be used.
The osmic solutions will soon begin to become blackened. When the
blackening has become pronounced, rinse out with distilled water, trans-
fer to a clean bottle, and cover with fresh osmic solution.
4. Wash water for 24 hours. It requires considerable
in running
experience to determine when to terminate the osmication process.
There is no way of knowing when anything whatever has been success-
fully impregnated. Trial by sampling is the only method. If the
osmication period was too short, nothing will be blackened; if too long,
the material may be too overblackened for critical investigation. Each
day after the four-day minimal period a few samples may be removed
from the bottle with a pipette (take every precaution not to let the
pipette be contaminated by other substances, particularly chromium
compounds), dehydrated, and embedded.
5. Dehydrate by any rapid process, and embed in paraffin. The
impregnation is relatively stable, and no unusual precautions are necessary.
6. Cut sections not over 6/u thick and mount on slides as usual. After
the sections have dried down, remove the paraffin with xylol, pass
through a change of absolute alcohol to ensure complete dehydration,
then pass through another jar of xylol, and mount in balsam. Or one
may bleach cautiously by the potassium permanganate-oxalic acid
method after taking the slides down to water. Counterstaining, h
desired, may be with eosin, erythrosin, orange G, fast green, or a similar
dye, made up with 95% alcohol.
The Weigert (Mann-Kopsch) Method,
1. Fix in corrosive-osmic:

1% aqueous osmic acid I part

Saturated solution of mercuric chloride in 0.75% aqueous sodium chloride 1 part

As soon as the material has become blackened, which with some plants
may be in 15 minutes, pour off the fluid, and add fre^h fixative, repeating
 SPECIAL METHODS 99

if necessary later on. The time of fixation is variable and can be deter-
mined only by trial. Periods of 1, 2, or 4 hours may be tried.
2. After fixation, wash in running water for 30 minutes.
3. Wash in 2% aqueous
distilled water, and begin the osmication with
osmic acid. It would be best to place the material in an incubator at
25°C. if the room temperature fluctuates too much. As in the Kolatchev
method, the osmication step is subject to wide variables, which should be
determined as described for that method.
4. Wash material for 24 hours in running water.

5. Dehydrate, embed, section, deparaffin, and mount, as described

for the preceding method.

The results are so variable and interpretation so problematical that
one should proceed with great caution when studying the resulting
preparations. Reference should be made to the papers of Bowen (1929,
for bibliography) and other reliable investigators.
Mitochondria. — Far more work has been done on the mitochondria
of animal tissues than on those occurring in plants, but the latter, despite
inherent difficulties, can be easily manipulated if the requisite attention
to details is exercised.
Ordinary technical methods do not reveal the mitochondria, since
the acids dissolve them. Special chemicals are required in the fixing
fluids, which should always be ones that give basic ’fixation images.
One of the most successful killing fluids consists of:

Cupric bichromate 5 g.
Cupric oxide 1 g.

10% aqueous acetic acid I oc.

Distilled water 100 cc.

The fluid should be made up at least a day before being used, and it
should be shaken frequently during that period. Fix from 36 hours to
six days, and wash out with two changes of 70% alcohol within 32 hour;
immerse for the same length of time in 80 and 95% alcolml. Complete
dehydration with two changes of absolute alcohol, allowing 1 hour in
each, clear in xylol or tertiary butyl alcohol, and enibid. Cut sections
not over lO/x in thickness. Stain wdth iron hematoxylin; as the mito-
chondria do not retain the stain so well as do chromatin-containing
structures, destaining should not be carried so far as is ordinarily done
when chromosomes are being studied (Fig. 1).
nuclei or
Another formula, which gives a similar fixation image, is

Chromic acid 5 g.

Glucinum carbonate 3 g.
Distilled water 200 cc.
 100 GENERAL METHODS

If the glucinum carbonate is not in excess, a little more should be

added. Dehydrate as described above.
Still another formula also gives beautiful results on occasion:

10% aqueous chromium sulphate 1 part

8% aqueous formalin neutralized with an excess
of calcium or lithium carbonate 1 part

If the washing is with 70% alcohol and the dehydration is carried

through rather rapidly, the cytoplasm is granular, and the mitochondria

Fig. 1. — Allium cepa: portion of a longitudinal section of a root tip fixed and stained for
mitochondria. Fixed with Zirkle’s reduced chromic fluid; stained with iron hematoxylin.
V

are preserved in nearly every part of each piece of tissue. If washing is

with water and dehydration proceeds slowly, the mitochondria are
dissolved from the epidermis and cortex, remaining only in the central
 SPECIAL METHODS 101

portions. Dividing nuclei and the cytoplasm are well fixed (the cyto-
plasm granular than after alcohol washing), and in the cytoplasm
is less

the vacuoles are so sharply delineated that their growth as well as their
behavior during mitosis can be studied.
Licent^s fluid also gives good results:

2% aqueous chromic acid 60 cc.

Neutralized formalin 30 cc.
Glacial acetic acid A few drops
10% aqueous nickel acetate 10 cc.

To this mixture 10 cc. of a saturated aqueous solution of mercuric chloride

may be added. Make up the fluid only as required.
Mitochondria are perfectly preserved by quinone (parabenzoquinone).
Treat tissues for 1 hour in quinone dissolved in normal saline solution,
then1. transfer to any other fixative (Bouin\s or Alienas B-15 Bouin). The
quinone may be at any concentration: 0.05% for delicate tissues and 0.5%
for tougher ones. Stain with iron hematoxylin.
The complicated staining schedules used by animal technicians are
practically useless on plant materials and fade quickly.
Milovidnov’s Method of Differentiating between Bacteria and
—
Mitochondria. It is frequently necessary to distinguish between bacteria
and mitochondria in sections of infected tissue: the following method
affords a means of differentiating between the two (Dufrenoy 1928).
Kill and fix for 24 hours in

1% aqueous chromic acid 50 cc.

1% aqueous potassium bichromate 50 cc.

Formalin, neutralized with powdered calcium carbonate. . . 8 cc.

2. Wash for 24 hours in running water, then dehydrate and embed

by the tertiary butyl alcohol method. Cut vsections as thin as possible,
preferably at 4 m.
3. Dissolve paraffin from slides, dip slides into a very thin solution
of celloidin in equal parts of absolute alcohol and ether, then run down
to water.
4. Stain in the following solution, heated to about 80®C.

Acid fuchsin 2 g.
Anilin water 10 cc.

(Anilin water consists of about 2 parts anilin oil to 100 of water, very
thoroughly shaken.)
5. Wash in running water.

6. Destain for a few seconds in

Aurantia 0 6 . g.

70% alcohol 100 cc.

102 GENERAL METHODS

7. Wash again in water.

8. Treat for a few minutes in

Phosphomolybdic acid 1 g.

1 % aqueous sodium hydroxide 10 g.

Distilled water 100 cc.

9. Rinse in water.
10. Stain for a few minutes in Unna^s polychrome methylene blue,
prepared as follows: a solution of 1 part methylene blue dye and 1 part
potassium carbonate in 20 parts 95% alcohol and 100 parts distilled
water is evaporated down to 100 parts. It may be used at once or
diluted with an equal part of anilin water for sections.
11. Rinse again, dehydrate very quickly with 95% and absolute
alcohol, pass through xylol to balsam.
Bacteria stain a deep violet blue, mitochondria and plastids red.
Freehand Sections. —The term ^‘freehand was originally
sections'^
applied to sections which were cut by means from material
of a razor
held in the hands or placed against a length of elder pith. Later it came
to include sections cut from live or preserved, but not embedded, material
by means of a sliding microtome. The present usage includes all sec-
tions, no matter how cut and whether embedded or not, which are handled
loosely and not attached to slides by means of an adhesive.
Few, if any, technicians nowadays use the primitive sectioning meth-
ods employed by the early botanical technicians, since the availability
of all types of sliding microtomes greatly facilitates sectioning.
To say the best for them, freehand sections of nonembedded materials
are rarely perfect insofar as the sectioning goes. The thickness, for
example, is generally very uneven, but when microtomed at 25/x or

Over, becomes better. The material must be fairly rigid, not soft or
it

succulent or composed of mixed soft and hard tissues. The most

difficult problem is to clamp the piece of mat/crial tightly enough to
prevent it from bending when struck by the knife yet not so hard that
it is crushed. Pieces of wood or woody stems give comparatively little

difficulty unless they are very small. *

Any type of sliding microtome may be .used for cutting freehand

sections. None, however, has a really satisfactory clamp, consequently
one must exercise some ingenuity to devise supplementary aids. For
instance, one may obtain a suitable cork, cut away sectors from opposite
sides, then bisect parallel to the flat sides and cut longitudinal U shaped
grooves down the center of the bisected sides. The depth of the groove
should be shallower than the radius of the piece of material. (Or use
two pieces of thick cork linoleum and cut a groove in each.) The material
is placed in the grooves and the whole placed in the clamp.
 SPECIAL METHODS 103

The operation of the sliding microtome should be carefully studied.

Most such microtomes require hand adjustment of the feed, although a
few models resemble rotary microtomes in having an automatic feed.
all

It is impossible to cut good freehand sections thinner than about 16m;

most technicians cut at about 30m. The knife should be oriented at a
wide oblique angle in order that as much of the length of the blade as
possible mlay be used for cutting purposes. The knife must, obviously,
be kept sharpened and free from nicks. With the material clamped,
adjust the feed until the material almost touches the underside of the
knife. If the material is fresh, moisten it and the knife with water by
means of a camcFs-hair brush; if preserved, use the preservative or 50%

alcohol. In some instances, a mixture of equal parts of 95% alcohol

and glycerin affords a better lubricant. As the sections are cut, move
them toward the back of the knife with the brush and leave there for
a few minutes to lessen danger of curling. Many technicians hold the
brush over the piece of material as a further precaution against curling.
Fresh material should then be transferred to watch glasses containing
formalin-aceto-alcohol or other desired fixing fluid; sections of preserved
material should be placed in 50% alcohol. One should cut about twice
as many sections as might actually be required, so that the best ones may
be picked out for mounting after the staining and dehydration are
completed.
Hard woods should be sectioned as described in the following section.
Celloidin sections are commonly treated as if they were freehand sections
(see Chap. XI). Paraffin sections of woody stems and other objects
from which essential portions will not drop out and become lost may also
be treated as if they were freehand sections. Sections of old Aristolochia
stems may, for instance, be placed in a watch glass of xylol to remove the
paraffin and then carried down to weak alcohol or water for staining.
The staining and dehydration may be carried out in watch glasses;
the liquids are removed by means of pipettes. Useful stain combinations
include: safranin and either Harris' hematoxylin, fast green, anilin blue
or crystal violet (the last dye especially for gyrnnosperm tissues), iodine
or methyl green with acid fuchsin, iron hematoxylin and orange G, or
any other stain recommended page 64), plus
for lignified cell walls (see
a contrasting cytoplasmic or cellulose cell-wall stain. Wide changes
between water and all alcoholic percentages can be made without much
damage to the tissues. When the staining is completed, commence
rapid dehydration according to the hygrobutol method (page 110),
no matter in what solvent the sections might lie. Give two changes of
pure hygrobutol. Mounting may be done directly in balsam, but it is
preferable to place first in dilute balsam and partially evaporate the
solvent. Xylol and similar hydrocarbons should be avoided since they
 104 GENERAL METHODE

desiccate the sections and cause them to become very brittle and to
curl up.
Hard Wood Sectioning. —Hard woody stems have long been exasperat-
ingly difficult to microtome. It is now possible to cut relatively thin
sections in a good sliding microtome by a simple expedient (Crowell
1930). Arrange a large Erlenmeyer flask over a gas flame near the
microtome; from the flask run a narrow-bore aluminum or monel metal
(not copper) tube, in which a few coils have been turned, to a position
above the block holder in the microtome. Adjust the heat under the
flask so that the water boils slowly. Below the coils in the tubing place
another burner (preferably with a fishtail attachment) in order to vaporize
all the water before it leaves the open end of the tube. No water of
condensation should leave the tube, but the steam should condense upon
the wood, keeping it hot and wet. Sections as thin as lO/z, free from air
and curling, may now be cut, and thereafter treated as freehand sections.
Maceration of Tissues. —Sections of plant stems, roots, barks, and
other organs rarely convey an accurate conception of the real nature
of the cells of which they are composed. The only method which reveals
cells in their entirety is the dissociation method. By this procedure
the stem or other organ is treated with chemicals which dissolve the
middle lamellae and allow the cells to become separated from one another.
Thickenings, pores, and other distinguishing characteristics of the cells

are clearly brought out.

Jeffrey's Method ,
—First cut the material (either fresh or dry) into
small slices or slivers about 300/i thick. Either boil and cool repeatedly
until free from air, or use the suction pump for the same purpose. Then
macerate in a solution of equal parts of 10 %
aqueous nitric acid and 10%
aqueous chromic acid. The solution may be heated in the paraffin
oven for woody tissues, but not for herbaceous stems. The time varies
according to the material, but cells should begin separating in about
24 hours. A thick glass rod with rounded end may be used to crush the
material very gently. If it does not crumble easily, replace the macerat-
ing mixture with fresh fluid and continue action. Wash very thoroughly
with water to remove the acids. The use of a centrifuge is advisable
in order to speed up the process. The material may now be stained with
any suitable basie stain; safranin may be recommended. Leave in 1%
safranin for about 6 hours; rinse thoroughly in water, then dehydrate
by the fairly rapid addition of hygrobutol. Give two changes of pure
hygrobutol, then infiltrate with balsam highly diluted with hygrobutol,
and evaporate down to a mounting consistency. Take care not to put
too much material on the slide (see Frontispiece).
Various other methods have been proposed Harlow 1928),
but Jeffrey’s method is the simplest and has been found to be entirely
adequate for all maceration purposes.
 8PEQAL METHODS 105

—
Preservation of Plant Materials. The permanent preservation of
alltypes of plant materials in their original colors has long been a per-
plexing problem. Considerable success has recently been attained with
many types of material, but others, such as the Rhodophyta, still give
plenty of trouble. In the second section of the present text, methods
are cited for many of the plants therein discussed, but the following
general methods may be utilized.
1. A saturated solution of boric cxid in glycerin is said to be excellent.
2. ConanVs Hot Method. —Prepare a stock solution of 50% acetic
acid saturated with cupric acetate. For use, dilute 1 part stock solution
with 4 parts water, place the material in this diluted solution, and boil
until the permanent ^‘copper chlorophyll” develops in the material.
3. ConanVs —
Cold Pack^' Method. Dilute 1 part of the stock solution
described in the preceding paragraph with 10 parts of 5% aqueous
4.
formalin. Place specimens in the fluid, and leave until the proper
chemical changes occur. This may require several weeks, but a more
natural green than that imparted by the hot method usually results.
For more delicate material, such as the Chlorophyta, add a few drops
of the stock solution to the following formalin-aceto-alcohol variation:

Foriualin 6.5 cc.

Glacial acetic acid 2 5
. cc.

5.
50% ethyl alcohol 91 .0 cc.

The standard formalin-aceto-alcohol solution may also be used,

adding 0.2% cupric sulphate (Blaydes 1937). Or the proportions of the
formalin-aceto-alcohol may be changed as follows, adding the same
percentage of cupric sulphate:

Formalin 10 cc.
Glacial acetic acid 5 cc.
70% ethyl alcohol 85 cc.

Material preserved in these mixtures may, if necessary, be embedded

for sectioning. Preliminary experiments show that formalin-propiono-
alcohol is even better than formalin-aceto-alcohol, since the acetic acid
of the latter occasionally causes artifacts.
Dissolve cupric acetate crystals in 50% acetic acid until no more are
taken up. To 1 part of the solution, add 4 parts water. Place the
material or specimens into this solution, and bring the latter to boiling
over a flame. The green color of the material will change first to yellow-
ish-green, then back to green. The boiling may take from 3 to 15
minutes. When the color appears satisfactory, remove the material,
wash in water, and store in 70% alcohol plus about 5% glycerin to
prevent excessive evaporation,
106 GENERAL METHODS

6. Keefers Preserving Fluid , —This solution

is supposed both to kill

and to preserve the green In the experience of most users the

color.
results are very inferior to Conant^s methods. Gymnosperm material
and fruits, particularly, are badly preserved. The color of flowers is
not preserved, but the fluid, if 10 g. copper acetate is substituted for the
copper chloride and uranium nitrate and if it is diluted somewhat with
water, is excellent for the Cyanophyta.

50% ethyl alcohol 1

. 90 cc.
Formalin t 6 cc.
Glycerin 2 6 . cc.

Glacial acetic acid 2.6 cc.

Copper chloride . 10 g.
Uraniuin nitrate • 1.6 g.

Frozen Sections. —The freezing microtome, so commonly used in

clinical has scarcely been employed in botanical micro-
laboratories,
technique because plant materials do npt lend themselves readily to thci
customary freezing methods.
Many types of plant materials containing both soft and hard tissues
may, however, be embedded in an agar matrix and excellent sections may
then be cut on a freezing microtome (Evenden and Schuster 1938).
Fix with formalin-aceto- (or propiono-) alcohol, rinse with water and
immerse in 5% agar in distilled water, and pour into heavy paper trays
of the sort used for paraffin embedding. Allow the*agar to solidify.
Block and place in the freezing microtome.
Some practice is required in judging the degree of freezing of the
material. is made, the material should be partially
Before each section
thawed to a depth approximating the thickness of the section to be cut.
This may be done by rubbing the finger gently over the face of the block
or moistening with 50% ethyl alcohol. The directions for operation
of the freezingmicrotome, which come with each machine or which (^an
be obtained from the manufacturer, should be explicitly followed,
particularly with regard to the attachment of the cylinder of freezing
gas.
The solidifying qualities of the agar decrease with age and remelting.
The stock solution should not be remelted more than three or four times.
If necessary to preserve the blocks, wrap in wet paper toweling arranged
with a wick and water reservoir to insure a constant moisture supply,
then place the whole under a bell jar. The blocks will keep in this
fashion for two or three weeks.
Those who wish to experiment further with freezing microtome
methods should consult texts on histological technique, clinical laboratory
methods, or recent research papers (e.g., Geschichter, et ai., 1931; Hjort
and Moulton 1931).
 SPECIAL METHODS 107

Special Fixation-staining Methods

^^nnins . —To show the distribution and nature of tannin-containing

vacuoles, fix in

Ferrous sulphate 2 g.
Formalin 10 cc.
Di stilled water 90 cc.

Wash in water after 48 hours^ immersion, embed, section at not over

remove paraffin from sections, and mount in balsam without staining.
lO/Li,

Root tips of Pinus are exceptionally favorable material.

Nucleoli. —
After fixation with a fluid giving an acid fixation image',
the resting nucleus is a hollow body with a centrally located, darkly
staining, spherical nucleolus, and a periphery composed of the (ihromatin
reticulum. The colorless halo surrounding the nucleolus is thus quit<j
evident. The halo surrounding the nucleolus has been reported by some
cytologists to be a distinct structure in the living cell, but others more
correctly consider the halo to be an artifact. After fixation in a fluid
giving a basic fixation image, the nucleus is a solid body composed of
fixed nuclear lymph in intimate contact with a centrally located nucleolus.
In this type of fixation image there is no halo.
The best fixing fluids for nucleoli are mixtures of bichromates and
acetates. The acetates seem to penetrate the tissues more rapidly and
thus primarily determine the fixation image. The bichromates harden
the image and enable it to pass through the dehydration stages relatively
unaltered. Most of the fixing fluids designed to fix chromatinhave no
cation except hydrogen. These solutions are satisfactory preservatives
of chromatin but are quite erratic in the fixation of nucleoli. When the
cation of a chrom-acetic mixture is one of the group of heavy metals

{e.g.j copper, mercury, uranium, or lead), both chromatin and nucleoli

are well fixed and mordanted (Zirkle 1928).

In the chapter on Staining Procedures the effects of different dyc^s
on nucleoli have been noted. To distinguish the nucleolus during
mitoses, staining in the following mixture for about 12 hours has been
recommended:

Distilled water 100 cc.

Iodine green 0 1 5 g.
.

Acid fuchsin 0.01 g.

Dehydration should be rapid to prevent excessive removal of stain.

—
Mycorrhiza. Mycorrhizas of forest trees occur on the smallest
rootlets and in the more superficial layers of the soil. Alnus growing
on the banks of streams furnishes an excellent source of material, as the
 108 GENERAL METHODS

mycorrhizas are easily recognized on roots growing in the water or along

the banks. Fixation of such types may be in 1% chrom-acetic or in
formalin-propiono-alcohol.
Another fixing fluid which has been recommended is 5% chromic
sulphate in 4%
formalin saturated with picric acid or preferably with
salicylic acid (Cohen and Doak 1935). The ectotropic type of mycor-
rhiza is more difficult to recognize; the roots of the orchid Corallorhiza
furnish the best material. Portions of the root may be fixed with the
fluid just mentioned or with chrom-acetic.
The ordinary safranin-fast green schedule gives a good stain on Alnus;
a triple combination is usually excellent on Corallorhiza; or the following
method might be tried. Stain sections for 30 minutes in 3% aqueous
acetic acid saturated with orsellin BB (a Grilbler dye), rinse with
water, pass through the alcohols to 95%, counterstain with 1% crystal
violet in clove oil (prepare by adding enough alcohol to 1 g. of the
dry dye in a flask to form a paste, heat the flask gently to drive oS
excess alcohol, then add 100 cc. clove oil) for 5 minutes, rinse with xylol,
then clear in another jar of xylol, and mount in balsam.
If it is desired to cultivate the mycorrhiza-forming organism, Arcu-
larius^ solution may be utilized:

Malt extract 30 g.
Dibasic potassium phosphate 8 g.
Potassium carbonate 6 g.
Agar 26 g.
Distilled water 1000 cc.

Plasmodesma. —None
of the usual embedding methods gives as good
plasmodesma as do temporary mounts of freshly
or as striking pictures of
stained tissue. Although the mounts are only temporary, the prepara-
tions give a clear and vivid picture of protoplasmic continuity between
cells (Crafts 1931). The following solutions should be made up in
advance :

Killing Solution: Potassium iodide, 0.75 g.; iodine, 1.50 g. water, ;

100 cc.
Mordanting Solution: Potassium iodide, 1.25 g.; iodine, 1.0 g.; 5%
aqueous sulphuric aqid, 100 cc.
Staining Solution: (1) 5% aqueous sulphuric acid; (2) crystal violet,
0.5 g.; distilled water, 100 cc.
Mounting Solution: Glycerin, 30 cc.; distilled water, 60 cc.; zinc
chloride, 2 g.; potassium iodide, a trace; iodine, 0.2 g.
The killing and mordanting solutions should be mixed and warmed
until the iodine has dissolved to the point of saturation. The mounting
solution should also be warmed a little and a small crystal of potassium
iodide added, which dissolves some of the excess iodine. The resulting
 SPECIAL METHODS 109

solution should be of a light tan color. All solutions containing iodine

should be kept in brown bottles in the dark when not in use.
Place fresh sections in the killing solution for 5 minutes; then swell
for 5 minutes in 10% sulphuric acid. Transfer to the mordanting solu-
tion for 5 minutes, followed by washing in the 5% sulphuric acid until
the iodine starts to fade. The staining solution isprepared by placing
1 cc. of the sulphuric acid in a small watch glass and adding the violet
solution drop by drop until a deep green color is obtained. This unstable
stain solution should be used at once, and discarded when it starts to
fade. Transfer sections to the stain and allow to remain until darkened.
Remove sections to a slide, brush with a camers-hair brush on l)oth
surfaces to remove any adhering precipitates, then mount in the mount-
ing solution. The sections are good for several weeks, and may even be
mounted in glycerin jelly for longer preservation.
The most satisfactory and easily available material in which to
demonstrate plasmodcsma is the pith tissue from the center of a potato
tuber. Ch-oss sections of the leaflets of Cycas are also excellent.
Statoliths. —The name is given to the starch grains found in the basal
region of the cells of the root cap. They are rarely found in root tips
grown consequently tips grown in soil should b(^
in nutrient solution,
used. Most fixing fluids so hydrolyze the statoliths that they are not
revealed by the subsequent staining. LaCour\s fluids ha-ve given good
fixation. When the sections are stained by a triple combination, the
statoliths usually but not always take up the violet dye; when a quad-
ruple combination is used, they absorb the green.
Cilia. —
Drop material into a concentrated aqueous solution of mer-
curic chloride; after 10 to 20 minutes, wash in water; mordant 10 to
15 minutes in 0.33 to 1.65% phosphotungstic acid (discard the mordant
after using once) wash well in water; stain in toluidin blue (a 1 1000 to a
;
:

1 5000 dilution) for 80 seconds to several minutes, depending on the form,

:

at 50 to 60°C. dehydrate through the alcohols, clear in xylol, and mount

;

in balsam. Use the centrifuge whenever necessary. Cytoplasm should

be light blue, cilia and basal bodies dark blue.
—
Antherozoids. Put 1 drop of water containing the antherozoids on a
chemically clean slide, and fix by inverting the drop over the mouth of a
bottle containing osmic acid. About 2 minutes is required for fixation.
Then set aside to dry. Place slides in a saturated aqueous solution of
tannic acid for several hours (Steil 1933). Remove and wash the slides
thoroughly. Almost any brilliant coal-tar dye may now be used:
safranin, the violets, and fast green all give excellent results. Iron-
acetocarmin is good for the sperms of all Pteridophyta. The stain may
be allowed to react for 10 minutes to several hours. After staining,
rinse the slide in water, dehydrate with two changes of absolute alcohol,
clear with clove oil, pass through xylol, and mount in balsam.
 CHAPTER IX
WHOLE-MOUNT METHODS
The present chapter deals with methods of making whole mounts of
entire small or more or less flat objects. There are many types of
material which are most satisfactorily mounted entire, i.e., without
sectioning, although some trimming of excess tissue, or redu(!tion of large
pieces to a more convenient size may often be required. Among the
subjects commonly mounted whole may be mentioned filamentous algae
and fungi, flat thalloid algae, fern prothallia, delicate bryophytes and
mosses, pieces of leaf epidermis with stomata and occasionally trichomes
and pollen grains.
or glands, sori of ferns, small flowers,
Whole mounts display many features not revealed by sectioning
methods and frequently permit a better comprehension of the nature of
certain organs than do sections. In well-prepared fern prothallia, for
instance, the development of the antheridia and archegonia can be
followed out far more easily than in sections. Again, whole mounts are
the only means by which the surpassing beauty of some of the most
fragile of plants can be preserved permanently. The red algae Anti-
thamnion and Ceramium are examples.
Certain types of preparations are actually whole mounts but are
commonly included under other classifications. Smears of pollen
mother cells are really whole mounts, but since they are objects intended
for specialized cytological investigation, rather than for general morpho-
logical purposes, they are classified and described elsewhere.
Freehand sections of woods or stems might also be included as whole
mounts, but their treatment is so different that they must be excluded.
Various staining methods applicable to the classes of material men-
tioned above are outlined in a special section at the conclusion of the
descriptions of the different procedures.

PROCEDURES
The Hygrobutol Method. —This method is probably the one most
certain to afford satisfactory results. Even beginners in microtechnique
have had complete success with the schedule, as it works well even with
such a supposedly difficult subject as Spirogyra. It is the least expensive
of all.

110
 WHOLE-MOUNT METHODS 111

It must be emphasized that each step, in its logical sequence, must be

carried out. Variations in or departures from any of the steps are likely
to result in disaster.
la. A weak or medium chrom-acetic killing fluid will probably, in
most cases, allow the most brilliant staining effects. The filamentous
Chlorophyta will come out better in such a fluid than from one containing
formalin. Washout the fluid thoroughly with running water.
6. The marinealgae of a filamentous or sufficiently thin thalloid type
may be fixed in a 1% chrom-acetic fluid, or in 10% formalin, both made
up with sea water; washed out thoroughly with sea water; and the trans-
fer from sea to distilled water carried out by gradual stages.
c. Formalin-aceto-alcohol works satisfactorily with all types of
material except those from aquatic habitats. Wash out the fluid with
distilled water instead of the customary 50 or 70% alcohol.
The material should be kept in deep solid watch glasses or low stender
or crystallizing dishes for this and all the succeeding steps. Fluids
may be removed with a giant pipette. All the steps may be carried out
in the one container, which procedure obviously lessens the danger of
damage resulting from too much handling of the objects.
2. Stain with an aqueous stain or preferably some hematoxylin or
carmiii mixture. Harris’ or iron hematoxylin or Mayer’s carmalum
are strongly recommended, the first and last particularly so. Differenti-
ate the dye sharply, taking care not to destain too far since the following
steps may sometimes carry the differentiation a litth^ further. Wash
out the differentiator thoroughly.
3. Dehydrate in 15, 30, 50,and 70% ethyl alcohol, allowing at least
20 minutes in each. Always keep the container covered between changes
of fluids.
4. l./eave overnight or for at least 18 hours in 85% ethyl alcohol. Tlu^
purpose of the long immersion is to harden the tissues; no change will be
actually apparent, but the hardening nevertheless occurs. The material
does not become rigid or brittle; in fact the apparent lack of change during
this period may lead incautious technicians astray. If the step is short-
ened, the material will become excessively brittle immediately upon the
addition of a stronger alcohol, and it generally will not be amenable to
further treatment.
5. Counterstain with any desired cytoplasmic dye dissolved in equal
parts of 95% alcohol and methyl cellosolve. After a hematoxylin,
erythrosin B is recommended for the Rhodophyta, orange G for the
Phaeophyta, and fast green for the Chlorophyta, since in each instance
the material will be given a more or less naturalistic coloration. On
some materials stained basically with a carmin stain, a counterstain
may be undesirable, but on others one will reveal otherwise obscured or
 ;

112 OENERAL METHODS

unstained details. With leaves or portions of epidermis, for example,

anilin blue or fast green provides a good contrast for Mayer^s carmalum.
The counterstain may be used in fairly strong concentration as there is
little chance of overstaining. The time should be about 15 minutes, but
one should make certain that the stain has thoroughly penetrated the
material. Differentiation of the counterstain is rarely required. The
hygrobutol, to follow in the next step, exerts a certain differentiating
effect at the same time that it prevents the 95% alcohol from extracting
too much of the stain.
6. Pour off the counterstain, and rapidly wash out excess dye with
95% ethyl alcohol. Replace with fresh 95% alcohol, and immediately
(even if the second 95% alcohol is tinged with the counterstain) begin
the addition of hygrobutol by the gradual substitution method. Tins
consists in adding a small amount of the hygrobutol every 30 seconds or
so, avoiding strong diffusion currents, and mixing th('. fluids by tilting
the container back and forth. Between every third or foiirth addition,
discard some of the mixture. Continue in this fashion until the propor-
tions are about 90 parts of hygrobutol and 10 parts ethyl alcohol. It is
unneciessary and probably undesirable to attempt to get rid of all the
ethyl alcohol, unless a very delicate counterstain which might he injured
by the ethyl alcohol has been used.
7. Pour off most of the mixture of butyl and ethyl ak^ohols, and replace

immediately with balsam (thick), diluted at least ten times with hygro-
butol. Uvse many times as much diluted balsam as the bulk of the
material. Set the container aside, exposed to the air, but in a warm,
dust-free place, until the solvent has evaporated sufficiently to leave t,h(‘

balsam of a mountable consistency.

The evaporation should not proceed too rapidly on delicate materials
2 hours is probably the safest minimum for the process. Watch the
progress of the evaporation; if the material should be in danger of
becoming exposed owing to an insufficient amount of diluted balsam
having been used at the beginning, add more of the diluted balsam.
Never add thicker balsam to a diluted mixture.
8. Mount in balsam. Circular cover glasses should preferably be
used since a more accurate judgment of the required amount of balsam
can be made.
Objects which are to be mounted singly can be manipulated without
difficulty. With filamentous algae or fungi, fine scissors should be used
freely. One should never attempt to pull filaments of Spirogyra, for
example, out of the mass of material, but the entire mass should be cut
into portions not over 5 mm. long. The mass can then be easily separated
and the individual filaments spread out in the drop of balsam on the
slide.
 WHOLE^MOVNT METHODS 113

To make the mount, put a fairly large drop (about the size of two
grains of wheat) in the center of a clean slide. Take up some of the
material with a needle or tiny spatula hammered out of a needle, and
place in the drop of balsam, taking care that the balsam covers the
material. Carefully spread the material to avoid overlapping. It
would be a good idea to trace the outline of a slide on a 3
5 piece of X
cardboard, then within the outline to trace around the size of coverslip
being used, allowing sufficient room at the left for the label (note the
first two slides in Fig. Pick up the cleaned coverslip with fine-
13).
pointed forceps, pass once through an alcohol flame to remove moisture,
then apply to the balsam and lower in a slanting position so as to avoid
trapping air bubbles. If the amount of balsam has been properly
judged, should just come up to the edges of the coverslip. Slight
it

pressure on top of the cover with the for(‘eps may be required if the
amount of balsam was somewhat insufficient, or if the material is curled
up a little. Finally place the slide on a warming plate for a day or two
to solidify the balsam.
The Dioxan Method. —The dioxan method is also easy but is a rather
expensive method. It has one serious disadvantage, viz., that only
aqueous or wf^akly alcoholic stains can be employed. But even this
disadvantage may easily be circumvented if a thorough knowledge
of the manipulation of the various dyes is already possessed by the
technician.
Only pure anhydrous dioxan can be employed in this method; the
practical and ordinary commercial types are useless.
1. Kill, fix, wash, and stain the material with aqueous stains. Care-
fully wash out all surplus dye with w^ater; if unattached dye particles
remain, they will be precipitated by the dioxan.
2. Dehydrate with the following mixtures of dioxan and distilled
water, allowing about 30 minutes in each: 50, 75, 80, 90, 95%, then leave
overnight in pure dioxan and follow by two changes during a J^^-hour
period the next morning. The critical period is between the 90% and
pure dioxan. If any plasmolysis (apart from any produced by the killing
fluid) occurs, return to the next lower percentage, and leave until turgor
is restored and upgrade again by a more gradual series.

3. Infiltrate with balsam highly diluted with dioxan, in the same

manner as described above in the hygrobutol method. The evaporation

of the dioxan will proceed somewhat rapidly, consequently progress
should be checked at frequent intervals. In mounting, do not use too
much balsam because a small amount of dioxan-diluted balsam will be
carried over with the material, and it will dilute the balsam somewhat
but not enough to cause bubbles to form under the edges of the coverslip
later.
114 GENERAL METHODS

—
The Creosote Method. While the two preceding methods should
prove satisfactory with most materials, one may occasionally prefer to
use for bulkier materials, such as fern prothallia with young sporophytes
attached, Nemalion and similar Rhodophyta, thick pieces of leaf epider-
mis or styles with microgametophytes, a clearing agent which renders
them more transparent than does hygrobutol or dioxan.
Beechwood creosote is excellent for the purpose. The brand employed
should be Hartmann and Hauer^s, as all other brands, most of which
are synthetic compounds, have failed completely to give even passable
results. Great care must be taken to avoid getting any creosote on the
skin as produces extremely irritating blisters.
it

Kill, fix, wash, and stain the material as usual. One may freely
transfer the material, if necessary, from water to 50% ethyl alcohol,
and vice versa, without appreciable harm. Pass through 70 and 80%
ethyl alcohol, allowing at least 10 minutes in each. Any counterstains
which are best employed in strong alcoholic solutions may be used at
this juncture. Next pass through the following mixtures of creosote and
85% ethyl alcohol, allowing 15 to 20 minutes in each: 1 part creosote
to 4 parts alcohol, equal portions of the two reagents, 4 parts creosote
to 1 part alcohol, followed by two changes of pure creosote. Since
creosote and thick balsam are perfectly miscible, one could theoretically
go directly into balsam diluted with creosote. The practical difficulty,
however, is that it would be many months or even a year before the
creosote would evaporate sufficiently to permit mounting the material,
and many more months would elapse before the balsam mounts hardened.
It is therefore better to remove most of the creosote with a more volatile
solvent which is perfectly miscible with both creosote and balsam.
Either hygrobutol or dioxan may be used, in the following proportions of
solvent and creosote: 1:5, 2:5, 3:5, 4:5, then through a change of the
pure solvent. About 15 minutes in each change suffices. Next place
the material in balsam highly diluted with the solvent, then set aside
to concentrate as usual.
The Glycerin-Xylol Method.
1. Kill, fix, and wash as usual. Stain with an aqueous basic stain,
such as one of the hematoxylins.
2. Put the material in 10% aqueous glycerin, place the container

where dust will not settle in (but do not cover), and leave for several
days until the diluted glycerin attains the consistency of pure glycerin.
The process may be accelerated by placing the container in the paraffin
oven on an upper shelf. Do not allow the material to become exposed;
add more of the diluted glycerin if necessary,
3. Remove the glycerin with several changes of 95% alcohol. The
thicker the mass of material, the more the number of changes of alcohol
 WHOLE-MOUNT METHODS 115

required to remove the glycerin, as it sticks to the mass rather closely.

If the material placed in a wire strainer and the latter placed in a

is

suitable dish so that about 1 cm. of space is left clear below the strainer,
the heavier glycerin will soon settle down.
4. Counterstain, if desired, with any suitable acid dye dissolved in
95% alcohol.
5. Complete dehydration
in a change of absolute alcohol.
Dealcoholize in a series of about six to eight intermediates between
6.

absolute alcohol and xylol; a longer series is preferable to a short one.

Start with a proportion of 9:1, then 8:2, e^t(5. The time in each mixture
may be between 5 and 10 minutes. It is not necessary to get rid of all
absolute alcohol as it will evaporate during the next step, but if there
seems to be danger that the alcohol may extract any stain, give two
changes of pure xylol.
Hygrobutol may be substituted for the xylol, and tluTo is less danger
of the material becoming hardened.
7. Transfer to balsam highly diluted with xylol (or with hygrobutol

if that reag(‘nt has been substituted), then set aside to eva[)orate to a

mounting consistency.
The Venetian Turpentine Method. —The following schedule repre-
sents a modernization and simplification of the old Venetian turpentine
method, long a favorite with the older botanists.
1. Kill, stain, and dehydrate gradually with ethyl alcohol until
absolute alcohol is reached. Give two changes of the absolute alcohol.
Make a 10% solution of Venetian turpentine in absolute alcohol,
keeping it in a tightly stoppered bottle. If the turpentine is too thick
to flow easily, place the container in a hot-water bath until it flows easily.
Prepare an exsiccator. A specially constructed exsiccator should be
available in most laboratories, but if one cannot be found, it is not at all
difficult to arrange a satisfactory substitute. In the regular exsiccator,
fill the bottom compartment half full of a mixture of equal portions of

sodium hydroxide (wdiite sticks) and calcium chloride. Or fused calcium

chloride may be used alone. In lieu of an exsiccator, secure a piece of
plate glass and a bell jar of as low a height as possible but wide enough
to cover a saucer and one or more watch glasses. Place the hydroxide-
lime (or chloride) in the saucer. Cover the top of the exsiccator or the
bottom of the bell jar with a thin but even layer of vaseline or petrolatum.
The transfer now ready to be made, and it should be performed as
is

expeditiously as possible in damp climates. Pour off the absolute alcohol,

place the watch glass or other container in the exsiccator, and fill with
the 10% turpentine. Then place the cover or bell jar in position, and
see that the vaseline has spread evenly, leaving no air leaks. Be careful
not to let any of the drier get into the turpentine. The concentration
 116 GENERAL METHODS

of the turpentine should not proceed too rapidly. The amount of drier
can be regulated so that the process takes four or five days. Do not
try to open the exsiccator until you are fairly certain that the turpentine
has become thick enough for mounting, which will be when it has the
consistency of pure glycerin.If the cover is taken off too soon, the
turpentine is practically certain to become cloudy.
After the drier absorbs sufficient moisture to become nearly saturated,
it is best to remove it, heat until dry and then replace; or fresh drier
may be used. If the turpentine shows any sign of cloudiness, it is

spoiled. If the material is too valuable to be thrown away, wash in

absolute alcohol until the cloudiness disappears, then begin again with
the 10% turpentine.
To mount, first cut the material into suitable portions with fine

scissors, if this should be necessary. Gather up a small quantity of the

material with a needle, and place on a clean slide. Add the requisite
amount of xylol-balsam, then the coverslip (circles are preferabl(‘).
The material may be mounted in the turpentine, of course, but experien(‘e
has shown that it is far better to mount in balsam as the turpentine is
rather prone to crystallize, and one cannot be perfectly certain of its
quality.
If it is not convenient to mount all of the material at once, it may b(‘

stored in vials which must be securely corked; the cork and upper part
of the vial should be sealed with melted paraffin as a precaution against,
the turpentine becoming too thick.
2. Proceed as far as the end of step 2 of the dioxan method. Make
a 10% solution of Venetian turpentine in dioxan and transfer the material
to this mixture. The solvent can be evaporated in the open air without
danger of the absorption of moisture. Mount in balsam as usual after
the turpentine has become sufficiently thickened.

STAINS AND STAIN COMBINATIONS FOR WHOLE-MOUNT MATERIALS

The selection of the stain or stain combination to be used on objects
intended for mounting whole depends entirely on the nature of the
material itself. The greater the clarity of internal detail that may be
required, the more transparent the stain or stains must be. Some dyes
give an opaque stain, which obviously tends to obscure finer internal
details. Most of the coal-tar dyes are in this category, but with certain
types of material there frequently results more differentiation than
might be expected. One can only try the stain or combination on a
small portion of the material and observe the result under the microscope.
Harris' hematoxylin is an unusually transparent stain eminently suited
for bidky materials and is unsurpassed for many types.
 —

WHOLE-MOUNT METHODS 117

The basic or nuclear stain should be used in rather dilute concen-

tration. Differentiation is not always easy.
Iron Hematoxylin. —For filamentous algae and many fungi of a
similar structure, this is particularly suitable. It is, unfortunately,
very difficult to apply to the Rhodophyta as these plants usually break
up in either the mordant or the stain. Nor is it suitable for pieces of
leaf epidermis, for Bryophyta, or for fern prothallia.
After the fixing fluid has been thoroughly washed out, mordant in 2%
aqueous ferric ammonium sulphate for 2 hours, no longer. Then wash
thoroughly in running water for about 20 minutes, and rinse in two
changes of distilled water. Stain for at least 2 hours or overnight in
0.2% aqueous hematoxylin, well ripened. Wash out all excess stain with
distilled water. Destain in 2% aqueous ferric ammonium sulphate
or ferric chloride until the stain, as observed under the microscope, seems
to be satisfactory. When the nuclei appear dark grayish in water, they
will look black or bluish-black when finally mounted in balsam. One
soon learns to recognize a grayish fluorescence in the material, which is
generally an accurate criterion of proper differentiation. Wash the
differentiator out very thoroughly with running water.
Iron Hematoxylin with Counterstain. —
Many excellent counterstains
for iron hematoxylin are available, the one to be selected being governed
principally by the nature of the material. Sometimes one dye works
better than another; only experimenting can determine this point.
Erythrosin, fast green, orange G, anilin blue, and safranin may all be
used in 1% solutions in 95% alcohol. In the case of the safranin add a
few drops of the 1% solution in the 95% alcohol in which the material
lies, and watch the material critically for the next 20 minutes or so.

When the stain is just right, wash out the excess stain in a change of
95% alcohol.
Delafield’s Hematoxylin with Iron Hematoxylin and Fast Green.
After washing out the ferric destaining solution, counterstain with
Delafield’s hematoxylin. With some materials this solution (which
should be used in highly diluted form) imparts a violet stain, with others
it merely acts as a sort of mordant for the fast green and causes the other-

wise faint or colorless cell walls to stain brilliantly. Apply the fast
green in 95% alcohol as usual. The combination is most vsuitable for
filamentous Chlorophyta.
Harris’ —
Hematoxylin with Counterstain. Harris' hematoxylin is
really superb for the Rhodophyta and for fern prothallia with sex organs.
The stain is decidedly transparent and clearly reveals the internal
cellular organization of even thick objects. The color imparted by
Harris' hematoxylin is similar to that of Delafield's but is less variable
and more precise.
118 GENERAL METHODS

For the Rhodophyta the best counterstain is ersrthrosin B; it may

be used in 95% ethyl alcohol as usual. If the dioxan method is used, a
1% aqueous solution of the erythrosin may be employed with safety
since the dioxan scarcely extracts the stain. Fast green should be sub-
stituted ior the erythrosin in the case of fern prothallia, leaf epidermis,
and similar subjects; the erythrosin will stain such material more evenly,
but the red color is too unnatural.
Safranin and Fast Green. —This combination is excellent for many
types of plant material, such as powdery mildews and similar fungi on
leaves, fern leaves with sori, and thick epidermis peelings. It would
be best to follow the glycerin dehydration method. When the dehydra-
tion with glycerin is begun, add a few drops of a 1 % aqueous solution of
safranin 0. As the glycerin concentrates, those structures possessing
an affinity for safranin become red. Wash out the glycerin as usual.
To the last change of absolute alcohol add a few drops of 0.2% fast
green in 95% Proceed to Venetian turpentine. (Unpublished
alcohol.
method of Dr. G. H. Conant.)
Anilin blue may be used in place of the fast green, but a saturated
solution in 95% alcohol is required.
If the dioxan or creosote dehydrating schedule is used, the fast
green or anilin blue should obviously, and as a
rule, be used in aqueous
solution. However, the fast green may be dissolved in 50% dioxan or
in pure creosote and used at the appropriate stage in the respective
schedule. It scarcely overstains, and only the surplus stain needs
to be washed out (with 50% dioxan or with pure creosote, as the case
may be).
Fast Green. —The
dye alone suffices for many classes of material,
especially thosewhich are naturally dark colored and do not lose the
color during the various processes. Maturing zygotes of Rhizopus or
ripe sporangia of many ferns are cases in point. The stain will also
reveal clearly the embedded foot of the sporophyte in whole mounts of
Anthoceros.
 CHAPTER X
THE GLYCERIN METHOD
Among the older botanists, and especially with the algologists, the
glycerin method was the only one followed in the mounting of algae and
similar plants. The method has fallen into disuse since the introduction
of more permanent methods, but it still has its occasional uses, and for
this reason it is being presented. When first proposed, the schedule
called for mounting in pure glycerin, but later improvements included
final mounting in some form of glycerin jelly, which added to the per-
manence of the preparations.
—
Unstained Preparations. Glycerin will frequently preserve the
natural colors of various plants, such as unicellular and colonial Chloro-
phyta, moss protonema, spores of fungi, Equisetum^ and ferns. Delicate
objects which might easily plasmolyze may be placed in a drop of 10%
aqueous glycerin on a chemically clean slide and the glycerin allowed to
coiu^entrate. When this has occurred, add a coverslip very cautiously
so as not to allow any of the glycerin to ooze beyond the edge, and later
seal as described below. If only temporary sealing is required, vaseline
will serve.
—
Stained Preparations. Kill and fix the material as usual. Only
aqueous stains may be used on material intended for mounting in glycerin
jelly. Iron hematoxylin with or without a counterstain will generally
be found wholly satisfactory. The staining completed, place the material
in 10%aqueous glycerin in a dish which permits the exposure of as much
surface as possible. Put the container where evaporation of the water
may take place. It may be put on an upper shelf or cooler part of the
paraffin oven, but care should be taken to see that evaporation does not
proceed too rapidly; otherwise some shrinkage may result.
When the diluted glycerin has become about as thick as pure glycerin,
the material is ready to be mounted. The best mounting medium is
Kaiser's glycerin-gelatin. To make it, place 1 part by weight of Knox's
or other pure gelatin in 6 parts by weight of water, and allow to soak for
at least 2 hours. Add 7 parts by weight of glycerin, and for every
100 g. of the mixture add 1 g. phenol crystals. Warm for about 15 min-
utes, stirring constantly and vigorously until all the flakes produced by
the phenol have disappeared. Filter through cotton while still warm.
The mixture is best stored in a small bottle that can conveniently be
119
120 GENERAL METHODS

warmed in a hot-water bath. When ready to mount, put some of the

material, freed from as much of the excess glycerin as possible by touching
to absorbent paper, on a chemically clean slide, and add just enough of
the warmed jelly mixture to come to the edge of the round coverslip
after the latter has been applied. If any of the mixture has oozed beyond

the coverslip, it is impossible to seal the mount properly. The moupts

may be sealed either as soon as the jelly has solidified, or they may first
be set aside for a week or 10 days.
Various cements and other media have been Used for sealing the
mounts. Gold size has been most commonly used, Canada balsam com-
ing next. Shellac, asphaltum. King's cement, Duco cement and plain
Duco, Hazen's cement (8 parts rosin and 2 parts anhydrous lanolin:
melt the rosin first, then add the lanolin; apply hot), and different kinds
of varnishes have all been employed.
Sealing may be done freehand or with the aid of a turntable. In
either case considerable practice is required to turn out a satisfactory
ring. Brush a thin coating of the cement over the margin of the cover-
slip and the adjacent slide. Wait for one coat to dry thoroughly before
applying the next. The number of coats required depends mostly
upon the character of the cement used, but two should suffice. In using
the turntable, place the slide in position, and center accurately. Dip
a fine-pointed brush in the cement, give the table a spin and touch the
tip of the brush to the slide as far from the coverslip as it is desired that
the ring shall extend. Approach the coverslip gradually but without
actually touching it. Dip the brush in the cement again, and give the
table another spin. Bring down the brush so as to touch the coverslip
gently, and bring the ring of cement into contact with the earlier applica-
tion. The cement should be extended over the coverslip about 1.5 mm.
from ^he periphery.
Glycerin or |lycerin jelly preparations should always be stored flat,
never on edge, otherwise the material will sink to the lower edge.
 CHAPTER XI
CELLOEDIN METHODS
To some schools of microtechnical philosophy, the celloidin method
is one that has lapsed into desuetude; to others it is the only procedure
worth following. The one considers the smear and paraffin methods
adequate for practically all purposes, while the other regards them
as distinctly inferior. The beginning technician will very soon find
himself confronted with this very sharp conflict of opinion and will
wonder why it should exist. The celloidin method as applied to plant
materials is practically the procedure developed by a single man and
his students, while the other methods were built up by numerous workers.
For plants one method is scarcely better than another, intrinsically;
it all depends upon the individual technician and his manipulative

ability, whether time is an important factor and cost no great object.

Despite the claims of its proponents, the celloidin method is a difficult
one to master in its details. It is also the most expensive one. Never-
theless, anyone who has had sufficient experience with both the paraffin
and the celloidin methods will readily admit that the celloidin schedule
will afford better results with certain types of materials. For example,
perfect sections of woody stems cannot always be cut in paraffin, but
really remarkable results are obtainable with celloidin. On the othei*
hand, root tips and buds for chromosome studies are better in paraffin
than in any other medium, if sections rather than smears are indicated.
Almost any sort of plant material may be embedded in celloidin, if
the nature of the material is carefully considered.
The treatment of the material preliminary to embedding is the same
as for the paraffin niethod. After the killing fluid has been washed out,
the material is gradually brought up to absolute ethyl alcohol, of which
at least two changes should be used to insure the removal of all water.
The material is then ready for infiltration.
By another method the material may be dehydrated with tertiary
butyl alcohol in the regular manner but carried only as far as the mixture
of 75% tertiary butyl alcohol and 26% absolute ethyl alcohol. From
this it is transferred to a mixture of equal parts of tertiary butyl alcohol,
absolute alcohol, and ether.
The pieces of celloidin should be removed from the bottle in which
they are stored in water, washed with water, then with 95% alcohol,
and finally spread on paper toweling to dry thoroughly. Equal parts of
121
122 GENERAL METHODS

ether and absolute alcohol is the solvent almost universally employed.

Make up five solutions of solvent and celloidin, each in a separate,
tightly stoppered bottle: 2, 4, 6, 8, and 10%.
From absolute alcohol the material is passed through an intermediate
stage of equal parts of ether and absolute alcohol, then transferred to a
wide-mouthed bottle and covered: with 2% celloidin. Subsequent treat-
ment may follow either of two methods, in one of which (1) the celloidin
is used cold, in the other (2) heed; is employed.

1. From the 2% celloidin transfer to the 4% and so on to the 10%

solution, allowing from one to many days for each percentage. The
length of time required depends on the nature of the material and can
be easily judged. Or one may partially expose the material in 2%
celloidin to the air and allow the solvent to evaporate slowly. In either
case, weeks are often required for the proper impregnation and embedding
of the material.
2. If heat is to be used to hasten the impregnation process, wide-
mouthed flanged bottles are needed. After placing the material in the
bottle and covering with 2% celloidin, insert the cork stopper securely,
and either wire or otherwise clamp the cork in firmly so that it will not
be blown out by the pressure exerted through the vaporization of the
solvent. Place the bottle on its side in a constant temperatun‘ oven,
with the temperature between 45 and 55°C. The change from one
concentration of celloidin to the next higher onemay be made about
every 24 hours. Always cool the bottle before attempting to open, and
clamp the cork tightly, as before.
After the 10% celloidin has been reached, it is impracticable to use
higher percentages because of the difficulty of holding back the material
when making transfers. The 10% solution may be thickened by the
occasional addition of small chips of dry celloidin. The thickening
process should be carried on until the celloidin mass appears more or
less stringy as it runs down the sides of the bottle when the latter is

quickly inverted.
Embedding is accomplished by removing the individual pieces of
material from the thickened celloidin with a pair of fine forceps and
holding in chloroform to harden. Care should be taken to see that each
piece of inaterial is surrounded by an ample coating of celloidin before
being immersed in the chloroform. The mass will in a few moments be
sufficiently hardened on the surface to be cut from the forceps with a
scalpel. After 24 hours' immersion in the chloroform, transfer the
and 95% alcohol,
pieces of material to a mixture of equal parts of glycerin
in which they may be left indefinitely.
A sliding microtome is required for sectioning materials embedded in
celloidin, although the new Spencer No. 820 rotary microtome may be
 CELLOIDIN METHODS 123

equipped with a special holder in which the knife may be oriented at an

oblique angle. The knives. used should be heavier in style than those
employed for paraffin sectioning and a keen edge must be maintained.
After the embedded material has remained in the glycerin- 95% alco-
hol mixture for several days, it is ready for mounting and sectioning.
One should not attempt microtoming the material without first treating
with the glycerin-alcohol mixture. One end of a block of wood, cut to a
convenient size, is dipped in 6% celloidin, then allowed to dry. Remove
excess celloidin from the embedded piece of material which is to be sec-
tioned. Then immerse both the wood block and the piece of material
in 6% celloidin, place the object in position upon the end of the block,
and set the whole aside to dry for about 10 minutes.
Place the mounted block in the object holder of the microtome.
Insert the knife, and clamp tightly. The edge of the knife and the
object are both kept wet with 95% alcohol, which may be applied with a
fine-pointed camers-hair brush. Held the brush on top of the object
(as is done when cutting freehand sections), taking care not to move it
or to allow it to project between the object and the knife, then move
the knife carriage forward with a steady stroke to cut a section. The
section may be moved toward the upper edge of the knife blade and left
there for awhile. If removed from the knife immediately, there is a
strong tendency for the sections to curl. Serial ribbon sections, such as
occur when one microtomes material embedded in paraffin, cannot be
obtained from celloidin material. The sections may presently be
transferred to a watch glass of 95% alcohol. After a section is cut, if the
sliding microtome is used, move the knife carriage back, and if the feed is
not automatic, adjust the object holder to the proper thickness for the
next section. The sections should be examined from time to time by
placing one on a clean slide and placing this under a low-power microscope
objective, paying attention to the proper thickness of the material and the
smoothness of the sectioning.
The angle at which the knife is oriented is a matter of importance
and is best determined by experience. As a rule, the softer the material
to be sectioned, the more obliquely the knife should be placed to the line
of movement of the knife carriage. For hard materials, for example, such
as tough stems or pieces of coal, the knife should be at a right angle.
Almost any desired combination of stains can be used on celloidin
sections. The celloidin matrix is usually not removed from the sections;
if it seems desirable to remove it, it is best to do so during the later stages

of dehydration or just before mounting. The matrix sometimes pre-

vents the dyes from being as brilliant as they are with freehand or paraffin
sections; the tones, in other words, appear flat. The matrix itself is
usually stained more or less but this does not interfere to any appreci-
124 GENERAL METHODS

able extent when the completed slides are examined under the micro-
scope. For woody sections iron hematoxylin and safranin form an ideal
combination.
To effect dehydration, transfer the sections to 95% alcohol for about
1 minute, then pass through two changes of absolute alcohol to which
about 5% of chloroform is added to prevent the alcohol from dissolving
the matrix. Clearing may be effected with either xylol or preferably
benzol. If it is desired that the matrix be removed, this can be done by
interpolating ether or a mixture of equal parts of ether and absolute
alcohol between the second absolute alcohol and the clearer. The 95%
and the first absolute alcohol will remove the excess stain, in case coal-tar
dyes have been used, from both sections and matrix. Dehydration
may also be effected by the use of beechwood creosote; the creosote
softens the celloidin just enough to prevent the sections from curling
when mounting and also removes excess stain from the matrix. Wash
out the creosote with xylol. When ready for mounting, the sections
may be transferred to a slide and arranged as desired. Place a few
drops of balsam, preferably dissolved in benzol and somewhat thicker
than that used for paraffin sections, on the sections, and add the cover-
slip. If the sections appear not to be sufficiently flattened, a lead weight
may be placed on top of the coverslip to press out the excess balsam.
Dry the mounts on the drying table as usual.

DOUBLE EMBEDDING
Suitable materials may first be embedded in celloidin and then in
paraffin. Various methods have been proposed for accomplishing the
result, but all differ solely in the nature of the reagents employed for
making the transfer into paraffin. In any case, the infiltration with
celloidin is carried out as usual, and the block is hardened in chloroform.
From the chloroform the celloidin-embedded material may be trans-
ferred to castor-xylol (1 part castor oil and 3 parts xylol) in order to
clarify and to complete hardening. Then place in melted paraffin in the
oven for a day or two. If desired, the material may be placed in either
pure xylol or pure cedarwood oil for 12 hours before going into the
paraffin. Finally embed, and section as for ordinary paraffin materials.
Another method is to transfer the material to 4% celloidin in clove
oil (an 8% solution of celloidin in absolute alcohol-ether diluted with an

equal volume of clove oil) for 2 to 24 hours (Tschernyachinsky 1930).

Place in chloroform for 30 minutes, then transfer to chloroform-paraffin
in the oven, and leave Next transfer through
until the material sinks.
several changes of pure paraffin and
embed.
finally
A third method, which involves the chance of considerable danger
from explosion if ovens with exposed heating elements are used, utilizes
 CELLOIDIN METHODS 125

benzene (not benzine). From the chloroform, transfer to equal parts

of chloroform and benzene, then to pure benzene, next to a mixture of
benzene and melted paraffin, and finally to pure paraffin.
Sections may be cut on a rotary microtome, exactly as plain paraffin
sections are made, and a ribbon will be formed. To mount the sections
on slides, prepare the following adhesive: mix 2 cc. acetone with 1 drop
methyl benzoate, and add 8 cc. distilled w^ter. Two or three drops of
Mayer^s adhesive may also be added and the whole mixed thoroughly.
Flood the slide with this mixture, and place the sections thereon. Use
little heat for spreading, as the sections begin to straighten out as soon

as they are placed on the mixture.

 CHAPTER XII

PARAFFIN METHODS
The parafl&n method the most popular one, and there is little
is still

reason to suppose that ever become entirely supplanted unless

it will

some as yet unknown substance can be found which has all the properties
of paraffin except that it does not require heat to transform it to the fluid
state and that it is miscible with all solvents. There is scarcely any
danger that either the smear or the celloidin method will supersede the
paraffin method since both have inherent faults not found in the paraffin
method, although it is freely admitted that the paraffin method has its
occasional difficulties. The different methods should, as a matter of
fact, be considered as supplementary to one another.
The older paraffin infiltration procedures called for the use of ber-
gamot oil, cedar oil, or xylol as a ^^clearing agent but the recent introduc-
tion of tertiary butyl alcohol as a substitute for these reagents has
permitted considerable refinement, shortened the time, allowed greater
latitude in the selection of materials to be embedded for microtoming,
eliminated excessive hardening of the material, and greatly lessened the
probability of failure, as well as making the method far less tedious and
expensive. The advantages of tertiary butyl alcohol over other fluids
are numerous, its disadvantages so few as to be negligible. However, for
the benefit of those who might wish to use them, several of the older
schedules will be described in sufficient detail.
Whether one uses xylol, tertiary butyl alcohol, chloroform, acetone, or
whatever other clearing agent, the preliminary stages of manipulating
the material are identical and may be discussed independently.

COLLECTING OF MATERIAL, KILLING AND FIXING

Collection as well as preparation of the material to be embedded is a
far more important step than it has usually been regarded. Indeed, with
the newer and highly refined schedules, the preliminary stage becomes
the one upon which everything else depends. If the material is not
selected with sufficient care and properly prepared at the beginning,
none of the succeeding steps will correct the initial errors.
In a single collection of material there should be included only one
specifier form, and only some particular organ or portions of this organ,
su<|i4^ anthers or sections of stem or leaf. If, for example, one wishes to

study meiosis in the anthers oi lAlium^ the anthers should be dissected

126
 PARAFFIN METHODS 127

out of the buds and partitioned into sections not over 8 mm. in length.
An entire series of stages in the development of the anthers should be
included in the first collection since one can learn onlyfrom personal
experience or by experiment the exact stage growth during which a
in
particular phase, such as diaphase, occurs. Then, in later collections, any
desired stage may alone be collected. Buds, ovaries, and similar organs
with enclosed air spaces of considerable size should be opened in some
innocuous way to facilitate penetration of the various reagents. Whea>
there is only one kind or type of material in a collection, far more uniform
fixation, dehydration, and infiltration are secured.
It is presumed that the material to be embedded has been accurately
identified. It goes without saying that anything whose source and exact
identity are unknown is not worth bothering about.
Some permanent record of the nature of the material, the procedures
followed, and other pertinent data, is necessary. A series of numbers or
other symbols may be written on fairly stiff white paper with waterproof
India ink, and the corresponding symbols on ordinary 3 X 5-inch index
cards or in some sort of record book. A number is cut off and placed
in the vial or bottle with the material and embedded with the latter
(note Fig. 4, A, C, and D), while all requisite information is written on
the card or in the record book. In a large research laboratory it is
necessary to keep rather accurate and detailed records; a printed form
(.will then be found most satisfactory.

The various killing and fixing fluids have been described in an earlier
chapter, together with notes regarding their suitability for various kinds
of material and directions concerning aftertreatment. In the second
section further suggestions with respect to the different groups or families
of plants are offered. It is well to bear in mind the fact that, simply
because a certain fluid gave excellent results with some particular
material, it does not follow^ that equally good results will be obtained
with almost anything else. To use the first available fluid and to expect
the material to be properly fixed a common practice and one severely
is

to be condemned. Some study should be made of the nature of the

material to be fixed, with special reference to exactly which structures
one particularly wishes to study, at the same time giving consideration
to suitable killing and fixing fluids. Formalin-aceto- (or propiono-)
alcohol, for example, gives perfect fixation of such diversified structures
as filamentous algae and woody stems, but usually gives atrocious
fixation of chromosomes and dissolves mitochondria.
For ordinary purposes shell vials about 2.5 cm. in diameter and
7 cm. in length, with tightly fitting corks, are wholly satisfactory. For
cbllectiing in the field or on long trips, where there is danger of crushing
the rathter thin shell vials, homeopathic vials of about the same size are
128 GENERAL METHODS

preferable. If the collections are bulky, corked or screw-cap bottles

of an appropriate size may be used. In general, all the processes of
killing, fixing, ’washing, dehydrating, and clearing can be carried out
in the one
vial or bottle. For infiltration other vials or bottles should
be used, since it is very diflficult to remove the paraflSn. Vials which
have once contained paraffin may be used for this purpose over and over
again.
V The amount of killing and fixing fluid should always be at least ten
times the volume of the material. A much greater volume, even to
fifty times, is necessary with material containing a high percentage of

Fig. 2. — Suction pump setup: A, bottle containing vial with material; B, stopcock;
Cy safety bottle; D. pump attached to faucet. The glass tubing at the right-hand side of the
safety bottle C should extend to within 1 cm. of the bottom of the bottle.

water. The water, obviously, dilutes the killing fluid, and allowances
should bemade for this fact. With the dehydrating and clearing reagents
one need use only a little more than enough to cover the material. All
fluidsshould be discarded once they have been used.
Someclasses of material contain too much air for them to sink into
aqueous killing fluids; this also occurs in some alcoholic fluids as well.
The Bryophyta are of this type. The use of a motor-driven suction
pump for long periods, as advocated by a few technicians, can hardly
be countenanced because of the artifacts which such a violent procedure
demonstrably produces. A pump attached to a water faucet can be
used safely (Fig. 2). The apparatus is rigged up as follows: connect the
side thbe of the pump by means of thick rubber tubing and a rights
 PARAFFIN METHODS 129

angled glass tube to a safety bottle fitted with a two-holed rubber stopper;
insert another right-angled glass tube in the other hole, then attach a
short piece of thick rubber tubing to this, followed by a stopcock and
another piece of rubber tubing attached to a right-angled glass tube
inserted in a one-hole rubber stopper placed in a bottle whose mouth is

wide enough to allow for easy insertion of the vial containing the material.
The rubber tubing should be inserted well over the ends of each piece of
glass tubing, and all connections should be airtight. Insert the vial
containing the material (or even several vials, if of a sufficiently small
size), plug the stopper tight and then turn on the water almost to full
force. Adjust the stopcock to prevent too rapid extraction of the air.
Exhaustion complete, close the stopcock, then turn off the faucet, and
wait a few minutes before opening the stopcock and then the bottle
containing the material. The safety bottle should be emptied of its
water occasionally.
Material which is heavily cutinized or suberized may take several
hours to sink. In fluids containing at least 50% alcohol, most materials
will sink either immediately or within a short time, but the air should
nevertheless be exhausted, or one might find the material floating when
put in the paraffin oven.

WASHING
It is almost always necessary to wash out the killing fluid thoroughly
before proceeding with the dehydration and infiltration. Some reagents
inhibit proper staining, others leave pre^cipitates which will later cause
trouble and annoyance, not to mention their being a source of error in
interpretation when the completed slides are examined, while still others
must be removed continued action damage the material.
lest their
In the chapter on killing and fixing fluids the nature of the washing
and the approximate time required for the process have been noted for
most of the solutions. The following general rules are, for convenience,
summarized here. Practically all aqueous fluids, particularly those
containing chromic acid, are washed out with water; an arrangement
if

to utilize running water cannot be set up, then the water should be used
in large volumes and changed as often as possible. In some sections
of the country the water frequently contains so much air that it would be
advisable to exhaust the air first by boiling or by using a suction pump,
in order to prevent the air from driving the material to the surface.
Alcoholic solutions should be washed out with plain alcohol of approxi-
mately the same percentage as that in the solution. Reagents containing
picric acid, whether in aqueous or alcoholic solution, should always be
washed out with alcohol, never with water unless the fluid also contained
some substance which fixes chromatin indissolubly. If mercuric chloride
130 GENERAL METHODS

is used in an aqueous solution, it should be washed out with water, but

ifemployed in an alcoholic solution, it should be washed out with 70%
alcohol. The deposits of mercury are best removed from the sections
as described elsewhere (p. 38).

DEHYDRATION
The washing completed, the next step is to remove the water from
the tissues. When xylol, chloroform, or an essential oil is employed
as clearer or paraffin solvent, it is necessary to insure removal of every
trace of water before beginning replacement of the absolute alcohol with
the clearer or solvent. With the introduction of the tertiary butyl
alcohol method, this necessity was removed: in other words, one may
begin the transfer to the butyl alcohol at a point where the dehydration
process is barely half completed. In any event, the transfer from one
step to another in the dehydrating process must be very gradual, other-
wise plasmolysis is certain to result. The process should never be
rushed, but, on the other hand, tissues become brittle if left too long in
the higher alcohols beyond 70%.
Dehydration with Tertiary Butyl Alcohol. —From the writer^s expe-
rience, the tertiary butyl alcohol method is the most satisfactory of all

(Johansen 1935, 1937-19^).

If the washing was with water, commence the dehydration process
with 5% ethyl alcohol in distilled water, followed by 11, 18, and 30%
ethyl alcohol. Two hours in each percentage is ordinarily sufficient,
but bulkier or tougher materials such as woody stems should be immersed
for a longer period. Material which has been washed with either 50 or
70% alcohol has already received sufficient preliminary dehydration.
However, if the washing has been with an alcohol of a percentage above
70%, it will be necessary to use the xylol, chloroform, or an essential
oil schedule after first dehydrating the material thoroughly with absolute

alcohol.
The following series of solutions of water, ethyl and tertiary butyl
alcohols should be prepared. The volumes are in cubic centimeters;
the various solutions may, for convenience, be designated by the approxi-
mate tofjd percentage of alcohol:

Approximate total percentage of alcohol 60/ 70 85 96 100

Distilled water . 60 90 16
96% ethyl alcohol 4o 60 60 45
Tertiary hutyl alcohol 10 20 96 66 76>
100% ethyl alcohol 26 ^
 PAR4^P^1N METHODS 131

In the last solution put enough dry erythrosin dye to give a red tinge;
can be easily oriented
this will stain the material superficially so that it
during embedding and microtoming. The dye will come out of the
sections readily after they have been brought down to alcohol when the
stainingis being carried out.

Transfer to the 50% solution from either 30, 50, or 70% ethyl alcohol,
depending upon the circumstances of the previous washing. After
2 hours or longer, pour off, and replace with the 70% solution, which
should be allowed to remain overnight. If the killing reagent contained
chromic acid, the longer immersion in the 70% solution tends to correct
certain irregularities in the fixation image. After the 70% solution,
an hour in each of the remaining percentages will suffice as a minimum
period for most materials. Following the 100% solution, there should
be three changes of pure tertiary butyl alcohol (one of which should be
allowed to remain overnight), by which time every trace of unbound
water should have been removed from the tissues.
The solutions should be used only once because the stock solutions
are readily contaminated by the many substances dissolved out of the
tissues by the two alcohols. If strict economy must be observed, the
used solutions should be kept in separate containers and not poured back
into the original bottles.
The material is now ready for infiltration. The transfer from the
butyl alcohol to paraflin should be gradual, but fortunately there is a

simple method of doing this.Transfer the material to a mixture of

equal parts of paraffin oil and tertiary butyl alcohol, in which it should
remain for at least 1 hour or somewhat longer for bulkier or tougher
materials. (Vials which have contained paraffin oil may be cleaned with
waste ethyl alcohol, then washed with water.)
Fill a vial or other suitable container three-fourths full of melted
Parowax (preferably Standard Oil Company of Indiana brand), and let
stand until the Parowax has solidified but not cooled completely. If
the Parowax is allowed to become cold and hard, the glass will break
shortly after the vial has been placed in the oven. Put the material
on top of the solidified Parowax, just cover with the butyl alcohol-
paraffin oil mixture, and place the container in the paraffin oven at once.
Do not place where the Parowax will melt too quickly; an upper shelf
is preferable to a lower shelf which is apt to be directly over the heating

elements. It is important that the oven be well ventilated in order

that the evaporating alcohol will be carried away; if there is no air cir-
culation, there will be no evaporation. The material will slowly sink
through the melting Parowax until it rests on the bottom of the container.
Infiltration is thus really a gradual process. The paraffin oil both pre-
vents any actual damage to the tissues by the heat of the oven, and allows
132 GENERAL METHODS

the Parowax to diffuse in gradually. About 1 hout (minimum period)

after the material has sunk to the bottom of the container, pour off
the entire mixture of Parowax, oil and what traces of alcohol remain,
and replace with pure melted Parowax. (A large beaker of melted
Parowax should always be kept ready in the oven, as well as another
beaker of the final embedding mixture.) Repeat the process twice
during the next 6 hours or so, discarding each change of Parowax.
Finally replace with a rubber-Parowax mixture (such as that described
on page 22) or with a good quality of paraffin melting at around 56°C.,
and the material will be ready for embedding within the next 30 minutes.
Small, delicate objects require much less time for infiltration than
do large and tough objects. Aquatic plants are more easily infiltrated
than xerophytic material. The heat of the oven does not do much
damage to plant tissues if paraffin oil precedes infiltration; consequently
the time element may to some extent be disregarded. The important
matter is to be rid of all the tertiary butyl alcohol and paraffin oil.

Just before making the final change of Parowax, there should be no

discernible odor of butyl alcohol. The customary practice is to place
materials in the oven the last thing before leaving for the day, to make
the change as soon as one arrives the next morning, and to get the
first

embedding completed by late afternoon.

Normal butyl alcohol may be used in place of tertiary butyl alcohol,
but is far less satisfactory. It causes some hardening. It is miscible
with water to the extent of about 8% by volume.
Dehydration with Xylol, Chloroform, Secondary Butyl Alcohol, or an
—
Essential OH. With all these reagents, the material must first be
thoroughly dehydrated through a graduated series of ethyl alcohol and
distilled water.
If the fixing fluid was washed out with water, a close series of dilutions
of ethyl alcohol (based on 95% alcohol) should be used: 2 }'2 ) 5, 7)^, 10,
16, 20, 30, 60, 70, 85, 95%, and absolute alcohol. In case the fluid
was an above the one used for
alcoholic one, begin with the alcohol next
washing. Several hours^ immersion in each percentage should be
allowed; the last three percentages should be used for at least 12 hours
each. The absolute alcohol should be changed two or three times. If it
is necessary to store the material for any length of time, it should be left
in the 70% alcohol.
The be as gradual as the dehydration. There should
clearing should
be a mixtures of clearing agent in absolute alcohol in the follow-
series of
ing percentages: 2J*^, 6, 10, 15, 25, 50, 76, and the pure clearing agent in
at least two changes. To either of the last two solutions add enough
erythrosin dye to impart a red coloration. The solutions may be used
 PARAFFIN METHODS 133

again two or three times. The vials or other containers must always be
kept tightly stoppered except when solutions are being changed.
Xylol hardens nearly all plant tissues more or less and causes some
shrinkage or plasmolysis; chloroform and secondary butyl alcohol have
only a slight hardening action. Bergamot and cedar oils have none, and
these two oils furthermore clear bulkier pieces of tissue better than most
other reagents; it is obvious that the different grades should be allowed
to react far longer than in the case of the more volatile fluids. For the
latter, 2 or 3 hours in each fluid is ordinarily long enough.
After the pure clearing reagent has been reached, the material is
ready for infiltration with paraffin! This process must also be gradual.
There are three methods from which to choose. The easiest, but one
which can be dangerous to the material if one tries to rush things, is to
add very small paraffin shavings from time to time to the clearing reageat
containing the material until it is finally saturated with partially dis-
solved paraffin. Wait until the first chips have dissolved before adding
the next one. A second method is to carve a small block of Parowax
into the form of a long, narrow cone and place it in the vial point end
down and resting at the bottom of the container. It will sink into the
reagent slowly as it dissolves. The third method is to cut coarse wire
gauze into squares just wide enough to be a fraction less than the diam-
eter of the vial or bottles. Bend the corners down to serve asjegs and
insert into the vial in such a position that the gauze serves as a table
above the material. Place chips of Parowax on the table; as they dis-
solve, the paraffin settles down upon the material. Whatever the
method that has been used, the preliminary steps should be carried out at
room temperature. As soon as the clearing agent has become saturated
with paraffin, the corks may be removed from the vials and the latter
placed on top of the paraffin oven or in a warm place. Shake or stir
the contents occasionally, and add more paraffin chips. In about
24 hours, the vials may be placed inside the paraffin oven. After the
vials have been in the oven about 3 hours, pour off the mixture of xylol
and paraffin and immediately replace with pure melted paraffin. It is
of no use to attempt to salvage the paraffin from the first change as
there is too much xylol present; Parowax is so cheap that it can be freely
used. Within the next 12 hours or so make at least two more changes;
it is necessary to get rid of all traces of xylol or chloroform, whichever

has been used, otherwise the paraffin will crystallize after embedding.
Crystallization can to a great extent be prevented by embedding in
rubber-Parowax. It is not so imperative that all traces of the essential
oils be removed before embedding. In the case of bergamot oil, the
^hfirapteristic odor can be detected in paraffin blocks that have not been
 134 GENERAL METHODS

cut for as long as 26 years, consequently it is probably impossible to

remove every trace of the oil.
If desired, one may go from the last change of xylol, chloroform, or
secondary butyl alcohol to a mixture of any of these fluids and paraffin
oil and carry out the infiltration process as described for tertiary butyl

alcohol.
Dehydration with Benzol. —Benzol may be satisfactorily substituted
in case absolute ethyl alcohol is unavailable (Kisser 1929). Dehydrate
the material as described in the preceding section. After 95% ethyl
alcohol, pass through each of the following mixtures of 95% ethyl alcohol
and c.p. benzol (benzene, water-free), allowing several hours in each:
3 parts 96% alcohol and 1 part benzol
2 parts 96% alcohol and 2 parts benzol
1 part 96 % alcohol and 3 parts benzol
Pure benzol, changing at least once.

Infiltrate as described in the preceding section.

Dehydration with Acetone. —Acetone constitutes a useful substitute
for ethyl alcohol when supplies of the latter cannot be secured (Sass
1932). The series of acetone in distilled water should be fairly close:
begin with 7 %, thence to 10 % and by 6% stages to 40 % and 10 % stages
thereafter until pure acetone is reached. The intervals can be less than
1 hour in each fluid. Clear in four grades of chloroform and acetone, and
infiltrate gradually.
—
Dehydration with Diozan. Transfer the material from water directly
into60% dioxan in distilled water, thence to 70, 95%, and at least two
changes of pure dioxan. Or one may transfer directly to pure dioxan,
then give two changes of the reagent. Add little chips of Parowax
gradually to the pure dioxan^ and place the container in a warm place to
accelerate the solution of the Parowax, as dioxan is a very poor paraffin
solvent. After the dioxan becomes saturated, place the container in the
paraffin oven, give at least four changes of pure Parowax, then embed in
rubber-Parowax.
Dioxan is irrational in its action and apparently works far better on
animal tissues than with plant materials, but it is worth trial. It should
always be borne in mind that dioxan is heavier than melted paraffin and
consequently is difficult to get>out of tissues during the infiltration process.

EMBEDDING
The infiltration of the material completed, th^
next step is that of
embedding. This consists of pouring the contenll of the vial or other
container (with the paraffin or rubber-Parowax in the liquid or melted
condition) into suitable receptacles, arranging the material in proper
%rder and then quickly cooling the entire mass.
 PARAFFIN METHODS 135

There are many kinds of embedding receptacles, or ‘Hrays^^ as they

are sometimes called, on the market. None of these, however, has any
great advantage over a simple folded paper tray. Use a fairly stiff paper
with slightly glazed surface (if a porous paper is used, the paraffin will
penetrate it). The size of the paper may be regulated to any dimensions,
and the side walls may be made as shallow or as deep as required. Using
the diagram (Fig. 3) as a guide, first fold over along CC' and DD', the
width of the fold being governed by the thickness of the material to be
embedded. This should be about 2 or 3 mm. more than actually needed
because the layer of paraffin, when cooled, is somewhat thicker along the
periphery than in the center. Next fold over on A A' and BB', the width
being twice that of CC' or DD'. Then
fold back along the middle of each of
these two flaps, as indicated by aa' and ^
bb\ Hold the paper in the fingers, and,
by using the nail of a thumb, make the
^
short diagonal creases. To complete
the folding, bring one end and one side
perpendicular, with the fold at the
short diagonal and turn the
crease,
resulting flap back of the end wall.
Bring up the opposite side wall, and
fold its flap back. Fold down, back-
ward, the upper flap of the end wall,
thus securely locking the entire end. B
Follow the same procedure for the
opposite end. The size of the central
area should be so regulated as to allow
a little more room than may be actually Description in text,
needed for the proper disposition of the
pieces of material. These trays are easily removed after the paraffin
has cooled and may be used over and over again if dried under pres-
sure to prevent curling. In fact, they actually improve with use. Trays
may also be made from thick cellophane (No. 450 M.T. cellophane). Or
one may use small porcelain trays or even solid watch glasses, first
coating the inside with a thin smear of glycerin.
Place the embedding dish at one side of the paraffin oven. A gas .

fiame should be placed near-by for heating the needle used for arranging
the material. Remove the vial or container from the oven, shake the
material to get it off the bottom and quickly pour into the tray. Add
more melted paraffin from the stock container if necessary; there should
be just enough to cover the material adequately. A superfluity of
paraffin prevents rapid cooling of the mass. With a needle, heated
136 GENERAL METHODS

slightly in the flame, quickly dispose the pieces of material into an orderly
arrangement; avoid overiapping and leave plenty of space between
neighboring objects. Many workers place the embedding tray on some
form of hot plate while arranging the material. This procedure is
scarcely to be recommended (except when the temperature of the room

—
Fio. 4. Methods of embedding various materials: A, root-tips by twos for transverse
sectioning; B, root tips (onion) for longitudinal sectioning; C, single stems; X>, leaves (pine)
in bunches of threes; single leaf portions; F, small buds in bunches, large ones singly.

is below 60®F.) because of the danger of overheating and the unnecessary

prolongation of the cooling process. Quick work and skillful manipula-
tion, gained after a little practice, are all that one requires. If the tray
istoo cold, it may be warmed by leaving in the paraffin oven for a short
time or by pacing through the flame.
 PARAFFIN METHODS 137

The accompanying illustrations (Fig. 4) indicate the correct methods

of arranging various types of plant material during embedding. The
arrangement of the material depends upon (1) the purpose for which the
material to be used, (2) the size of the pieces, and (3) their nature.
is

Leaves should be arranged individually, but small ones, as well as needles

of conifers like Pinus, can be embedded in bunches of three or more.
Root which are to be sectioned longitudinally, such as those of Allium
tips
cepa, are likewise embedded separately, but tips which are for cytological
purposes should be embedded in bunches for transverse microtoming.
Buds are in practically the same category as root tips. For studies of
the meiotic chromosomes, buds should be embedded and cut in bunches
until they become of a size too large for this method; the chances of
getting the reduction divisions are thus increased and the amount of
work as well as the quantity of slides, covers, and reagents is lessened.
Some types of ovaries may be treatc^d similarly, but it is safer to embed
and section such organs singly if critical studies of megagametogenesis
are being undertaken. If soft enough to section easily, stems may be
embedded in bunch(»s, but most stems should be treated individually.
The paraffin usually begins to cool first at the bottom of the receptacle.
As soon as the tray can be moved without disturbing the arrangement of
the pieces of material, transfer to a vessel of cold water. (Do not leave
the faucet running, if the tray is in the sink, and avoid splashing water in
from above. Attach a piece of rubber tubing to the faucet, and run it to
the bottom of the dish in this manner running cold water may be uti-
:

lized.) Let the tray float until the surface of the paraffin becomes suffi-
ciently firm to permit plunging the tray slowly beneath the surface of
the water. The tray may be weighed down by placing a heavy steel
scalpel or similar object across the ends. As soon as the paraffin is firm
enough, it may
be removed from the paper tray by unfolding the latter.
The paraffin blocks will usually float out of watch glasses and similar
dishes after a while, but may need to be loosened by inserting the point
of a scalpel at some place where there is no material that might be dam-
aged. Always leave the blocks in the water for hour or until thor-
oughly cooled.
The rapid cooling of the paraffin is a point which needs to be empha-
sized. If the paraffin cools too slowly it may crystallize: the blocks
become white spots and patches. Such crystallized paraffin
full of fluffy
cannot be microtomed. The only remedy is to cut the material out and
reembed it. In warm or hot weather ice water should be used for cooling
the paraffin.
The finished blocks should be stored and not allowed to remain about
tobecome covered with dust. The small boxes in which slides come are
very convenient for storage purposes, Po not store one block upon
 138 GENERAL METHODS

another without inserting a piece of stiff paper or thin cardboard between

them. Excess paraflSin should be trimmed away first; it may be melted
down and used over again. If scraps of tissue are present in used
paraffin, the latter should be filtered through cotton before being reused.

MICROTOMING
The material is ready for sectioning as soon as the paraffin has
thoroughly cooled. Some materials seem to section better if cut at

Fig. 6. — Method of cutting up paraffin blocks: make a straight cut across entire bloek
with tip of scalpel, then break apart. Next make similar cuts between individual pieces
of material and break up.

once, while others cut badly under the same circumstances. Little
or no phenomenon has been collected.
definite information concerning this
A piece of tissue may be removed from the block of material by
inserting the point of a sharp, narrow scalpel and cutting a nearly straight
furrow across the block (Fig. 5). The furrow should be at least 2.5 nun.
deep, or deeper for thick blocks. Hold the block firmly in the hands and
break apart along the cut. Trim down the piece until surrounded by
about 3. mm. of paraffin except on the bottom, where at least 6 mm.
should xemain. The photographic. diagrams show the proper method of
tiimiffing down the material (Hg. 6).
 PARAFFIN METHODS 139

The round metal holders which are furnished with most microtomes
are cumbersome, too large, and generally unsatisfactory. Hardwood
blocks are far better and can be made in any desired end dimensions
and in any quantity (Fig. 6). The wood should be of a kind not readily
crushed by the pressure exerted by the holder of the microtome. The
length should be at least 2 cm., while the endsmay be either square or
rectangular and of different dimensions to receive paraffin blocks of
various sizes and shapes. Make a large and assorted quantity of such

—
Fig. 6. ^Method of mounting material embedded in paraffin on wooden blocks. Lower
row shows hardwood blocks of various widths and thicknesses, with mound of paraffin on
top. At paraffin material roughly trimmed and ready for mounting; material mounted;
C, material trimmed down, ready for microtoming.

blocks only a few sections are to be cut at first; the whole can be set
if

aside of removing the pieces of embedded material, as

and there is no need
is the case with the metal holders.

Heat waste paraffin very hot, immerse one end of the wooden block
for a depth of 2 mm. until saturated with paraffin, then stand on the
other end until all the blocks have been dipped. With a pipette, build
up a mound of paraffin on top of each block, using paraffin that is barely
liquid. When ready to attach a piece of embedded material to such a
block, heat an old scalpel or a similar flat instrument with nonmetallic
handle in a gas flame, and touch first to the paraffin on the wboden block
then to the bottom of the embedded piece, bringing the two together
140 GENERAL METHODS

while the paraffin is still more or less melted. Reheat the instrument and
cautiously melt together the two paraffins so that they form a single mass
on the wooden block. Avoid touching the plant tissue with the hot
instrument. Work quickly. Next stick a needle (inserted in a holder)
in the unused end of the wooden block, and put the latter in a dish of cold
water to cool the paraffin.
The next step is to trim away the excess paraffin (Fig. 6). Use a
sharp scalpel with a long, straight blade, not a curved one. It is abso-
lutely essential, if perfect sections are to be cut and the ribbon is to be
straight, that the two longest edges be exactly parallel to each other. The
face may be square, of course, but the rectangular form is preferable.
Do not cut away paraffin until the object is exposed on the sides, but
leave at least 1 mm. of paraffin surrounding it. Some types of material
are very liable to swell up and break apart when the sections are warmed
to straighten out the kinks in the ribbon; the preventive of this sort
of disaster is to leaveenough paraffin around the material, particularly
at the short ends. The
rather gelatinous marine algae are likely
to swell if fixation has not been thorough. If only one or two sec-
tions are to be mounted on a slide, the margin of paraffin can be
much wider than if as many sections as possible are to be mounted on
each slide.
The student should now familiarize himself thoroughly with the
mechanism and operation of the rotary microtome, which is preferred
by the majority of technicians for sectioning materials embedded in
paraffin. Note particularly the following points: (1) the clamp or holder
for the wooden block and the method of orienting it at all angles; (2) the
feed mechanism and the method of setting it to give any desired thickness
(the scale is in microns) (3) how to reset the mechanism after it has run
;

its course; (4) the points at which oiling should occasionally be done;
(6) the knife holder and the method of adjusting both the holder as a
whole and the knife itself. Note that the holder is adjustable so that
the knife can be oriented at different angles to the vertical. This is to
allow for differences in the bevels of different knives. An ordinary knife
should be inclined at an angle of about 8® from the vertical, which will give
ample clearance. Safety razor blades in holders should be set nearer to
the vertical, but take care to see that there is nothing in the way which
might scrape or be struck by the paraffin block. The blade should never
scrape against the paraffin at any point df it is almost vertical it will
;

scrape rather than cut.

For one^s own peace of mind, on© ^should possess his own knife, or
furnish his own safety razor blades if a holder is available, rather than
depend upon knives in common use. The latter are too apt to have an
edge like that of a saw.
 PARAFFIN METHODS 141

It is of the utmost importance that microtome knives be sharp.

Never begrudge time spent in sharpening knives, for it is time well spent.
A regular microtome knife, kept in perfect condition, provides the best
of all cutting edges, no matter what the claims of those who favor safety
razor blades. Begin cutting at one end of the blade and gradually work

—
Fig. 7. Arrangement of table for microtoming: A, microtome; B, black cardboard on
which to place ribbons; C, bottles with adhesive and flooding solution; D, warming plate;
E, absorbent paper towel folded over, for draining excess flooding solution. Box of slides
is at one end of the warming plate. Lamp should be so placed that the light is directed in
front of the microtome knife.

over to the opposite end. Trim down unwanted parts of the material
on a used part of the knife, then move the latter forward to a new place
when the desired sections have been reached. When the whole of the
edge has been used, remove the blade, and strop, or resharpen and strop.
Insert the wooden holder in the clamp and screw down securely (see
Fig. 8). The part of the wood projecting from the clamp should not be
more than 3 mm. in length. First look down upon the paraffin block
142 GENERAL METHODS

from above, and adjust so that the face of the block is parallel with the
blade; move
the carriage forward until the blade barely touches the
paraffin at its lower edge. (If the object is not oriented so that it is

perfectly perpendicular to the blade edge when examined from above,

adjust properly, cutting away any excess paraffin with a sharp scalpel.)
Then cautiously bring the block down toward the knife by turning the

Fig. 8. — method of operating microtome and of holding ribbon away from

Illustrating knife
by means of a needle in order to prevent telescoping of sections.

wheel, and examine from directly in front. If the lower (longer) edge
of the paraffin block is not parallel horizontally with the edge of the knife,
adjust the holder until the edges of block and knife coincide. The
vertical adjustment should be permanently fixed; there is no excuse for
not trimming down the front of the paraffin block so that when it is placed
in the holder the object is perpendicular to the knife when viewed from
either side. If the embedded object is nearer to one lonig edge of the
paraffin block than the other, onent with the nearer edge toward the knife
 PARAFFIN METHODS 143

to avoid knocking the object out of the paraffin or tearing the upper edge
of the sections.
Set the micron scale at the desired thickness. On most modern
rotary microtomes a single notch in the geared wheel is equivalent to
1/x in thickness, but on many sliding and a few older rotary microtomes

each notch is equal to 2/i. One can easily determine which is the case by
studying the indicator scale. The scale should be carefully adjusted.
Do not leave the indicator between points on the scale; this will cause
sections of very uneven thickness to be cut and will wear down the

Fig. 9. —Good and V)ad microtoming: 4t what happens when opposite edges are not
parallel; B, when —
the narrow edge is toward the knife an example of careless trimming
—
and orientation of the block; C, result of leaving a hard stem too long under water all
sections torn, some curled; D, mediocre ribbon from a woody stem, but some of the sections
can be salvaged; B, perfect ribbon of old Ariatolochia stem.

mechanism quickly. There is no such thing as fractional microns;

however, specially constructed machines which will cut at half-microns
are available. (If one is uncertain at which thickness to cut, run off a
few sections at 12 m, mount on a slip and examine under the low power
of the microscope to see whether they are too thick or too thin.)
One is now ready to commence cutting (Fig. 8). Move the wheel
with a steady, even stroke. The first few sections are invariably worth-
less and should be discarded, except in the case of materials of which
complete serial sections are required. If the material was such that it
could not be trimmed down sufficiently before being killed and fixed, the
excess tissue is easily cut away; use for this purpose any part of the knife
 144 GENERAL METHODS

that has already been used. On the rotary microtomes the sections form
a ribbon as they are cut one by one. There is a very slight heating of the
lower edge of the block as it strikes the knife; this is what makes the sec-
tions stick together to form a ribbon. If the cutting is stopped, the
ribbon is likely to become broken. The ribbon sometimes exhibits an
annoying tendency to curl up and under the paraffin block. To avoid

Fia. 10. —A perfect example of bad microtoming. A dull knife full of nicks caused both
sharp cuts (near bottom) and corrugated tears (above middle and elsewhere), while there
was so much compression that breaks parallel to the plane of sectioning were produced
(upper left) and portions of the section were also moved out of position. (Material is bud
of Botrychium.)

cutting it, leam to hold a needle or small camel’s-hair brush in one h&nd
and keep the ribbon from getting under the block or from becoming
telescoped on the knife (Fig. 8). The ribbon should be straight (Fig. 9F).
If it is not, the opposite edges of the paraffin block are not parallel; less
often, the object is of uneven hardness, or the paraffin may not have
solidified homogeneously. In any case, the remedy is to trim away care-
fully a tiny wedge from the thick edge, cutting a few sections to ascertain
if the ribbon has become straightened or if not cutting away a little more
 PARAFFIN METHODS 145

paraffin. Of course, if the sections are to be mounted singly,

it does not

matter if the ribbon not strictly straight. It will be observed that

is

the side of the ribbon toward the knife is smooth and glossy while the
other side is dull and slightly rough keep the glossy side down and mount
;

likewise in order to secure greater adherence to the glass slide.

MICROTOMING OF REFRACTORY MATEMALS

Many plant tissues, although soft and easily cut with a scalpel at the
time of collecting, are rather tough and difficult to section easily after
having been embedded.

11. —Illustrating a common fault resulting from failure to keep the back of the
microtome knife free from debris. The tear extends across an ovule, rendering the mega-
gametophyte useless for study.

The remedy is easy of application. Cut away enough of one end

of the paraffin block and through the piece of material to be cut so that
the latter is exposed. Place the material in a vial or bottle full of sterile

water, cork tightly, and set aside overnight. Of course, several pieces of
material may be softened simultaneously in the one container. The
water will penetrate the cut end and soften the material. Some woody
stems may require a month or longer to become sufficiently softened, but
no specimen should be left in the water any longer than the time required
to soften it, otherwise maceration will set in. The rate of penetration
may be gauged by the increased opacity of the paraffin around the
material. If a number of sections are cut and then trouble is encountered,
146 GENERAL METHODS

return the material for further soaking. Once material hoe been placed
under water, it cannot be dried and then resoaked; it should all be sec-
tioned and the ribbons stored if all are not to be mounted.
Some objects, such as root tips, which have little paraffin in them, cut
better and one also obtains less wrinkled sections if the block with its
mounted object is4mmersed in ice water for a few minutes before being
microtomed. Do not leave in the ice water too long, otherwise the
sections will fail to ribbon. If this occurs, warm the knife on the drying
plate, but do not attempt to warm the paraffin itself.
It has frequently been claimed that better microtoming can be done
during rainy than during dry weather; this may be true for some geo-
graphical regions but is otherwise merely a superstition. One should
easily be able to arrange things so that microtoming can be done at any
time in any sort of weather. If the atmosphere is very dry and the
sections become so full of static electricity that they stick to everything,
the remedy is to keep a beaker of water boiling in the immediate vicinity
of the microtome. /

The newer safety razor blade holders are so constructed that either
ice or lukewarm water may be run through them. For ordinary purposes
and under average weather conditions, let ice water drip through some-
what slowly; for very thin sections or during very hot weather, run the
ice water through rapidly; dilping very cold weather or for very thick
sections, which are prone to curl and refuse to form ribbons, lukewarm
water may be run throqgh the holder. A little experimenting will reveal
the optimum temperature fOY different sorts of material under various
atmospheric conditioife'.

MOUNTING
Place the length of ribbon on a piece of *«tiff paper dr thin cardboard
with a smooth, more or less glossy surface. A piece of black poster
board, as used by artists and sign painters, is excellent for the purpose,
as the sections are more readily detected against the dark background.
Do not put the ribbons on paper towels or similar soft papers. Be
careful to avoid drafts and not to blow or to breathe heavily.on the fragile
ribbons* The ribbon may be cut into sections of a convenient' length;
use a sharp scalpel for cutting, and cut by drawing the scalpel rather
than by pressing down. •

The number of sections to be mounted on each slide is governed, by

the nature df the material. Sections of stems (if not too wide), leaves,
roots, and similar subjects are best as a series of not more than three,
since one section is about identical with the others. Longitudinal
sections of root tips are usually mounted five to a slide, but cross sections
of root tips are usually all nidunted <m one sl^^^^ (see Figs. 12-14),. In
 PARAFFIN METHODS 147

the case of ovaries the sections should be examined with a hand lens;
those showing ovules or ovules with clear spaces indicating the presence

Fig. 12.—Illustrating various mounting methods: A, sections of root, rhisome and stipe
of the same plant mounted together; Band C, single large sections; D, three serial sections
of material embedded in bunches; Et throe different growth stages of the same type of
material.

of megagametophytes should be selected and the rest of the sections dis-

carded. With such materials there are invariably a large number of

worthless sections, and it is only a waste of time and reagents to run all
 GENERAL METHODS

of them up. Always make allowances for the fact that the sections
expand when warmed to flatten them out.

PI

mW'P

Fio. 13.— Commercial preparations, showing how materials are mounted ‘'upside
down/’ in the case of both whole mounts and sections, so that they will appear in the
natural position when observed under the microscope.

In still other cases, as in Ricdocarpus, Anthoceros, and other Hepaticae,

only certain sections are really worth anything. The difficulty is to
locate such sections. Fill either half of a large Petri dish half full of
 PABAFFIff METHODS 149

water; cut the ribbon into sections about 1.5 or

distilled (or air-free)
5 cm. in length), and
2 cm. less than the length of a slide {i.e., about

Fio. 14.— Methods of mounting research materials; A,

serial
buds; Z®"®'
B, serial sections of a single bud; C. serial sections of bunched f
sections of a s g
'
y
single bud for raegasporogenesis; E, serial longitudinal
embryogenesis.

dish on the
on the water in the Petri dish. Place the
place the sections
straightene out,
warming plate, and leave until the sections have wholly
and set aside for
whereupon the dish should be removed from the heat
 150 GENERAL METHODS

the water to cool since the ribbons would be irreparably damaged if

handled while still warm. Smear adhesive over the entire surface of a
slide, flood with water, take up a length of ribbon with a scalpel and place
on the slide. Drain off excess water, then examine the sections under the
microscope. Do not let all the water evaporate under the ribbons.
The desired sections may then be selected and cut away with a scalpel,
and the worthless ones rejected; then reflood the slide -with water, pick
up the sections with a scalpel, transfer to other slides smeared with
adhesive, and warm again.
Haupt’s adhesive is unexcelled for affixing sections to the slides.
Place a small drop of the adhesive on the slide, smear it evenly pver the
surface with a finger, and wipe off any excess adhesive; only a barely
perceptible film should remain. Place the slide on the table, or better
still on the bottom half of the box in which 24 X 50 mm. coverslips come,

%
and immediately flood with 3 formalin in distilled water. The adhesive
must not be allowed to get dry before the formalin is added. Touch the
point of the scalpel in the water, and insert the tip of the blade under a
section or portion of the ribbon. The drop of water will cause the
section to stick to the scalpel so that it can be lifted. Place the section
on the flood, then put the slip on the warm plate to flatten out the wrinkles
in the paraffin and incidentally to generate fumes of formalin which will
serve to coagulate the gelatin of the adhesive. The temperature of
the warm plate should not be over 43®C,, as it is important not to melt
the paraflfin. If a thermostatically controlled electric warm plate or
slide warmer is not available, hold the slide cautiously over an alcohol
flame and watch the sections to avoid melting the paraffin. After
sections have stretched out, take the slide off the warm plate and set
aside for a few minutes until the water cools. Do not try to move the
sections while the water is still warm, as the paraffin is easily damaged
at this stage. After the slide has cooled, hold the sections in place with a
needle, drain off the excess water, and touch the slide to absorbent paper
to get rid of as much water as possible. Then move the sections to the
position it is desired that they occupy,and set the slide aside to dry
completely. the sections show a tendencjjr to move around, watch
If
them, and restore to the proper position before the slide gets completely
dry. Do not attempt to move the sections from a wet to a dry place
on the slide: this is usually impossible without damage to the sections.
Set the mounted slide away in a dustproof place for several hours
or a day for the drying to be completed. The slides should not be left
on the warm plate to dry as untidy stained backgrounds frequently
resultfrom this practice. Mounted slides may be stored for as long as
*

several years not convenient to stain them within a few days.

if it is

As soon as the slides have dried thoroughly, they are ready for staining.
 PARAFFIN METHODS 161

STAINING
Before the sections can be stained the paraffin must be removed.
Xylol is used for the purpose by the majority of technicians. Place
the slides, either singly or as a series in a rack, in a jar of xylol for about
5 minutes or somewhat longer. There should be sufficient xylol, as well
as enough of all reagents subsequently to be used, to cover the slides.
^ There is no economy in using as small quantities as possible since they
will soon become so badly contaminated that replacement becomes
necessary more frequently than is the case when ample quantities are
used.
From tl5SP%tJ«lGl withdraw the slide slowly, and transfer to a jar con-
taining equal parts of absolute ethyl alcohol and xylol. The slides
should not be pulled out of solutions quickly as this procedure causes
them to carry over a large amount of fluid, which contaminates the next
solution. Withdraw the slide somewhat slowly, thus carrying over a
minimum amount of fluid, but not so slowly as to permit drying, or
^b^j-ption of moisture from the air.
Thin sections and the average nonwoody tissues will stick safely
to the slides, but sections of woody tissues, those containing mucilage,
fats,and waxes, as well as those which are overchromated, are retained
only by the exercise of great caution in avoiding diffusion currents.
Sections may stick until water is reached, whereupon they float right
off the slide. It is therefore advisable to play safe at all times by covering
the sections andwith a protective coating of thin celloidin. The
slides
carmin dyes arc the only ones which have any tenden(*y to stain the
celloidin, but, fortunately, these dyes are scarcely ever used on sections
of plant material.
After 5 minutes in the absolute alcohol-xylol mixture, transfer the
slides to a mixture of equal parts of absolute alcohol and ether (anesthetic
ether should be used) to which has been added sufficient of any celloidin
solution to make the strength about, but not over, 1 %. Between 5
and 10 minutes in this fluid is long enough.
’
Withdraw the slides, and allow them to remain in the air (which
should be warm and dry) until the sections show signs of bottoming
whitish-opaque, which indicates that the celloidin film is nearly dry.
Then plunge the slides into either 95 or 70% ethyl alcohol for 5
minutes to harden the celloidin. Next transfer to 70% alcohol if

95% was used, otherwise to 35%, for 5 minutes. If an alcoholic

stain is to be used, pass to this stain from the 70% alcohol; if the
stain is aqueous, go through 35% alcohol to water before placing in the
aqueous stain. «

Staining procedures are outlined in Chap. VII,

162 GENERAL METHODS

DEHYDRATING AND MOUNTING

After the sections have been stained, they must be dehydrated
preparatory to mounting in balsam. More or less of the dehydration
may be accomplished during the staining procedure. If aqueous staining
fluids alone have been employed, proceed to 35% alcohol, thence to
70 and 95%. After alcoholic stains the slides are usually in 95%
ethyl alcohol, or may
be transferred to that percentage. Counter-
staining of most plant materials is done with the dye dissolved in clove

Fig. 16.— Arrangement for counterstaining with clove oil solutions; A, slides in 96 %
alcohol in flat dish (covered with large crystallizing dish or similar cover); B, dropping
bottle with counterstaining solution; C\ dropping bottle containing used clove oil solution
for washing off excess stain into waste jar D\ E, jar containing differentiating solution; F,
xylol wash; O, first xylol jar; H, second xylol jars. Also shown are forceps and cloth for
wiping off underside of slides.

oil diluted with absolute alcohol, and frequently also with methyl cello-
solve (Fig. 1 5) . The slides, in such cases,
are taken from the 95 alcohol, %
the lower side swiftly wiped dry with a clean cloth, the counterstain
applied from a small bottle equipped with a pipette stopper, allowed to
remain for a few seconds, and then poured back into the bottle. Used
clove oil solutions should be saved up; put a portion in a bottle with a
pipette stopper, and dilute with one-fourth its volume of a mixture of
equal parts of absolute alcohol and xylol (if the slides were too heavily
coated with celloidin and it becomes stained, add a little ether to the
mixture). Use this diluted clove oil mixture to wash off excess stain
into a waste bottle, taking care not to squirt the fluid directly against
 PARAFFIN METHODS 153

the sections with the pipette, and thus avoiding too rapid contamination
of the clove oil differentiator with stains. The differentiator is made by-
mixing 1 part U.S.P. clove and
part of a mixture of equal parts of
oil 1
absolute alcohol and xylol. Move the slide slowly back and forth in
the differentiator for about 10 seconds, then transfer to a jar of xylol
to which a trace of absolute alcohol has been added to take up any
moisture inadvertently brought over. (Always keep the clove oil mixture
covered except when actually in use, as it absorbs moisture readily.)

Fig. 16 . —A typical example of the damage done to a section when there is little balsam
under the coverslip and the latter is moved around.

Move back and forth for a few seconds, then place the slide in a jar of
pure xylol. A large number of slides can be stained and left in pure
xylol forsome time or until convenient to mount in balsam. Just before
mounting, place for a short time in a third jar of xylol.
Remove a slide from the xylol, wipe the xylol from the underside with
a clean, dry, lint-free cloth and also wipe it from around the sections
(taking great care not to come too close to them) in order that, if too
much balsam should inadvertently be applied, it will not become dis-
solved and run over the slide. Next place a small drop of thin balsam
on top of or at the left of the sections. Pick up a cleaned coverslip
(an entire box of coverslips should be cleaned at one time and placed
154 GENERAL METHODS

in an empty slide box: to clean them, dip in a mixture of equal parts of

96% alcohol and xylol, touch to paper toweling to absorb excess fluid,
then polish quickly with a scrupulously clean cloth), with fine-pointed
forceps, pass quickly through a clean alcohol flame to remove surplus
moisture, hold so that the forceps are at the right, and bring the left
edge of the coverslip down on the slide, still holding up the opposite edge
of the coverslip with the forceps. Let the coverslip come down gradually,
allowing the balsam to spread to the opposite edges and then to be
pushed ahead as the coverslip is brought lower and lower. At times a
littlepressure exerted with the nail of the left index finger on top of the
coverslip will be helpful, especially if bubbles have become trapped.
Do not lift the coverslip up once it has balsam under half of its area.
After three-fourths of the area under the coverslip has been covered
with balsam, withdraw the forceps (also the finger if it has been used),
and allow the uncovered area to become covered with balsam by the
weight of the coverslip. The size of the drop of balsam must be learned
by experience; it is determined first by the thickness of the balsam itself
and secondarily by the thickness of the sections. Thin sections require
less, as well as thinner, balsam. The area of the coverslip does not
enter into calculations to the extent that might be imagined, unless the
balsam is quite thin. Thick balsam should be used on sections, no
matter what their number or size, that are over 20m iu thickness. Some
kinds of material tend to lift the cover during the drying process, thus
admitting air bubbles or canals. Sometimes these can be forced out
by gentle pressure on the coverslip, but in other cases one can only run
in more balsam from one edge of the cover, or put the slides into xylol to
dissolve the balsam and to loosen the coverslips, later remounting.
When is first applied, it should be held in the exact
the coverslip
position desired, and after coming down completely, it should never be
moved sidewise m any direction, otherwise there is great danger of
damaging the sections (Fig. 16). The less balsam there is under the
coverslip, the greater the chance of damage. Sections containing loose
cell contents, such as very starchy tissues, are more liable to distortion.
The slides are now ready on a warming table or in an
for drying
incubator heated to not over 60®C. The balsam does not become
completely solidified throughout the area under the coverslip for several
months; consequently the slides should be removed from the warming
plate after a day or two (by which time the balsam will have become
sufficiently hard around the edges of the coverslip), lest the stains
become damaged by heat. The slides may then be placed in slide boxes,
which come in various types and capacities.
 CHAPTER XIII

SMEAR METHODS
The more refined of the various smear methods afford, in a relatively
short time, preparations which are of the greatest value in counting
the monoploid and diploid chromosomes and in studying the chromo-
somes themselves. In fact, smear preparations arc so useful that at
the present time many critical cytological studies arc based principally
upon such slides. The student consequently would do well to master
as soon as possible the technique of smearing either plant or animal
material or both, t(t gain some familiarity with the different methods of
staining smears, and to jiid^O correctly what finally results.
The smear method is limited to some extent in applicability to cells
which are not firmly united to one another, as by middle lamellae. In
the higher plants this refers to the microsporocytes after they have begun
to round up, and in animals to the celH of the testis at the corresponding
stages. The microsporocytes of some plants (e.gf., those of Acacia and
some orchids, which are cqUected together into pollinia) and the testis
cells of many animals cannot be satisfactorily smeared but should be

prepared by other methods. In the present chapter only plant material

will be considered.
The essential idea underlying all smear methods is to spread the cells
out into a single layer in order that they may be killed instantly and
fixed evenly and uniformly, without distortion or the production of
artifacts. Practically all cells which can be smeared will adhere to the
slide;hence the necessity of using a cementing agent (which might itself
be stained) is obviated. The slips can be carried through the various
staining and dehydration processes with the loss of only a few cells,
provided, of course, that the changes are not too violent. Special pre-
cautions will occasionally be noted in the procedures listed below.
Technique of Smearing—The slides upon which the smears are to
be made must be chemically clean. New slides should be given a long
immersion in the sulphuric acid-potassium bichromate mixture (page
13), rinsed in running water, placed for a short time in strong alcohol
to which a ammonia is added, rinsed again, and finally dried with
little
an absolutely clean cloth free from starch and lint. It will save time
if an entire box or two of slides are cleaned at one time. Slides which
have previously held sections should never be used for smears.
165
 166 GENERAL METHODS

The fluid which is generally considered to be superior to all others

for the killing and smears is Navashin^s. The two portions
fixing of
composing mixed together just before using. It may be
this fluid are
necessary in, some cases to vary the proportions of some of the ingredients,
particularly the acetic acid; this can be judged only by trial.
The most satisfactory vessel in which to carry out the fixation of
smears is the square Petri dish recently placed on the market (obtainable
from E. H. Sargent and Company, 156 East Superior Street, Chicago,
111.). Place slender glass rods, cut to the proper length, at opposite ends
of the lower half of the dish to keep the slide off the bottom. The dish

Fiq. 17 .^Trillium ovatum: low-power view of the microsporocytes at the first division;
permanent iron-acetocarmin smear preparation. {Smear and photomicrograph by Dr, H. E,
Warmke.)

willhold two slides placed one beside the other. Pour the killing solu-
tion into the dish just before
making the smears; it should be more than
enough to cover the rods (approximately 40 cc.).
Different technicians have methods of making smears adapted to
their own needs. Most of them remove the anthers from the buds,
cut off both ends, or cut each anther into short segments if it is over
2 mm. in length, and place near one end of the slide. Previously a mark
should be made in the upper right corner of the slide with a grease pencil
(or etched with a diamond) in order that one may know on which side
the smear is made. With a clean scalpel, which should be honed flat
and smooth, quickly and evenly crush and spread the anthers over the
qenter of the slide so that the microsporocytes are in a single layer over
the greater part of the slide. Immediately invert the slide and place
 SMEAR METHODS 157

in the Petri dish in such a way that the entire smeared surface comes
into instantaneous contact with the killing fluid. If the slide is brought
into the fluid in any save a perfectly horizontal position, most, if not
all, of the material will be washed off. The time elapsing from excising
the anthers to placing the smear in the killing fluid should not exceed
4 seconds. As the capacity of the Petri dish is two slides, a second
smear may now be made, using anthers from another bud if desired, and
placed beside the first. Although the slides may be turned smear side up
after about 10 minutes, it will be safer to leave them as they are for the
duration of the time necessary for proper fixation, which with Navashin’s
fluid is about 4 hours.

Fig. 18 . —Nicotiana alata: smear preparation of second division in microsporocytes.

The anthers contain a dense jicriplasmodium which absorbs some stain. Fixed with
Navashin’s fluid; stained by Johansen’s methyl violet method.

Another method smearing the anthers or pieces of anther is to

of
place them and to
in the center of a slide, to hold a second slide crosswise,
exert just enough force firmly to extrude and to spread the microsporo-
cytes. Immediately invert both slides in the dish containing the killing
fluid.

Other killing fluids may, of course, be tried. The following has proved
excellent for chromosome structure:

10% aqueous acetic acid 10.0 cc.

10% aqueous chromic acid 1 .0 cc,

2% osmic acid in 2% chromic acid 7.5 cc.

Distilled water 41 6 cc.

.
168 GENERAL METHODS

To add approximately 1% saponin; the amount may

the mixture
have to be adjusted for different species. LaCour^s 2BE fluid sometimes
gives good fixation, but frequently leaves the cytoplasm in such a con-
dition that it is too dense to permit the chromosomes to be clearly
outlined by stains.
The and with forceps
fixation completed, turn the slide right side up,
remove anther fragments and other thick pieces of debris that might
unduly elevate the coverslip when mounted. Then place in low staining
dishes, and wash for about 15 minutes in gently running water. The
slides should be examined under the microscope and any undesirable ones
rejected. The smears are now ready to be stained. There are available
a number of staining schedules, each adapted for particular needs and
each giving a different picture of the chromosomes and their structure.
Johansen’s Methyl Violet Method.—The principal attributes of this
method are its brevity, ease of tnanipulation, and the certainty of obtain-
ing sharply stained chromosomes from diaphase onward. It will ordi-
narily prove useless for prediaphase stages, except possibly in certain
plants.
1. Transfer smears from water to a 1% aqueous solution of methyl
violet 2B, preferably freshly made. This should should not have been
boiled, otherwise precipitates will be left in the cells. The staining
requires 15 minutes or less but may
be increased if necessary.
2. Transfer slides to water, in which they may be left for as long as

hour. The slides should hereafter be handled individually.

3. Differentiate in 70 and 95% alcohol, to each of which is added

0.5% picric acid (if the crystals come in the moist form, add a little
more to make up for the weight of the water). Hold the slide with
broad forceps, and move back and forth gently in the vessel containing
the alcohol. About 10 seconds in each alcohol is long enough. Do
not use the series after they have become saturated with dye, nor should
used solutions be allowed to stand unused for more than a few days,
lest a greenish-yellow color be imparted to the cytoplasm.
4. Immerse the slide for 15 seconds or so in 95% alcohol containing
about 4 drops ammonia to each 100 cc. alcohol.
5. Complete dehydration in absolute alcohol for about 10 to 12

iieconds.
6. Complete differentiation in pure clove oil: this is the critical step
in the procedure. Eight to fifteen seconds is generally long enough.
Keep the slide moving back and forth to ensure even differentiation.
7. Wash in xylol containing a trace of absolute alcohol.
8. Leave the slides in pure xylol for about 2 hours, but not longer
than 3 hours, before mounting: this will improve the sharpness of the
 SMEAR METHODS 159

stain. The slides may be examined under the microscope and any
unsatisfactory ones rejected before mounting in balsam.
In general, a counterstain is undesirable with smear preparations,
and few if any cytologists use one. However, any of the usual cyto-
plasmic counterstains may be used if occasion demands.

Trillium ovatum (above) and T. chloropetaluin {h(i\ow) propha»i' stages in

:

ineiosis. Permanent iron^acetooarmin smears. {Prsparaticms atid. photomicrographs by

Dr, H. E, Warmke.) ’

—
Newton’s Gentian Violet-Iodine Method. Newton originally added
1 g. each of potassium iodide and iodine to each 100 cc. of solution of each
of the dehydrating alcohols, passed the slide through the series, washed
thoroughly in absolute alcohol, cleared in xylol, and mounted in balsam,
Any degree of depth of the stain could be obtained in this fashion.
Present-day workers, however, have eliminated the chemicals from all
160 GENERAL METHODS

the dehydrating alcohols save either the 70 or the 85% one. The
procedure is as follows:
Pass from water through 30 or 35% alcohol to the 70% containing
1.

1 each of potassium iodide and iodine to each 100 cc. of solution.

g.
The slide should be kept immersed until no more color comes off, which
will be in from a few seconds to a little over 1 minute. The smears
should look quite black.
2. Remove the iodine by washing in 70% alcohol.
3. Pass quickly, depending on how much stain is coming out of the
sections, through 85, 95%, and absolute alcohol.
4. Complete differentiation in clove oil. This should be rather
brief, or if may be omitted altogether.
necessary
5. Wash
thoroughly in xylol, clear in pure xylol for 2 hours or longer,
then mount in balsam.
Newton^s method requires more experience than does the preceding
one and, furthermore, demands greater accuracy in judgment in order
to secure the optimum results. Most technicians use the gentian violet
(crystal or methyl violet may also be used) at a strength of 1 % (which
is almost a saturated solution), while others use 0.75 down to 0.2%.
If the stain appears to be too strong, it is a simpler matter to dilute it
with water than to cut down the time. The time depends upon the
material, but the optimum period is about 15 minutes. The method
does not succeed with many plants and is said to be inferior to other
procedures in the staining of the diaphase and earlier stages. The
chromosomes often appear so transparent that this has called forth
objections. It has been claimed that the transparency can be greatly
reduced by judicious exposure of the completed preparation to sunlight.
The stained slides last about 1 year, then begin to fade.
Sax’s Variation of Newton’s Method. — Make smears as usual, and
fix preferably with Navashin's fluid for 1 to 2 hours (Sax 1931). Wash
in 10 to 20% ethyl alcohol for 12 to 30 minutes, then stain with 1%
aqueous crystal violet for 1 to 5 minutes. Rinse in water, and immerse
in 30% alcohol for 15 to 20 seconds, followed by the same length of time
in 50% alcohol. Iodize in 80% alcohol containing 1% iodine and 1%
potassium iodide for 30 seconds. Destain and complete differentiation
with absolute alcohol, clear with clove oil, pass through xylol, and mount
in balsam.
—
LaCour’s Method. It has been found (LaCour 1935) that, if the
anthers are smeared, immediately placed, smear side down, in a dish
containing 3%
aqueous cane sugar for a second or two, then laid flat
over an open dish containing concentrated ammonium hydroxide or
concentrated nitric acid for a few seconds, and next placed in the fixing
fluid (preferably a Flemming medium solution plus osmic acid) for 2
 "

SMEAR METHODS 161

hours, the spiral structure of the chromosomes is more clearly revealed.

The main precaution in order to prevent loss of cells is always to keep
the slip flat and to move it horizontally. After fixation, there is much
less danger of the cells washing off. Wash, bleach if necessary, and stain
as preferred. ^
' '

'

:
,

Fis. 20
. —
Trillium chloropetalum: permanent iron-acetocarmin smear preparations of
{Preparation and
miorosporocytes; A, diaphase, showing chiasmata; B, early anaphase.
ph(^tomicrograph by Dr, H. E. Warmke.)

Kauftmaxin’s Iron-Hematoxylin Method.

1. Prepare smears, and fix in Navashin^s or a chrom-osmo-acetic

fluid,

2. Mordant in2% aqueous ferric ammonium sulphate for 1 hour.

3. Wash carefully in running water for a few seconds and rinse in
distilled water.
1624. GENERAL METHODS
6.

1 hour or longer in 0.5% hematoxylin.

Stain for
Rinse in water, then destain under observation in the same solution
that was used for mordanting, which is discarded upon completion of the
differentiation. When the stain seems satisfactory, transfer the slide
to water.
6. Wash in running water for at least 1 hour to remove the iron alum.
7. Dehydrate by stages of not over 10%, with 2 or 3 minutes in each
stage.
8. Clear in mixtures of absolute alcohol and xylol, of which about
four intermediate stages suffice. Then leave in pure xylol for a short
time before mounting in balsam.
—
Tuan’s Modified Hematoxylin Method. This is a method requiring
considerable patience, but the preparations are usually of such surpassing

Flo. 21 . —
Trillium ovaJtum: permanent iron-acetocarmin smear preparations of initoses
in the young microspore; A, metaphase; B, anaphase. {Preparation and photomicrograph
by Dr. H. E. Warmke.)

beauty that the extra effort is amply repaid. When properly carried out,
it is possible to study the internal structure of metaphase chromosomes.

1. Kill and fix the smears in Navashin^s fluid, or in a chrom-osmo-

acetic mixture, for about 20 minutes.

2. Wash in running water for 20 minutes.

3. Bleach if necessary with diluted hydrogen peroxide, then wash

again.
4. Mordant in 2% ferric ammonium sulphate for 20 minutes.
5. Wash in running water for 6 to 10 minutes, then rinse in distilled
water.
6. Stain in 0.5% hematoxylin for 20 minutes.
7. Wash out excess stain with water.
8. Differentiate in a saturated aqueous solution of picric acid. The
time varies according to the species and to the stage of development
of the naicrosporocytes or microspores: 20 minutes for divisions in pollen
 SMEAR METHODS 163

grains, 40 minutes for tetrad stages, and 100 to 120 minutes for dividing
9.
chromosomes. Examine occasionally under the microscope to note
progress of the destaining. It must be carried considerably further than
usual if one wishes to observe internal chromosomal details.
Wash in running water for 30 minutes, then proceed to the dehydra-
tion and mounting in balsam.
Capinpin’s Brazilin Method. —Belling's original brazilin method
lacked certain desirable qualities, one being permanence. It was better
adapted for the Liliaceae than for dicotyledonous families, in the opinion
of many critics. In the Onagraceae, and particularly for Oenothera,
it was for years impossible to make really first-class smear preparations

of the peculiar rings ofchromosomes found at the diaphase of many

species. A number improvements over Belling^s method have been
of
devised (Capinpin 1930), and it is possible to secure results of unusual
excellence.
1. Make smears as preferred, using Navashin\s fluid for fixation.
2. Transfer to a mixture of 1 part of Solution A of Navashin^s fluid
(the chromic acid part) and 4 parts water. Turn slides right side up,
and remove the thicker fragments and other debris. Leave for about
15 minutes in order to remove the formalin of the fixative.
3. Pass through 15, 30, and 50 to 70% alcohol, and allow to remain

overnight.
4. Mordant overnight in a freshly prepared 1% solution of ferric
ammonium sulphate in 70% alcohol.
5. Wash about 45 minutes in several changes of 70% alcohol.
6. Stain in a ripe 0.5% solution of brazilin in 70% alcohol. (The
brazilin solution should be ripe a week after being prepared.) The
time varies between 2 to 6 hours.
7. Wash briefly in 70% alcohol.
8. Differentiate in 1% ferric ammonium sulphate in 70% alcohol.
This process takes from 5 to 10 minutes. When examined under the
microscope, smears rightly stained show chromosomes brownish-black
or black, cytoplasm pink or straw-colored, and cell wall colorless.
9. Wash in two changes of 70 and one of 95% alcohol, 10 minutes in
each.
10. Dehydrate in absolute alcohol, and pass through the following
mixtures:

Equal parts of absolute alcohol and cedar oil

Equal parts of xylol and thin cedar oil

9 parts xylol and 1 part cedar oil

11. Complete the clearing in two changes of xylol, then mount in

balsam.
m GENERAL METHODS

—
Backman’s Method. This procedure combines the stain with the
fixing fluid (Backman 1935). It has not been thoroughly investigated
but being presented because of its possibilities.
is

Prepare smears as usual.

1.

2. Fix for 3 to 5 minutes, depending upon the density of the cyto-

plasm, in the following mixture:

Bouin^s fluid, using anthraquinone in place of the picric acid 100 cc.
Alizarin Red S 0 126 g.
.

Saponin O.lSOg.

Rinse for 2 to 5 minutes with tap water.

3.
Dehydrate through a rather close alcohol series up to 95% for
4.

about 1 minute in each.

5. Differentiate by flooding the slide with 0.5% sulphuric acid in

95% alcohol saturated with picric acid until the chromosomes become
clear; a few seconds generally suffice. A too prolonged treatment with
the acid solution is inadvisable. If the differentiation is diflBlcult, shorten
the time in the fixative.
6. Wash thoroughly with 95% alcohol to remove the acids. A further
neutralizing effect may be obtained by adding 0.5% potassium hydroxide
to the alcohol.
7. Intensify the stain, if desired or if the preparation is required for
immediate by washing for 30 seconds to 1 minute in 95% alcohol
use,
plus 4% by volume of cymene. (The originator does not specify what
type of cymene should be employed.)
8. Complete dehydration with absolute alcohol.
Clear for 2 minutes in each of the following mixtures: 5 parts abso-
9.

lute alcohol and 3 parts xylol; 3 parts absolute alcohol and 5 parts xylol.
10. Wash for 5 minutes in each of two changes of xylol, then mount in
balsam.
Belling’s Iron-acetocarmin Method. —
Really superb results have
been secured by the use of this method. Much care is required to obtain
successful preparations, and the method is not applicable to every
organism. For example, beautiful preparations of the reduction divisions
in the ovaries and testes of certain Orthoptera, or in anthers of Triticum
or Nicotiana are easily made, but it is difficult to get even passable slides
of meiosis in the microsporocytes of Lilium and impossible in Oenothera
and related genera. The preparations, as a rule and at their best, do
not remain in good condition for longer than a few months. Despite
its disadvantages, the student will find the iron-acetocarmin method
well worth trial and will feel more than repaid when good preparations
are secured.
Belling^s formulae for the preparation of iron-acetocarmin have
varied somewhat. The one which the writer prefers is to add 90 cc.
 SMEAR METHODS 166

glacial acetic acid to 110 cc. distilled water. Heat to boiling, remove
from the flame, and immediately add 1 g. (roughly) certified carmin dye.
Cool in the ice box, Add a few drops of an aqueous solution
then decant.
on standing is a dark wine red. Be careful
of ferric acetate until the color
not to add too much iron or the carmin will be precipitated. Keep in a
well-stoppered bottle.
There are several methods of using iron-acetocarmin. The method
to be used with a particular type of material depends upon the material
itself. Four methocfe are outlined below.
Place a few small anthers, or small pieces of large anther, in a drop
1.

of acetocarmin on a slide. After a few minutes withdraw the fluid with

absorbent paper, and replace with a fresh drop of dye. Press out the
microsporocytes with a scalpel or by other means, and remove anther
walls and debris. Put on a large coverslip, withdrawing any excess
fluid with absorbent paper. Then seal as soon as possible by covering
the edges of the coverslip with melted paraffin (of a low melting point)
wdth a warmed metal instrument, say a flattened broad needle. Allow
to stand for several days for the stain to penetrate thoroughly. Aqy
particular microsporocyte may be flattened out, or the cytoplasm and
chromosomes may even be forced from the cell, by careful pressure on
the coverslip.
2. Anthers cut into small sections or very tiny pieces of testis or

ovary are put into a test tube with at least fifty times their bulk of
acetocarmin. After about five days, depending upon the species,
preparations are made by putting a piece of the material on a slide in a
drop of acetocarmin, squeezing out the microsporocytes or flattening or
teasing the animal material, adding a coverslip, and sealing.
3. Smears of anthers or testis may be made on a coverslip in the
usual way and the latter immediately placed upside down on a drop of
acetocarmin on a slide.
4. If it is desired to stain the nuclei or chromosomes in microspores,
add just enough crystals of chloral hydrate to a few drops of acetocarmin
to clear the material. If too much of the chloral hydrate were added,
plasmolysis will ensue. for two days or so.
Leave in the stain Indi-
vidual cells may be squeezed or partially crushed to give better views of
their contents.
Whenviewing iron-acetocarmin^ preparations under the microscope,
a green should be inserted between the microscope and the source
filter

of illumination. The bluish-red chromosomes will then appear nearly

jfet black,
—Propionic
,

McCallum’s Iron-propionocannin. acid gives better

fixation and staining than does acetic acid. The amount of propionic
acid in the staining solution need not be so critical as with acetocarmin,
 : e

166 GENERAL METHODS

therefore the amount may be varied to give the optimum results for
each type of material (G. A. McCallum, unpublished). Incidentally,
the propionic acid solution dissolves more carmin dye and gives a ihdf
intense reaction, together with greater clarity of the cytoplasm.
Kill and fix the material in 1 part propionic acid and 2 parts absolute
alcohol. Place the material in iron-propionocarmin (prepared in exactly
the same manner as iron-acetocarmin, substituting the same volume of
propionic acid for the acetic acid) for 2 to 3 minutes, then transfer to a
slide in a drop of the staining solution, and flatten under a coverslip.
If the materialis difficult to flatten, heat the slide slightly. Float thc^
coverslip off in 50% aqueous propionic acid, then if necessary destain
in the same solution. Heat may be applied to accelerate the destaining.
Next dehydrate through the following series of mixtures, allowing about
five minutes in each

tertiary butyl alcohol, propionic acid, water

tertiary butyl alcohol, propionic acid
% tertiary butyl alcohol, yi propionic acid
Jio tertiary butyl alcohol, propionic acid
Pure tertiary butyl alcohol. (In the original method, tertiary butyl alcohol was
employed, but it is probable that hygrobutol would work better.)

Mount in balsam dissolved in tertiary butyl alcohol or hygrobutol.

McClintock’s Permanent Acetocarmin Method. Place anthers —
directly in a bottle containing part glacial acetic acid to 3 parts absolute
1
alcohol. Material can be kept for 12 hours to several weeks (the stain-
ability of the chromosomes is definitely improved by the fixative). For
longer storage transfer directly to 70% alcohol.
Transfer anthers from either killing fluid or 70% alcohol to a small
drop of acetocarmin. Carefully squeeze or tease out contents of anther.
Remove all debris, then add coverslip. Heat the slide over an alcohol
flame for about 1 second.This should be repeated four to five times
until the cells are somewhat spread and flattened. The solution must
not be allowed to boil. Examine slides at this juncture, and reject all
undesired ones. Be careful not to shift the coverslip or the sporocytes
will be loosened. Place slide in a Petri dish filled with 10% aqueous
acetic acid. The coverslip will gradually rise somewhat from the slide,
carrying some of the sporocytes with it. Push the coverslip gently
to the edge of the slide so that it can be grasped with forceps, but wait
until enough solution has run between the slide and coverslip to allow
the latter to float freely. The sporocytes stick to both slide and coverslip ;

consequently both must be handled in the solutions that follow. Care-

fully transfer both to a jar containing equal parts of acetic acid and
absolute alcohol. Then pass through the following mixtures, allowing
a few minutes in each :
 SMEAn methods; 167

• 1 part acetic acid to 3 parts absolute alcohol

1 part acetic acid to 9 parts absolute alcohol
Equal parts of absolute alcohol and xylol

The slide and coverslip are recombined directly from the last solution
in xylol-balsam. A pure xylol solution must not be used on fresh slides
as it will cause a distortion of the sporocytes.
Byone variation of the above method (Steere 1931), the anthers
are smeared on clean slides, then immediately fixed and stained by
placing the slides, smear side down, in a Petri dish full of steaming
acetocarmin for from 1 to 10 minutes. The slides are then transferred
rapidly through the following mixture: 2 parts glacial acetic acid plus
1 part absolute alcohol; t part acetic acid plus 2 parts absolute alcohol;
1 part acetic acid plus 9 parts absolute alcohol. Complete dehydration
by immersion for a few minutes in absolute alcohol, then clear for 2 to 3
minutes in a mixture of equal parts of absolute alcohol and xylol, and
mount in balsam from this solution, but work rapidly during the mount-
ing in order to prevent absorption of moisture from the atmosphere.
Zirkle’s Methods. —
These methods have been designed to combine
the acetocarmin staining mixture with inert substances which solidify
on standing and seal the mounts, thus avoiding the difficulties usually
attendant on transferring materials from the stain to a permanent
mounting medium (Zirkle 1937).
1. The is macerated on a slide in a drop of acetocarmin,
material
then several drops of the following solution are added:

Acetocarmin 80 cc.
Karo com sirup (dextrose) 10 cc.
Cert/O (pectin) 10 cc.

Next heat the preparation, then press down on the coverslip until the
smears are flattened to the desired extent. Excess solution oozing from
the coverslip need not be removed.
2. The following mixture combines in one operation fixing, staining,

and mounting. It may be used as made up, or it may be diluted with

acetocarmin in various proportions as required by the nature of the
material. If diluted, more fluid should be allowed in order to compensate
for the greater amount of water and acetic acid that evaporate.

Glacial acetic acid. 60 cc.

Water 50 cc.

Glycerin 1 cc.

Gelatin (powdered) 10 g.

Dextrose 4 g.

Ferric chloride 0.05 g.

Carmin dye To saturation

 ;

168 GENERAL METHODS

Dissolve the gelatin in the water, then add the other ingredients. Boil
and filter the mixture, exactly as is done with plain acetocarmin. The
medium becomes firm like balsam as the aqueous portions evaporate
it will not liquefy when heated, once it has solidified.
Warmke’s Method. —
The schedule affords permanent smears of
root tipsand gives exceptionally beautiful and useful preparations
(Warmke 1935). It may also be used on microspores in order to study
the first postmeiotic division.
1. Kill and fix tissues as directed in McClintock’s method.
2. Remove from
the killing fluid and place for 6 to 10 minutes in a
mixture of equal portions of 95% alcohol and concentrated hydrochloric

Kig. 22. TiHXliwn chloropetaluTn: cell from a root-tip smear prepared accordi 2 i|{ to
Warmke's method. (Preparation and photomicrograph by Dr. H. E. Warmke.)

acid. This serves to dissolve the pectic substances of the middle lamellae.
The may be varied considerably: 3 parts acid to 7 parts
proportions
alcohol may be used, but the time must be prolonged.
3. Transfer to Carnoy’s fluid (with chloroform) for 5 minutes or
longer. The purpose is to harden the tissues again after the acid treat-
ment, which more or less softens it.
4. Cut off a small piece (less than 0.5 mm. long) from a root tip or
piece of anther, and place on a clean slide in a drop of acetocarmin.
It will be difficult to flatten the attempted to
cells sufficiently if it is
use too large a piece of material. Press directly on the piece with a
flat scalpel: the cells will separate and float free in the stain. Place a
coverslip over the drop of stain, and apply gentle pressure. The size
of the drop of acetocarmin is governed by the dimensions of the coverslip.
If too much stain is used, it will flow out from under the coverslip and
 ;

mEAU METHODS 169

carry many of the cells with it. Small square coverslips will be found
more satisfactory than large rectangles.
5. Heat the slide cautiously by passing three or four times through
the flame of an alcohol lamp (take great care not to let the solution boil)
this serves to clear the cytoplasm.
6.The preparation may be preserved temporarily by covering the
with a mixture of equal parts of Parowax and gum
edges of the coverslip
mastic heated together. Such mounts will keep in good condition for
about a week. Permanent mounts can be made as described under
McClintock^s method, beginning with the placing of the slide in 10%
aqueous acetic acid.
Hillary’s Method. —
Fix the root tip with any suitable fluid, then
wash out the fixing fluid thoroughly, and apply the Feulgen technique
(Hillary 1938). Sufficient time should be allowed for the stain to pene-
trate thoroughly. Dehydrate through three changes of dioxan. Place
a tip in a drop of balsam diluted with dioxan on a clean slide, and divide
the tip into small longitudinal sections by means of sharp needles or a
very small scalpel. Finally add more balsam if necessary, then apply a
coverslip. Further pressure applied to the coverslip aids in spreading the
cells.

This method may also be used on anthers that are difficult to smear
before the microsporocytes have rounded up.
 CHAPTER XIV
CYTOLOGICAL METHODS

Microtechnical methods employed in the field of cytology are the

most critical of all, and before a person enters this domain, he should take
the pains to become fairly well grounded in the basic principles and
procedures of general botanical microtechnique. Only in this way is it
possible tobecome suflSicieritly proficient to devise the special variations
in schedules demanded by the differences in structure and chemical
composition of the tissues of diverse plants. No two species react exactly
alike to the identical technical schedule, and the beginning technician
must learn how to adapt the composition of killing and fixing fluids and
the staining procedures to the particular plants under investigation. It
is impossible to interpret and to judge the final results accurately and
adequately unless considerable preliminary experience has been gained.
The common practice of attempting to learn microtechnique and to
investigate a cytological research problem simultaneously cannot be
too strongly condemned. Those who do this usually never become
other than mediocre technicians and poor they are
cytologists, since
unable to master the finer points of microtechnique and consequently
cannot appreciate the value, from the standpoint of interpretative
cytology, of working with first-quality preparations.
Cytological technicians in the past have been too content to adopt
pragmatism as their sole philosophy. The rationale of their methods
has too often mattered little or not at all, provided the final result was
according to their notions. As one critic succinctly put the matter: ^*As
a result, textbooks of cytological and histological technique have tended
to resemble the pharmacopoeas of the Middle Ages.^^ A change in this
viewpoint is a vital necessity if cytological technique is to progress.

A
complete understanding of the behavior of the different substances
entering into the composition of killing and fixing fluids, particularly
with regard to their effect upon both resting and dividing chromatin, is
the first and probably the most important point upon which the

embryonic cytologist should become thoroughly grounded. Next in

order of importance is the ability to manipulate stains and staining

schedules to obtain the optimum optical contrast, bearing in mind the

fact that cytologists never employ general staining, as is commonly used
on purely anatomical materials, but always aim to procure the sharpest
170
 ;

CYTOLOGICAL METHODS 111

possible staining of particular structures. The fact should incidentally

be emphasized that not only do stains differ in their action upon different
species, but even upon different stages in the same piece of material.
Many cytologists use only one dye and concentrate on some one phase
under specific investigation, such as the chromosomes at the metaphase
stage in either mitosis or meiosis. The more critical cytologists employ
more than one killing and fixing fluid and different staining methods in
order to obtain confirmatory evidence through diversity of methods.
The beginning cytologist further needs to become proficient in the
use of iron-acetocarmin or iron-propionocarmin, whether smears or
crushed materials are used. The majority of the most competent tech-
nicians always make a preliminary study of their materials by means of
temporary mounts stained by some carmin method.
Cytological methods, on the whole, are the most painstaking of all,
but the final results, when successful, more than justify the trouble
entailed in their preparation. John Belling, one of the most critical
and competent of cytologists, thought nothing of spending weeks and
even months preparing a single perfect preparation: every step was
carefully considered in advance, and nothing was left to chance or guess-
work. One would do well to emulate his brilliant example. Except for
temporary carmin-stained preparations, a passable cytological slid(^
cannot be made in a hurry, nor by lackadaisical methods.
not possible to write precise directions, step by step, for cytologi-
It is
cal methods. The best that can be done is to enumerate the methods
which innumerable cytologists have found by sustained experience to
afford the optimum results: using these methods as a guide, a person with
sufficient preliminary training and experience will find himself able to
adapt them to the peculiarities and idiosyncrasies of his particular
materials.
Smears versus Sections. —The beginner who has followed the litera-
ture, especially thatbetween 1925 and 1932, will doubtless have noted a
sharp divergence of opinion between those cytologists who depend prin-
cipally upon smears (or crushed and spread tissues) for their observations
and those who favor sections. No one method, however, can
possess any inherent superiority over another since none alone can be
wholly sufficient by itself. Each method has its good points, as well as
its disadvantages, consequently each should be considered as supplemen-

tary to the others. Each supplies evidence of a type not readily afforded
by another; taken all together, each reveals a part of the whole. There
are circumstances in which one method is more useful than another, but
thisdoes not necessarily rule out the applicability of the others. To cite
a few instances: the pollinia of Aca^a and the orchids cannot be smeared
the microsporocytes of the Onagraceae are easily smeared but cannot
 172 GENERAL METHODS

always be well stained and show only a few metaphase stages at a time;
the microsporocytes of the Liliaceae and related families are equally good
smeared or sectioned but only smears will show the entire nucleus or
chromosome garniture because of their large size; buds of many species
are too small to be handled for smearing, and so on.
The rule to follow should be to make smears whenever satisfactory
ones are permitted by the nature of the material and its staining capacity
and always to make sections, checking the observations on the one against
those on the other.
For smears, consult the chapter on Smear Methods (page 155).
One should commence with temporary carmin-staining schedules, pro-
gressing to permanent carmin methods, thence to other permanent
methods. For paraffin sections, the tertiary butyl alcohol method of
dehydration is recommended. Celloidin methods are rarely satisfactory
for cytological studies since it is so troublesome to arrange serial sections,
but this difficulty can be partly circumvented by double-embedding.
—
Manipulation of Material. Materials intended for cytological inves-
tigations must be more carefully handled than is generally recognized.
Post-mortem changes must be completely avoided; the time from the
removal from the plant to placing in the killing fluid should be as short a
period as possible, and the plants should also be healthy and turgid,
unless pathological material is being investigated (such as parasitic
fungi, mosaic diseases, etc.). Wilted portions should never be fixed,
otherwise the completed preparations will reveal excessive plasmolysis,
clumping of chromosomes, cytomixis, and similar artifacts.
Root tips may be obtained from plants grown in nutrient solutions
or in not too large pots. Aquatic plants or those grown in nutrient
solutions may simply be lifted out of the water and the tips cut off
behind the meristematic region (which is whiter and more opaque than
elsewhere) by means of a small sharp scalpel or pinched off with fine-
pointed forceps. If the plants are grown in pots, they should have been
allowed to grow until the space between the soil and interior of the pot is
beginning to become filled with roots. Invert the pot, holding the stem
between the middle and index fingers of the left hand with the hand itself
flat on the soil, knock the edge of the pot against something solid, and
the mass of roots and soil generally comes out readily. With the whole
thing still held in the hand, snip off the tips with fine forceps, and place
immediately in the killing fipid. To be on the safe side, as many tips
as possible should be removed from the potted plants. The tips from
such a source rarely have particles of grit adhering, but care should be
taken not to carry any over. Sometimes there may be no root tips
visible; in such cases, the soil may be carefully washed away under a
faucet, when it is generally possible to find tips near the crown. Plants
dug put of gardens scarcely ever have tips in good condition.
 CYTOLOGICAL METHODS 173

Bulbs of Allium cepa may be grown in the dark in suitable containers,

using a somewhat diluted Knop\s or other nutrient solution. The tips
will be long enough for removal in about three days. Bulbs of Hyacin-’
thuSy CrocuSy Fritillariay and similar genera are better grown in a cool, dark
place in finely sifted soil (with no sand, if possible) or in shredded peat in
pots or boxes, since they require from two weeks to a month to attain
sufficientgrowth for removal of the root tips. Remove the bulbs from
the container, wash quickly under a gently flowing faucet, then cut off
the tips with a scalpel.
It is a simple matter to remove the buds from dicotyledonous plants.
Anthers from one or two may be removed and smeared in iron-aceto-
carmin to determine the stagt‘ of microsporogenesis, but ovules can
only be collected blindly. If the anthers have a yellow or reddish color,
the pollen grains have be(‘n formc^d; as a rule, meiosis has already taken
place by this time in the megasporocytes. If the buds are large, either
r(‘mov(' th(^ anthers and ovaries individually, or reduce the entire buds
to conveni<'ni small portions. With small buds the procedure depends
on whether the ovaric's are superior or inferior, and in the case of the
latter whether botli anther and ovary portion is to be removed as an
(uitity or separately. If the ovaries are superior, separate from the plant
by cutting bet ween the base of the ovary and the apex of the pedicel, then
cut off the overlapping sepals at the top in order to ])ermit the killing
and infiltrating reagents to enter more readily. In the large buds of
most Asteraceae bisect each capitulum in the median vertical plane; it
would be a good idea to do the same thing with other plants whenever
th(‘ nature of the material permits it. In the case of flowers with inferior
oA'aries one may remove the whole thing if it is small enough, and simply
nmiove the sepal tips. Otherwise separate the ovary and anther portions,
and run up independently. If the flowers come in small dense bunches,
it is the usual prac’tice to treat the whole group as an entity, but care

must be taken to insure complete penetration of the killing fluid by mak-

ing a few incisions in places where they will do no damage. All superflu-
ous tissue should always be removed.
The remarks in the preceding paragraph refer to buds that are readily
visible externally. In many monocotyledons, particularly in the Lilia-
ceae, the buds are concealed in bulbs or similar structures and must be
dissected out. It is necessary to have some knowledge as to the time of
year at which the various developmental processes are undergone. In
Trilliumy for example, meiosis in the microsporocytes occurs during
September and early October; in Hyacinthus it may occur as early as
August in some species or varieties, in October in others.
Plant groups other than the angiosperms, which have been considered
above, may be treated as suggested in the special sections devoted to the
pertinent groups.
 174 GENERAL METHODS

—
Choice of Fixing Fluid. If a proper fixing fluid, with the ultimate
purpose for which the preparations are required always kept in mind, is
not chosen at the beginning, little or nothing can be done afterward to
remedy initial errors. Of course, there will be occasions when one will
be forced to use materials which from necessity were fixed in the only
available fluids (as on collecting trips to regions remote from a labora-
tory) in such instances it becomes a matter of adapting staining sched-
;

ules to obtain the desired result. There is, however, no excuse, when
adequate opportunities are at hand, for not using a carefully adjusted
formula for the killing fluid. In determining the constituents of such a
fluid and the proportions of the various ingredients, the factors which
require to be taken into account include: (1) the nature of the material,
(2) the size or bulk of the latter and its penetrability by fluids, and (3)
the dye or stain conbination to be employed for staining. a violet
If

dye, for example, is to be used, the fluid should contain chromic acid;
if a

triple combination is to be used, the fluid should contain both chromic

and osmic acids. If the bulk of the material is somewhat large, the fluid
should either be somewhat stronger than usual or should contain ingre-
dients of strong penetrating power. The same precautions should be
observed if the material is heavily cutinized or suberized, since such
tissues impede penetration of fluids. For cytological purposes aqueous
fluids are always preferable to alcoholic mixtures, since alcohol is a poor
preservative of dividing chromatin.
Statements in the literature to the effect that such-and-such a fluid
^
without specifying under what conditions such
Ogives the best results,
results were obtained, are meaningless. Conditions are not the same
everywhere at all times. In England, for example, LaCour\s fluids are
popular and apparently afford excellent results, but on the Pacific Coast
the fixation is and staining is most difficult. Climatic con-
atrocious,
ditions apparently have a definite bearing on fixation results and certainly
have one on the subsequent staining. This statement refers more to
terrestrial plants than to those growing in aquatic habitats, as the latter
have a more nearly uniform milieu throughout the world when growing
in the same type of water. It is both desirable and necessary to experi-
ment with several fixatives and with variations in these fixatives in
order to ascertain which provides exactly the desired results. One
should not blindly accept some writer ^s statements, except insofar as
these provide clues as to which formula might be most useful.
The majority of cytological technicians prefer killing fluids giving an
acid fixation image. The more they concentrate on dividing chromatin,
the more acid are the mixtures that they employ since their sole purpose
is to observe the chromosomes during mitosis and meiosis. Fixing
fluids which give basic images are employed only when cell contents and
 CYTOLOGIC AL METHODS 175

inclusions are being studied; such fluids are useless for studies on dividing
chromatin since they dissolve it more or less.
Navashin^s fluid, or some variation thereof, is generally considered
to be the most useful for cytological purposes. It is usually made up in
two parts. Few workers change the proportions of the chemicals in the
first part, but most vary the proportions of the formalin of the second

part in accordance with the idiosyncrasies of their material. Many

technicians, furthermore, especially when working with thick materials
or when these are heavily suberized or cutinized or covered with dense
hairs that prevent the material from sinking into the killing fluid, first
immerse the material in Carnoy\s fluid for from 5 to 10 minutes, then
pour it off (some also give a quick rinsing with water) and replace with
Navashin’s fluid. The beginning cytologist cannot go far astray if he
first tries Navashin’s fluid, later making changes in the proportion of the

formalin if this seems desirable or necessary.

For reasons which have been amplified elsewhere, the simultaneous
use of potassium bichromate and chromic acid in a mixture is irrational.
Tissues fixed in such mixtures are always overchromated and staining is
difficult. It is usually necessary to resort to bleaching, which at times is
dangerous since it may alter the fixation image.
If the morphology of the chromosomes is being studied, particular

attention should be paid to the amount of chromic acid in the fixing

fluid, siiK^e it has a direct effect upon accurate preservation of chromo-

somal structure. Fixatives somewhat weak in this acid will reveal

constrictions that are not evident after fluids strong in chromic acid.
Infiltration and Embedding. — Standard methods of infiltration with
paraffin are employed by most cytological technicians. It is very seldom
The tertiary butyl alcohol method
that any difficulties are encountered.
isrecommended since it produces the least variations in the fixation
image and permits easy microtoming. The periods during which
materials remain in the dehydrating series and in the paraffin oven
should not be too prolonged.
Cytological materials are generally embedded differently than ana-
tomical or other ordinary specimens. They are so embedded that the
largest possible number of sections can be mounted on each slide (note
Fig. 4A, F). For instance, root tips are embedded in bunches of from
two to as many as a dozen, for microtoming transversely, and small
buds are embedded in groups depending on their size. If the buds are of
different sizes, one may either follow the less desirable method of grouping
together buds of various sizes, particularly if the exact stage when meiosis
occurs is not known and it is desired to ascertain this fact, or the buds
may be all the same size. The group of buds should not exceed 15 mm.
in any direction, but it does not matter whether they are arranged as
 176 GENERAL METHODS

squares or rectangles. If they are to be cut longitudinally, they should

lie flat on their sides, all preferably pointing in the same direction; if

they are to be microtomed transversely, then the end which is to face

the knife should be pointed down. Not all types of buds, however, can
be oriented with reference to any particular plane, so they may simply
be grouped together. Separate anthers, ovaries, etc., may be treated
in the same fashion. All materials should be grouped together as closely
as possible, since it is exasperating to have to move the slide across wide
empty spaces when it is being examined under an immersion lens. Unless
a special electrically heated embedding plate is used, do not try to
arrange too large a quantity of material at one time lest the paraffin
become too solid before the task is completed.
—
Microtoming. Although most plant materials for general study are
sectioned at from 10 to 12/1 cytological materials are not cut at any
,

standard thicknesses. Materials fixed in fluids giving the basic fixation

image are cut rather thin; some technicians consider that 2 and 3jLt are
none too thin, while others use sections as thic^k as lO/x. Mitochondria
are usually studied in sections cut at between 6 and S/x; th(' cytoplasm
in sections anywhere from 2 to 10m, depending upon the diamet(‘r of the
cells in which it is being investigated. In other words, the smaller the
mean diameter of the cells, the thinner the s(‘ctions must be cut.
In secitioning root tips and buds for the study of the somatic and
meiotic (ffiromosomes, the material should be microtomed at about thr(‘(‘-
fourths of the average diameter of the cells. Mi(;rosporocyt(\s and
megasporocytes increase enormously in size at a very rapid rate at about,
the time meiosis is occurring in each, consequently sections should b(‘

somewhat would judge the thickness to be before this

thicker than one
stage is reached. Serious mistakes in the counting of chromosomes have
been made when too thin sections have been employed. A commercial
concern once advertised onion root tips allegedly sectioned at l/x (which
isextremely doubtful) and designed especially for chromosome studies.
Such sections are very dangerous to use for that purpose since th(^
chromosomes have been cut into innumerable portions scattered over
as many as a dozen sections, and it is utterly impossible to match the
portions, much less to count the chromosomes accurately. The optimum
thickness for onion root tips, the more or less standard material upon
which beginning cytological technicians work, when microtomed longi-
tudinally is ll/x; when cut transversely it should be 13 to 14 m. The
majority of root tips should be microtomed transversely. There are
very few which are as satisfactory as the onion when cut longitudinally;
among these may be mentioned Vicia /a6a, Hyacinthus^ Trillium^ Liliuniy
and Zea mays. It is problematical whether there is any species in which
the chromosomes can be accurately counted in side views (i.e., in longi*
 CYTOLOGICAL METHODS 177

tudinal section) of the spindle at either metaphase or anaphase of mitoses;

ki any event, cytologists are skeptical of counts made under such circum-
stances. These sections, however, are excellent for studying the struc-
ture of the chromosomes. Very small root tips (using the onion for
comparison) with a comparatively short meristematic region should be
sectioned transversely at about lOju. In very large tips the diameter of
the cells should first be ascertained, then the appropriate thickness may
be calculated. In Fritillaria rneleagris^ for instance, the chromosomes
are so large and numerous that sections 30 to 36/i thick are required.
Such thick sections are better cut with safety razor blades held in a
Craig-Wilson holder through which lukewarm water is allowed to run;
this will cause them to form a ribbon, which they will not do on an
ordinary knife. In Crocus the cells are larger than in the onion, but the
chromosomes are mostly smaller and more numerous; hence sections
should be (*ut transversely at 12 to 14iu.
The ])lane in whi(*h ovari(‘s so small that it is irnpracti(^able to dissect
out the ovules for individual treatment are sectioned depends entirely
on the orientation of th(» ovules. It is invariably necessary to section
the ovules in the longitudinal plane perpendicular to the raphe or funicu-
lus; (consequently the technician should become familiar with the arrange-
ment of the ovules while th(' material is being prepared for fixation. In
general, tin' ovules are arrange^d either horizontally or vertically, and thus
the ovaries may
be sectioned transversccly in the first case and longitudi-
nally in the latter instance. When they are in mixed positions, i.e.,
radiating from a common center in all directions, one can only cut blindly
and trust to luck to find ovul(\s that w^ere sectioned longitudinally. This
obviously means that a larger (juantity of material must be sectioned in
order to afford a sufficient number of satisfactory sections. The mega-
sporocyte differs greatly in size among different species, and there is
little between the size of the ovule and the diameter of the mega-
relation
sporo(jyte. To contrast two genera: ovaries of Lilium and Oenothera of
approximately the same size may be taken; those of Lilium are sectioned
transversely at 24iut and those of Oenothera longitudinally at 12/i. The
ovules of Lilium wall be found to be rather large wdth few, large cells;
those of Oenothera will be found to be small with numerous rather small
cells. The megasporocyte of Oenothera averages less than one-fourth
the size of that of Lilium and the chromosomes are even smaller.
One piece of material should first be cut, stained, and examined under
the microscope. If the majority of the cells containing mitotic or meiotic
figures show that all the chromosomes are contained in either uncut
cells or in cells from which thin slices have been cut from either top or

bottom or both, then the thickness is correct, and the remainder of the
material may be sectioned at that thickness.
 178 GENERAL METHODS

Staining Methods. —Cytological staining must, above all, be sharp,

precise, and brilliant, 'with the clearest possible differentiation betwe^
structures. Sloppy staining is worse than useless. Many beginners in
cytological technique overdo the staining and therefore should exercise
rigid control over this aspect of their work. Cytological staining is at
the same time the most simplified and the most diflScult, yet if one takes
the necessary pains it can be easy. The secret of success is not to attempt
to do more than one thing at a time. To make this point clearer, it
will be recalled that in the average morphological preparation it is the
customary aim to attempt to stain as many structures as possible cell —
walls (whether cellulose, lignified, cutinized or suberized), cytoplasm,
nuclei, nucleoli, chromosomes, chloroplasts, sclereids, etc.; in cytological
preparations, on the other hand, one should not attempt to stain anything
besides the chromosomes, or the mitochondria, or the cytoplasm, as the
case may be. If, for example, anything besides the chromosomes is

stained, it should be looked upon as an accidental occurrence; if it does

not obscure the chromosomes, it is of no moment, but if the chromosomes
are rendered less visible, it is a serious matter that must immediately be
remedied.
Most root tips take an adequate stain with iron hematoxylin for all

stages of mitosis; others do not. Those taken from plants which natur-
ally grow in alkaline soils are especially unsatisfactory with this stain, as
are tips with very dense cytoplasm. Instances are known when certain
chromosomes became completely destained long before the remainder
were sufficiently differentiated, thus leading to inaccurate counts of the
chromosomes. The tips of some species destain rapidly, but most of
them destain somewhat slowly, and in many such cases great caution
niust be taken not to differentiate too far. A few cytologists destain
the hematoxylin until only the outlines of the chromosomes remain
visible, but this practice is hardly to be recommended. If the cytoplasm
is very dense, one can only judge from experience as to when the differen-

tiation should be stopped. Few cytological technicians use a counter-

stain on root tips in which the chromosomes are to be studied since it is
liable to obscure their morphology. Sometimes, however, one may. be
required to reveal the otherwise invisible cell walls or to intensify the
cytoplasmic background; orange G may be recommended for the purpose.
Practically all root tips take a sharp and brilliant cytological stain
by one of the violet methods (page 89 et seq.). Most present-day
cytologists prefer the violets, crystal or methyl, as they can be sharply
differentiated and have a high specific affinity for dividing chromatin.
The always so good for resting chromatin as is iron hema-
violets are not
toxylin. There is considerable variation between species in the degree
to which the violet is concentrated in the dividing chromatin and remoyed
 CYTOLOGICAL METHODS 179

from other structures. When the slide is removed from the differentiating
fluid, the stain may seem to be still present in the sections generally, but
if the slide is examined under the microscope while still wet, it will

usually be observed that the stain is quite dilute in the cytoplasm and
cell walls and sharp enough in the chromatin. In still other species every
vestige of the violet is removed from the cytoplasm and other structures,
and the dividing chromatin alone (if lignified tissues are present, they
are usually also stained) stands out, brilliantly stained. It will therefore
be seen that the macroscopic appearance of the sections is useless as a
guide to the adequacy or nature of the staining, and it should not bo
depended upon. As a rule, the thicker the sections, the more generalized
the stain appears to be, but macroscopic and microscopic examination
are two very different things. Very few cytologists use counterstains
after the violets, but if it appears desirable to add one, either erythrosin
or orange G would be preferable.
Not all the violet dyes on the market are satisfactory for cytological
staining. Some are, in fact, worthless for the purpose. Gentian violet,
as explained in the chapter on stains, is not a single dye, but a mixture of
crystal and methyl violets in different proportions. Crystal violet
should be used whenever gentian violet is specified and is probably the
most satisfactory for use by Newton^s method. Either crystal violet or
methyl violet 2B may be used in Johansen^s method. Certified samples
should always be used.
Sections thicker than lOfi cannot be satisfactorily stained with iron
hematoxylin, Vjut the violets give sharp staining at all thicknesses.
Other stains may be used on root tips, but none is of greater value
than the violets or iron hematoxylin. Tips of Allium cepa are commonly
stained with safranin and fast green or by the Cajal-Brozek method, and
both are very useful in elementary classes; hence one might experiment
with these two. If neither a violet nor iron hematoxylin stains ade-
quately, resort may be had to a triple combination; StockwelPs modifica-
tion is particularly useful, especially when the sections appear to be
overchromated.
Buds for the study of meiosis are commonly stained with a violet,
exactly as for root tips, followed by orange G. There is, however, far
more difference in the case of buds with regard to the extent that they
retain the violet stain. Sometimes it may be rather erratic, and in such
cases mordanting of one type or another is indicated. This may consist
in the application of 1 % aqueous chromic acid for at least 1 hour, or of
differential acidification previous togoing into the staining solution.
Iron hetnatoxylin is frequently very sharp on buds, and it is to be pre-
ferred for the prophase stages. The violets commonly do not stain the
prophase stages at all.
 180 GENERAL METHODS

Feulgen’s reaction is of the utmost value in cytological investigations.

The application of this chemical test appears to be rather complicated,
but once it has been mastered, it is a simple procedure and should be
adopted as a routine method.
The violet methods cannot be used on materials sectioned in celloidin,
but iron hematoxylin is generally quite satisfactory. Apparently no one
has yet applied the Feulgen reaction to celloidin sections.
A great many methods have been devised by animal cytologists for
the staining of mitochondria, but the stains all fade within a short time
and have never been any too successful on plant materials. Innumerable
staining methods for other elements making up animal tissues have also
been devised, but one need merely recall that animal and plant tissues
are radically different both chemically and physically, consequently these
methods are useless on plants.
Judgment of Results. —From the statements of many authors the
conclusion is inevitable that the problem giving them the greatest concern
is that of artifacts. The
on the other hand, is inclined to the
writer,
opinion that the been considerably ovf^remphasized.
question has
Modern technical methods, moreover, have gone far to remove the
commonest sources of artifact production.
An artifact has been described as ^^an appearance that arises from
treatment^’ (Darlington 1932). Consequently, when a living organism is
subjected to chemical treatment, artifacts are so liable to be introduced
that it could probably be stated categorically that no treated material
is entirely free from such appearances. Artifacts, on the other hand, are
rarely purely such. Those artifacts which are introduced extraneously
and as such are ‘^pure artifacts, include deposits from certain fixing
fluids (c.pr., those containing mercuric chloride) and certain stains. The
hematoxylins, especially when they have become aged and turn color, are
notorious for leaving deposits when carelessly handled. All pure arti-
facts, fortunately, are promptly recognizable as such.
Since artifacts may be considered as always being present, the prob-
lem becomes simplified into one of determining the extent of their signifi-
cance. The majority of artifacts are dependent upon the nature of the
fixing fluid, but others have different causes. The appearance of cyto-
plasm following the use of a fixing fluid giving the basic fixation image is
more nearly natural and normal; its appearance after a fluid giving an
acid fixation image is always an artifact. To cite another cause of arti-
facts, if the chromosomes appear clumped following the use of Bouin^s
fluid, the blame rests with the dehydrating fluids. The significance of
the artifact depends greatly on how far it was carried along to being what
it i|s. The chromosomes may show only a slight rounding up after
Bouin's fluid, in which case they may serve for counting purposes if they
 CYTOLOGICAL METHODS 181

can be clearly distinguished each from the others, but they are in no
condition for any other purpose; if they are badly clumped, the prepara-
tion is worthless.
Appearances will frequently be encountered where it is necessary to
determine whether an apparent artifact is natural or merely characteris-
tic. If a presumable artifact appears after only one fixing fluid (generally
also when the same staining method is used) and not after others, then
it is a genuine, noncharacteristic artifact. If, on the contrary, it appears
after a variety of fixing fluids and staining methods, it is a characteristic
artifact. Again, if it can also be observed in the living material, it can

scarcely be considered to be an artifact. From this discussion it becomes

evident that the artifact (|uestion must be approached with caution, and
it ill behooves a critic to call an appearance an artifact and thereafter
ignore it. Its nature must be investigated; it may turn out to be a matter
of greater significance than was at first realized.

Cytology texts should be (‘onsulted by those wishing to go further into

the artifact question, since a discussion of the subject from the cytological
standpoint is beyond tlu^ scope of the present text {tnde, e.g., Darlington
1932, Appendix Such texts should be considered in a critical vein
I).

since cytologists tlunnselves are not yet in agreement on many points.

The final problem to l)e considered is that of cytomixis. It is a
difficult one to answer. Most plant cytologists seem to consider the
extrusion of chromatin during meiosis in microsporocytes to be the result
of bad fixation or faulty tecflinical treatment, but evidence has been
presented to show that the extrusion of nuclear material regularly and
constantly occurs in certain plants. It is also likely to be found in
material collected during very warm periods of the daytime.
 CHAPTER XV
MICROCHEMICAL METHODS
The term microchemistry’’ connotes two distinct phases: purely
chemical microchemistry and botanical microchemistry. We are not
concerned with the first phase, but with the latter only. In botanical
methods qualitative reactions alone are almost exclusively employed,
whereas in chemistry quantitative methods are of greater importance.
Those who wish to pursue the chemical aspects may consult the standard
works (c.gf., Emich 1932, Haas and Hill 1928, Klein 1931-1933).
To many botanists microchemical tests are of significance only when
correlated with either anatomical structures or, to a lesser extent, physio-
logical appearances. These tests, unfortunately, are of a fugitive nature
for the most part, in but few cases is it possible to make permanent
and
preparations demonstrating microchemical reactions.
No attempt has been made to present an exhaustive list of procedures
or to include all substances for which tests have been devised.
—
Preparation of Sections. Only strictly fresh material should be
used, as a rule. Wilting of the tissues can sometimes produce changes in
reactions, and in some cases plant material that has been kept indoors
away from light and subjected to the influence of laboratory odors or
reagents will give a very different reaction from that of material freshly
collected outdoors.
Generally speaking, slightly tliick sections are best for use in micro-
chemical tests. Even those cut at around 50/i are not too thick. Very
thin sections present too little tissue, especially if the substance for which
the test is being made occurs in quite small amounts, and there is thus
danger of obtaining a negative reaction. Such thin sections, moreover,
are dijficult to handle.
Sections can be cut with safety razor blades, razors, or sharp scalpels
with thin blades. Clean the cutting implement thoroughly before each
operation to avoid contaminating the sections. In most tests the
sections are to be placed directly in the reagent, but if specific directions
concerning the immediate treatment of the sections are not given, it is
understood that they are to be placed in a few drops of pure distilled
water.
Jtiicrochemical Reagents. —The majority of the reagents are simple
dilutions of various salts, dilutions of different acids, etc., and their
182
 MICROCHEMICAL METHODS 183

preparation is given in sufficient detail upon each occasion. Some general

reagents in common use, however, are being described at this juncture and
their ingredients and preparation described.
1. When the term used with reference to an acid, it usually
dilute’’ is
means a solution in water of not over 5% strength. Dilute
distilled
solutions of salts may be of a strength not over 10% by weight.
2. lodine-Potassium Iodide .
—
Under certain circumstances variations
in the proportions of these two chemicals are required. When such are
not specified, the general formula may be employed. Dissolve 1 g.
potassium iodide in 100 cc. distilled water, then add 1 g. iodine flakes.
3. Millon^s Reagent .
—
Dissolve 1 g. mercury in 9 cc. concentrated
nitric acid of sp. gr. 1.52, and dilute the solution with an equal volume of
water. The reagent does not keep long, but its usefulness may be pro-
longed by the addition of a few drops of potassium nitrite solution.

ALDEHYDES
Formaldehyde. —Sections of the fresh tissue to be tested are placed
directly in a few drops of a 1 % solution of diphenylamine in concentrated
sulphuric acid on a slide. Heat slightly over a flame. A permanent
green color, representing a condensation product of formaldehyde and
diphenylamine, appears. With other aldehydes the green color shortly
changes to red.

ALKALOIDS
The number of known is now so large that tests for each
alkaloids
of the individual alkaloids cannot be given here. Reference should be
made to manuals or monographs on the group (c.gr., Henry 1924, Tun-
mann 1931). Alkaloids generally occur as colorless crystalline solids,
but a few are liquids. As a rule, they are insolubfe in water but will
dissolve in neutral organic solvents. Most alkaloids do not occur free,
but combined with some acid in the form of a salt.
Alkaloids, like most other substances having a nitrogen base, are
precipitated by the salts of the heavy metals. The best of such reagents
are 5% aqueous solutions of either gold or platinum chloride.
Parallel tests should be made on fresh sections and on sections which
have been freed of their alkaloidal contents. To extract the alkaloids,
put the sections in 5% tartaric acid in 95% ethyl alcohol for several days,
then wash, and test. Iodine-potassium iodide solution gives a chocolate-
brown precipitate with alkaloids; proteins give a yellow to brown color.
Innumerable color reactions for different alkaloids are described in
monographs.
Occurrence . —
Alkaloids are widely distributed. Saps of the Papa-
veraceae contain several different alkaloids. In the Berberidaceae,
 184 GENERAL METHODS

berberin forms tufts of needles of berberin nitrate when sections are placed
in 2% nitric acid.
Any may be used in tests for nicotine: with
species of Nicoiiana
phospho tungstic deep yellow colors becoming yellowish-green are
acid,
produced; with saturated mercuric chloride the color is white;
with platinum chloride (2%), yellowish-white; with iodine solution,
brown-yellow.

AMINO ACIDS AND AMINES

In testing for most of the amino acids, it is best first to crystallize
them out of absolute alcohol. Fresh sections are placed directly in a drop
of the alcohol on a slide, and the alcohol allowed to evaporate. In addi-
tion to the crystals of various amino acids, crystals of potassium nitrates
may appear. These crystals may be identified by their form, specific,
reactions, and optical characters. Add to the slide a saturated aqueous
solution of asparagine (about 0.5 g. in 29 cc. of water). The water-
insoluble needle crystals of tyrosine remain; water-soluble plates of leiiciiu^

crystals dissolve; potassium nitrate crystals, which have one right angle,
dissolve; asparagine crystals, which have a 51° acute angle and a 129°
obtuse angle, are not readily soluble but increase in size. Transfer i h(^
sections again to absolute alcohol, add a coverslip, allow the alcohol to
evaporate, then heat cautiously to 170°C. Leucine crystals sublime on
the coverslip; potassium nitrate crystals are unchanged; asparagine
crystals become transformed into foamy oil-like drops which are readily
soluble in water.
Arginine, Histidine. —The addition of a few drops of a dilute solu-
tion of picrolonic acid to fresh sections produces a yellow crystalline
precipitate.
Asparagine, Glutamine. —Quinone gives a red color to these two
amino acids: first crystallize the acids out of absolute alcohol then add 1
drop quinone solution. The asparagine crystals take a red color along
their boundaries, then dissolve slowly,and produce a red solution.
In order to distinguish between asparagine and glutamine, place the
sections in absolute alcohol, and dry for several days in a desiccator.
The glutamine crystals dissolve readily on the addition of water to
the sections.
A further test for asparagine may be carried out as follows: place
fresh sections in a drop of 7% aqueous cupric acetate on a slide. In
about 15 minutes begin adding traces of alcohol until ultramarine-blue
aphaerocrystals of copper asparagine appear.
Occurrence . —
Asparagine is of very widespread occurrence in plants;
it is easily demonstrated in etiolated seedlings of Lupinus and in tubers of
Dahlia,
 MICROCHEMICAL METHODS 185

Glutathione. —Heat thin sections of fresh tissue in dilute acetic acid.

Wash in a saturated solution of ammonium
sulphate, then place in a
watch glass containing about 5 cc. of this solution. Add 5 to 1 5 drops of
a 5% aqueous solution of sodium nitroprusside. Agitkte thoroughly for a
short time, add about 1 cc. ammonium hydroxide, and at the same time
watch the sections carefully. On the addition of the last reagent, a color
between pale pink and magenta will flash in the cells, lasting usually for
only a few seconds. The intensity and time of duration of the
color reaction vary, presumably with the amount of glutathione
present.
Leucine. —As crystallized out of absolute alcohol, crystals of leucine
are in the form of tliin plates arranged as rosettes, with angles of 70 and
1 10°. It commonly happens that leucine and tyrosine crystallize together
as needles ^and planes arranged in sphaerite form. If the sections are
covered with a coverslip and heated to 170°C., the leucine crystals sublime
on the coverslip.
Occurrence. —Seedlings of most Papilionaceae, and those of Cucumis
and Chenopodium; buds of Aesculus; tubers of Solarium tuberosum and
Dahlia.
—
Tyrosine. l"o sections crystallized out of absolute alcohol, add a
drop of Millon\s reagent. The tyrosine crystals are red at first but soon
dissolve into a red solution.
Place fresh sections in absolute alcohol on a slide, then after the
alcohol has evaporated, heat the sections in a few drops of a freshly
prepared solution of 0.01 g. sodium molybdate in 1 cc. concentrated
sulphuric acid. This dissolves the tyrosine crystals, giving a deep blue
color which soon turns violet.
With a solution of 2 drops acetaldehyde plus 1 drop concentrated
sulphuric acid, tyrosine gives a red color. When heated with a mixture
of approximately equal parts of formalin and sulphuric acid, a green color
is produced by tyrosine.

Occurrence. —
Shoots and tubers of Dahlia; very young Lupinus
seedlings.

CALLOSE
There are several color reactions distinguishing callose.
1. Permanent preparations of suitable small objects are easily made.

Place the material for 30 minutes in a freshly prepared and not too strong
aqueous solution of anilin blue. Transfer to a slide, add a small drop of
levulose sirup (10 g. levulose to 8 cc. warm distilled water) and cover
with a coverslip. After the sirup has evaporated slowly until it becomes
thickened, ring the coverslip as for glycerin mounts. Callose is stained
blue.
 186 GENERAL METHODS

2. may be employed in place of anilin blue.

Rosolic acid (corallin)
Use a 1% 4%
solution in aqueous sodium carbonate. A red color is
imparted to callose membranes.
3. Place the sections in a drop of a 1:2500 solution of resorcin blue.

After about 15 minutes, callose takes a brilliant blue stain. Transfer

to a drop of glycerin or levulose sirup on a slide for examination.
Callose, whose exact chemical nature is not yet definitely known, may
be distinguished from other membrane substances by its solubility
characters: (1) callose is insoluble, cellulose and the hemicelluloses
soluble, in copper oxide ammonia; (2) callose is soluble, cellulose and
chitin insoluble, in glycerin heated to 280°C.; (3) callose swells but does
not dissolve in the alkaline carbonates and in ammonia; pectic acid (or
pectic substances changed to the acid form) dissolves.
Callose is also readily soluble in 1% aqueous potassium^ or sodium
hydroxide and in calcium or stannous chloride solutions.

CARBOHYDRATES
Sugars. —Sucrose is the principal sugar stored in tissues, but other
sugars are also present, notably fructose and glucose. It is necessary
to ascertain whether such sugars are actually present before tests for
sucrose are made. The Fltickiger test may be employed for this purpose.
In a drop of 15 to 20% aqueous sodium hydroxide on a slide dissolve a
small quantity of copper tartrate, place the sections therein, and add a
coverslip. Fructose immediately gives a yellowish-red precipitate of
cuprous oxide. On gentle warming, glucose gives cuprous oxide crystals.
On heating the sections for about 20 minutes, dextrin causes the forma-
tion of cuprous oxide crystals. No precipitate is given by sucrose. Upon
the addition of 95% alcohol the fructose and glucose are dissolved, leav-
ing the insoluble dextrin in the tissues.
The osazone test, when properly performed, is the most satisfactory
test for sugars(Mangham 1911). Its only disadvantage lies in its com-
parative slowness. Make up the following solutions: (1) pulverize
phenylhydrazine hydrochloride in a mortar, then dissolve 1 part of the
powder in 10 parts glycerin, filter, and store in a brown bottle; (2) make
a solution of 1 part sodium acetate in 10 parts glycerin, and likewise
filter and store in a brown bottle. On the slide mix 1 drop of the phenyl-
hydrazine solution and 2 drops of the sodium acetate mixture, then
place the sections in the reagent, and add a coverslip. Avoid using too
much of the reagent. Keep at a temperature of about 20®C. In from
7 to 12 hours the formation of osazone crystals indicates the presence of
fructose; in two days glucose gives identical osazones. The difference
in the rate of crystallization makes it possible to distinguish between
fructose and glucose by the forms and general appearance of the crystals.
 MJCBOCHEMICAL METHODS 187

The appearance of yellow droplets in the cells indicates the beginning

of the reaction. In the case of glucose these droplets crystallize slowly
and produce dense, deep yellow to orange sphaeroclusters of needle
crystals. With fructose the clusters of crystals are less dense and clear
yellow in color. These distinctions are obtained only if there is more
than 1 % of the sugars present.
Maltose gives an osazone, which is soluble in 75 parts of boiling water
and can be crystallized from this solvent in rosettes of plates or broad
needles resembling sword blades. In the sections the osazone rosettes
are dense, lemon-yellow in color, and broader and larger than those
obtained with fructose and glucose.
Another procedure is to heat the slide carefully on a water bath for
15 minutes. The fructosazones are formed during the heating. Set the
slide aside to cool; the glucosazones form in about 30 minutes after
cooling.
Methylphenylhydrazine gives osazones with fructose and sorbose
only. Since the sorbose osazone is soluble, the appearance of osazone
crystals indicates the presence of fructose alone. Prepare the reagent
by putting 1 part methylphenylhydrazine and 10 parts glycerin in a
brown bottle, add acetic acid until the pH is about 4.7, and shake the
bottle at intervals for several hours until the mixture becomes homo-
geneous. Employ the reagent with sodium acetate as described for
phenylhydrazine hydrochloride above. The crystals appear in about
24 hours in the cold or in 15 minutes upon heating the slide on a water
bath.
Sucrose can be inverted by dilute acids, but since the acids would
also hydrolyzeany glucosides and hemicelluloses that might be present
in the tissues, this test is scarcely to be recommended. Instead, inver-
sion by means of the enzyme invertase is preferable. Invertin, a water-
soluble powder containing invertase, should first be prepared: mix
enough water with a cake of yeast to make a paste, keep at 40°C. for
12 hours, then press to extract the enzyme, filter, and precipitate with
95% alcohol. The resulting white powder is invertin. (1) Put sections
of the fresh material in a drop of invertin solution on a slide, and keep
at 40°C. for 3 hours or for a longer period if the tissues are somewhat
impenetrable, then test for glucose. (2) Put more sections in 5% citric

acid on a slide, heat for 10 minutes, then test for glucose. If much
glucose is found to accompany the sucrose, test for sucrose as follows:

perform the Fliickiger test (page 186) for glucose and fructose; wash the
sections with 5% tartaric acid; add warm magnesium chloride solution
to dissolve the cuprous oxide precipitate; wash again with the tartaric
acid, then test for glucose. The test should react negatively; if so, invert
the sucrose, and test.
 188 GENERAL METHODS

Amylodeztrin. —Amylodextrin is intermediate between maltose and

starch and is present in solution in storage organs where
commonly
starch is being hydrolyzed. It gives a red color with iodine-potassium
iodide solution.
—
Inulin. ‘Inulin occurs as sphaerocrystals in roots and tubers. Put
small pieces of fresh tissue in 70% ethyl alcohol for two to four days,
then cut off smaller portions, place on a slide in alcohol, add a eoverslip,
and examine. To bring out the concentric layers of the ciystals, add a
drop of chloral hydrate solution (5 parts hydrate to 2 parts water) to
the sections.

To identify the inulin crystals, locate some in the sections under the
microscope, and add a drop of a 15% solution of thymol in alcohol and a
drop of concentrated sulphuric acid. The crystals become carmine-red
immediately and soon pass into solution.
Inulin is readily hydrolyzed to fructose. Consequently, to show
typical crystals in the cells, a nonhydrolyzing killing fluid must be used
when it is desired to make permanent sections by the paraffin method.
Occurrence, —
Tubers of Dahlia (in autumn); in Taraxacum, Helian-
thus tuberosus, Allium, Galanthus, Leucojum, etc.
Starch. —The familiar iodine test is still the most satisfactoiy one,
even if other substances also give a blue color reaction with iodine.
Use a weak solution (0.3 g. iodine, 1.5 g. potassium iodide, 100 cc. water).
The blue color disappears on heating but returns on cooling. Small
starch grains may be better observed if the chlorophyll is removed from
 MICROCHEMICAL METHODS 189

the sections with alcohol before applying the iodine. The grains will
appear black. Heat the slide, then place under the microscope, and
watch for the return of the color.
Different types of starch may
be distinguished by their behavior
under polarized appearance of potato starch in Fig. 23).
light (note the
—
Glycogen. Glycogen occurs in animal tissues and in certain fungi,
such as Saccharomyces cerevisiaey in Myxothallophyta and in the Cyano-
phyta. With iodine solution, glycogen gives colors which vary according
to the amount present. If a definite iodine solution (0.1 g. iodine, 0.3 g.
potassium iodide, 45 cc. water) is used, a fair determination of the relative
amount of glycogen present can be made. A brown, orange, or yellow
color indicates traces of glycogen, while considerable amounts give a
wine-red color. Glycogen is soluble in Water and will soon disappear
if the tissues are the reagent.
left in
Glycogen may
be coagulated and then stained with any basic coal-tar
dye {e,g.y safranin, methylene blue, or Bismarck brown). Put the
material in strong alcohol for about 15 minut(‘S, then transfer directly
to a 10% aqu(‘ 0us solution of tannin. After 10 minut(‘S transfer the
material directly to 1 %
aqueous potassium bichromate for a few minutes,
then to 10% aqueous potassium bichromate for 10 minutes. This pro-
cedure renders the glycogen insoluble in water.

CELLULOSE
There are two kinds of cellulose, vf 2;.,.true celluloses and hemicelluloses,
that are readily distinguished by the fact that the true cellulos(\s are
not hydrolyzed by aci^j^^whereas the hemicelluloses are hydrolyzed by
both 3% and concentrated (sp. gr. 1.19) hydrochloric acid and by 3%
sulphuric acid. (Hydrochloric acid of a specific gravity other than
the one noted dissolves cellulose immediately.)
Cellulose is hydrolyzed by 70 to 75% sulphuric acid into a colloid
substance, hydrocellulose, which forms a blue adsorption compound
with iodine solution. Place sections in a drop of iodine solution (0.3 g.
iodine, 1.5 g. potassium iodide, 100 cc. water) on a slide, cover, and
observe the localization of the blue color reaction. Allow a drop of
75% sulphuric acid to diffuse in from one side of the coverslip, and watch
the swelling of the cellulose membranes. A few other plant substances
also give a blue color reaction, consequently the localization of any blue
color which appears before the addition of the acid should be noted.
Hemicelluloses are discussed below.

CHITIN
In an open beaker heat 100 cc. of a saturated aqueous solution of
potassium hydroxide to boiling. Put the sections in the boiling solution,
 190 GENERAL METHODS

cover the beaker, and continue the boiling for 20 to 30 minutes. Remove
the sections, and wash in 90% alcohol. The chitin has been transformed
into chitosan. Test for the latter with a weak iodine solution (0.2 g.
iodine, 2 g. potassium iodide to 100 cc. water) a red-violet color demon-
:

strates the presence of chitin.

Occurrence .
—Principally in the higher fungi, but also in a few of the
lower; however, there is some question whether the substance actually
is chitin.

CUTIN, SUBERIN

A IV in 70% alcohol imparts a red color

saturated solution of Sudan
to all Leave the fresh sections in the stain for 20 min-
fatty substances.
utes, wash with 50% alcohol to remove excess stain and transfer to a
drop of glycerin for observation. Fats may be distinguished from the
other colored substances by the fact that they exist as drops within the
cells. Fuelgen^s reaction also distinguishes cutin, suberin, and lignified
elements (Margolena 1932).
To differentiate suberized and cutinized cell walls from cellulose
membranes, concentrated sulphuric acid or copper oxide-ammonia may
be used; these fluids dissolve the cellulose.
All membrane substances except cutin, suberin, and chitin dissolve
quickly in the cold in 50% chromic acid; however, if the acid is heated, it
membranes after a time.
will dissolve all
The fact that suberin contains phellic acid and cutin does not provides
the method of differentiating between the two substances. After several
hours^ maceration of the sections in concentrated potassium hydroxide,
suberin and also lignin become yellow. Upon slight warming, the layers
of suberin swell, and the yellow color becomes darker. Raise the tem-
perature to boiling: granular masses of potassium phellonate, which
become larger as the heating is continued, appear. Let cool, wash thor-
oughly with water, and add a drop of chlorzinc-iodide. The potassium
phellonate acquires a reddish-violet color.

ENZYMES
There are but few microchemical localization tests for enzymes, and
these are not always either satisfactory or conclusive. Fairly good tests
for proteases, oxidases, peroxidases, and catalase can, however, be
obtained.
Oxidases. —These enzymes are probably universally distributed in
plants. Place sections in a drop of 1% benzidine in 60% alcohol. The
reaction will appear in about 16 minutes. If the tissue is acid, oxidase-
cont^aining cells turn blue; if the sections are alkaline, the color is brown.
 MICROCHEMICAL METHODS 191

A second test for oxidases is to place the sections in a drop of a 10%

solution of guaiaconic acid in strong alcohol. On oxidation there appears
a blue to blue-black precipitate, insoluble in water, but soluble in chloro-
form, ether, and benzol. The presence of tannins or weak acids retards
the reaction.
—
Peroxidases. The two reagents described as tests for oxidases are
oxidized by peroxidases only if hydrogen peroxide is added.
Ursol tartrate may be used as a reagent. Prepare it by mixing a
saturated alcoholic solution of ursol with a saturated aqueous solution of
remove the precipitate, wash it with alcohol, and
tartaric acid; filter to
from ether. On a slide put 1 drop of this reagent, add 1
recrystallize
drop of hydrogen peroxide, and place the sections in the mixture. The
immediate appearance of a green color, which changes to blue and finally
to gray, indicates the presence of peroxidases.
Catalase. — The prompt evolution of gas bubbles when hydrogen
peroxide is added to sections in a drop of 1 % gum arabic or gelatin solu-
tion demonstrates that catalase releases oxygen from the peroxide.

FATS
True always readily distinguished by their affinity for the
fats are
specific dyes, Sudan
III and IV. The majority of lipoids do not take up
these dyes, nor are they absorbed by vacuoles. To distinguish fat
globules from vacuoles, neutral red dye, which is taken in by the latter
but not by the former, may be used.
The stain solution is prepared by dissolving 0.5 g. of the dye in 100 cc.
of 70% alcohol. Leave the sections in the stain for 20 minutes, wash
carefully but quickly with 50% alcohol, and transfer to glycerin for
observation.

GLUCOSIDES
—
Amygdalin. Tests for this substance are based upon the production
of hydrocyanic acid during its decomposition.
Guignard^s test is one of the easiest. Immerse the sections in 1%
aqueous picric acid for 30 minutes, wash with water, and place in 1 drop
of 10% aqueous sodium carbonate on a slide. A red color appears if
hydrocyanic acid is released.
If the test is made quickly, the Berlin blue reaction is fairly good.
Place the sections in a potassium hydroxide solution composed of 20
parts of 20% aqueous potassium hydroxide and 80 parts 90% alcohol, for
a few minutes. In a small watch glass mix equal parts of a 2.5% aqueous
ferrous sulphate solution and 20% aqueous ferric chloride solution, and
heat to boiling. Transfer the sections to this mixture; after 5 to 10
minutes, transfer the sections to a slide holding a drop of 20% hydro-
192 GENERAL METHODS

chloric acid. A deep blue precipitate indicates the presence of hydro-

cyanic acid.
A test that requires more skill in manipulation and interpretation is

the mercurous nitrate reaction. Place the sections on a slide in a few

drops of 3% aqueous mercurous nitrate for 1 wash in dis-
to 2 minutes,
tilled water, and place in glycerin for observation. The performance
must be carried out rapidly, and it would be well to moisten scalpel and
object with the reagent. If the sections are heated, other substances will
give the reaction.
Occurrence—Seeds . of Amygdalus, Pyrus, Crataegus^ and related
genera; leaves of Primus laurocerasus.
Anthocyanin. — The color of most r(‘d and blue flowers is due to
anthocyanin. If petals of such a flower are held in ammonia vapor for a
minute, the color changes to green. Anthocyanin is red in acid solutions,
blue violet when there are traces of alkali, and green in alkaline solutions.
Put the petals or portions of su(^h in a drop of water under a coverslip
on a slide, and crush out the colored liquid. Some anthocyanins form
liquid globules on evaporation; others produce crystals. Or the petals
may be placed in a small drop of glacial acetic acid or 10% hydrochloric,
acid and the acid (which extracts the anthocyanin) evaporated slowly in
a moist chamber. The anthocyanin forms various sorts of red crystals.
Arbutin. —
Place sections in 10% nitric acid. A dark orange color
immediately appears in cells containing arbutin, but it soon changes to
yellow and slowly disappears.
Arbutin on hydrolysis is transformed into glucose and hydroquinone.
Heat the sections, placed dry or in a drop of water on a slide; the arbutin
sublimes in crystals. The latter become red-brown upon the addition of
ammonia, or pale green with ferric chloride solution.
Occurrence —Ericaceae and Pyrolaceae.
.

Saponin. —Sections placed directly in 1 drop concentrated sulphuric

acid on a slide exhibit a characteristic sequence of color reactions, begin-
ning immediately with yellow, changing to red within 30 minutes, and
finally becoming violet, or blue-green in a few instances. This test is
merely an indication of the presence of some type of saponin. To deter-
mine the localization of the saponin, put the sections in a saturated solu-
tion of barium hydroxide for about 24 hours. A practically insoluble,
compound^is formed by the barium and saponin. Wash the
colorless
weak aqueous solution of calcium chloride, then place in 10%
sections in a
aqueous potassium bichromate. The compound first formed is broken
down, and the barium unites with the chromium to form barium chro-
mate, yellow in color. Tannin-containing cells become brownish-red
during the reaction. ^
Occurrence —
Roots of Saponaria^ hoQthoe {Chlorogalum)^
.
etc.
 MICROCHEMICAL METHODS 193

Tannin. —
The word tannin does not denote a singles substance but is a
generic name covering a whole group of substances having certain charac-
teristics in common. They are mostly colloidal substances and occur
either in solution in the cell sap or not infrequently in distinct vacuoles.
The amount of tannin present varies with the state of growth, time of
year, physiological condition, and other factors; but in some organs, such
as root tips of Finns, the variations are within rather narrow limits
(McNair 1930).
Tissues, such as root tips, suspected of containing tannin localized in
vacuoles or circumscribed cell areas, may be fixed in an aqueous solution
containing 3 to 5% formalin and 10% ferrous sulphate for 24 to 48 hours,
washed, dehydrated, sectioned, the paraffin removed, and balsam and a
coverslip applied. The iron compound both fixes and stains the tannin.
If sections of fresh tissue are placed in 10% aqueous ferric chloride'
plus a little, sodium carbonate, a blue-green color is given by tannins.
Tannins are readily oxidized. In many types of prepared slides tlu‘
presence of dark red or reddish-brown, more or less isolated cells indicates
the occurrence of phlobaphene, which results from the oxidation of
tannins.
Occurrence .
—Tannin is rather commonly present. All parts of
Quercus spp., the Crassulaceae, and the Coniferophyta readily react for
its presence.

HEMICELLULOSES
Hemicelluloses are divided into two groups: (A) those entering into
the constitution of permanent cell walls, and (B) those occurring in

storage organs and which are consumed during growth. The distinction
is rather weak, since the same substance can occur in both groups. No
really distinctive localization reactions have been reported for the
hemicelluloses.

A. Skeleton Hemicelluloses Occur Chiefly in Woody Cell Walls and in

Seed Coats.
Galactase . —There no really satisfactory
is test for this substance.
Araban, Xylan —The phloroglucin-hydrochloric
. acid test may be'
carried out. Place sections in a drop of 1% phloroglucin solution (0.1 g.
phloroglucin, 10 cc. 95% alcohol) on a slide, add 1 drop hydrochloric acid,
and observe. Lignin gives an immediate violet-red color. Heat cau-
tiously for about 10 minutes to hydrolyze the xylan to the pentose xylose,
which takes a cherry-red color.
Methyl Pentoses .
—
Place the sections in 1 or 2 drops acetone on a
slide, add 1 drop concentrated hydrochloric acid, and warm gently for 15

minutes. The appearance of a red or violet color indicates the presence

 194 GENERAL METHODS

of methyl pentosans (rhamnose, fucose, quinovose, etc.)* The methyl

pentoses occur associated with pentoses as cell-wall constituents. The
pentoses also give a violet color by the above reaction, but this color fades
within 1 hour.

B. Storage or Reserve Hemicelluloses Are Found Principally in the Cell

Walls of Endosperm and in Very Young Bast Fibers.

Amyloid —
This occurs mostly in fatty seeds. It gives a blue color
,

with iodine solution: by adding 2% hydrochloric acid and warming, the

intensity of the reaction is increased.
Mannan , —Mannan is of somewhat widespread occurrence. Hydro-
lyze rather thin sections in a small watch glass in 2% hydrochloric acid
for 30 minutes or slightly longer.Transfer the sections to a slide, neutral-
ize with a few drops of 2% ammonia, then test for mannose as described
for the osazone reaction for glucose. Colorless plate-like crystals of
mannose hydrazone should appear within a short time.
Galactan .
—
This reserve sugar usually occurs accompanied by other
sugars. If mannose or amyloid is found to be present, galactan is pre-
sumably also present.

LIGNIN
Sections are placed on a slide in a large drop of a solution of 0.1 g.
phloroglucin in 10 cc. of 95% alcohol and covered with a coverslip. Allow
part of the solution to evaporate, then let a little 25% hydrochloric acid
diffuse in at the edge of the coverslip. The appearance of a red-violet
color indicates the presence of lignin.
To a 0.001% aqueous solution of methyl red, add just enough alkali to
render it yellow. A permanent color resembling that imparted by
phloroglucin is acquired by lignified cell walls in either fresh or paraffin
sections.
To test for the lignin oxide present in the walls of young bast fibers:
cover the sections on a slide with 1% neutral aqueous potassium per-
manganate for 15 to 20 minutes (the Maule test). Manganese dioxide is
deposited on the woody tissues. Wash thoroughly, and place in 2%
hydrochloric acid (sp. gr. 1.06). The acid reacts with the manganese to
produce chlorine which in turn produces a chlorination. After the
dioxide has been dissolved, wash the sections thoroughly with distilled
water. Add a few drops of either ammonium hydroxide or sodium
bicarbonate solution. A deep red color develops in the lignified elements
of deciduous plants, an indefinite brown in those of coniferous trees. This
test is said to afford a means of distinguishing between deciduous and
coniferous (except Ginkgo) woods.
 MICROCHEMICAL METHODS 195

Solubility tests may be used to distinguish lignified membranes from

other membrane substances:
1. Suberin, cutin, and chitin are insoluble in 50% chromic acid;
lignin and all other membrane substances are dissolved.
2. Lignin is not soluble, cellulose and the hemicelluloses are soluble, in

copper oxidfe-ammonia.
Most oxidizing agents, such as hydrogen peroxide, potassium chlorate,
nitric acid, etc., also dissolve lignin from membranes.

LIPOIDS
Lecithin. —The same dyes, Sudan III and IV, for which fats have a
marked affinity, are also taken up by lecithin. It is therefore necessary
first to rid the sections of fats by placing them in a vial of acetone for
about 12 hours. The sections may then be stained with Sudan IV or
with 1% aqueous osmic acid. With the latter reagent, the lecithin
acquires a light brown color.
Ph3rtosterol.— Thick sections of tissue are placed in concentrated
sulphuric acid: they first show a red color, then the phytosterol dissolves

into a foamy mass whose particles turn olive green in 3 or 4 hours and
finally become colorless. Or the tissues may first be placed in chloroform
and the sulphuric acid then added: the phytosterol is first rose-red and
finally brown. A second reaction is to add a trace of iodine-potassium
iodide solution after the sections have been placed in concentrated sul-
phuric acid. The color at first is red, then after the addition of the
iodine solution it changes to violet, next to blue, and finally to yellowish-
red or brown. A third method is to place the sections in strong tri-
chloracetic acid (9 parts acid to 1 part water), heating slightly or adding
a little hydrochloric acid: the resulting color is violet or reddish-purple.

MINERAL SUBSTANCES
—
Calcium. The most sensitive test is the formation of calcium oxalate.
Place the sections on a slide in an excess of 2% aqueous oxalic acid.
Leave uncovered. After about 30 minutes, withdraw some of the acid,
add a coverslip, and very carefully place 1 drop alcohol at one edge of the
coverslip. Very small but readily recognizable crystals of calcium
oxalate will indicate the presence of calcium.
When it is often accompanied
iron occurs in plants, by calcium.
Fixing fluids containingmight dissolve calcium
acids compounds;
formalin should also be avoided. Calcium is stained by hematoxylin
and has an affinity for silver and otHer metals. The latter fact permits
the use of a simple method involving the reduction of silver nitrate.
Bring fresh sections from distilled water into a weak solution of silver
nitrtCte in distilled water, and leave for 5 minutes. Wash with distilled
 196 GENERAL METHODS

water, then transfer to a weak solution of a photographic developer such

as pyrogallol, where the sections may remain until the calcium-containing
portions become blackened. Then wash out with absolute alcohol, and
pass through xylol into balsam. Keep such slides in the dark.
—
Calcium Oxalate. Small pieces of tissue or sections are treated with
a saturated aqueous solution of cupric acetate. The crystals of calcium
oxalate, if present, dissolve, and the oxalic acid diffuses into the inter-
cellular spaces where cupric oxalate crystals are formed. To test for
the dissolved oxalate, a solution of ferric sulphate (made by adding 5 g.
ferric sulphate and 20 cc. acetic acid to 80 cc. water) may be used, result-
ing in the formation of yellow crystals of ferrous sulphate.
—
Magnesium. If th(^ cells arc presumed to contain both magnesium
and a phosphate, the addition of a little concentrated ammonia to the
sections in 1 drop of water on a slide will cause the appearance of crystals
of ammonium-magnesium phosphate. Or if phosphates are absent, one
may be added in the form of sodium phosphate. Prepare the reagent
by adding a few drops of free ammonia to a saturated aqueous solution of
ammonium chloride, then sufficient sodium phosphate to make a 0.1%
solution. Put the fresh sections in 1 drop of this reagent, and warm the
slide a little. In about 10 minutes add a coverslip, and examine.
Occurrence.— Magnesium is found in the meristematic tissues of most
plants and in latex (Ficus, Euphorbia) and resinous secretions (Pinus).
Potassium. — Sectionsof tissue, which should be neutral or slightly
acid, may
be placed directly in a 10% aqueous (some workers prefer an
alcoholic) solution of platinum chloride: yellow octahedron crystals of
potassium chloroplatinate appear. If ammonium is present, similar
crystals of ammonium chloroplatinate will be formed.
A second indicator of the presence of potassium is by the formation
of crystals of potassium-cobalt nitrite. The reagent is prepared by dis-
solving 20 g. cobalt nitrate and 35 g. sodium nitrite in 65 cc. water plus 10
cc. glacial acetic acid. A precipitate of fine yellow crystals appears if

potassium is present.
Occurrence r — Potassium is found in the cytoplasm of most plants; it
is absent in nuclei and chloroplasts. Favorable objects are the stem
apices and storage tissues of Daucus carota, Solanum tuberosum, and Beta
vulgaris.
—
Sodium. Place the sections in a saturated aqueous solution of
uranium acetate on a slide, add a small drop of hydrochloric acid, then
place the slide, without a coverslip, in a desiccator so that the fluids
may evaporate slowly. In two to eight hours pale yellow tetrahedral
and rhomboidal crystals of sodium-uranium acetate appear. If mag-
nesium should be present, this will be indicated by the formation of large
rhomboidal crystals of uranium-magnesium-sodium acetate.
 —

MICROCHEMICAL METHODS 197

Occurrence, —Sodium is abundant in marine and strand plants and

also in many cultivated plants. Among the latter are Solanum tuberosum^
Spinacia oleracea^ Brassica oleracea and B. rapa.
Amm onium. The least complicated test is to observe the formation
of crystals of ammonium-magnesium phosphate. The sections may be
placed in a solution of sodium phosphate and magnesium chloride slightly
alkalinized with sodium hydroxide.
Occurrence, —
Stems of Helianthus tuherosus; bulb scales of Allium
cepa; leaves of Tradescantia,
Iodine. —Tests for iodine are indirect, and all are based upon the

ability of iodine to color starch grains blue. Iodine occurs in the Phaeo-
phyta, particularly in Laminaria, Place fresh sections in a small watch
glass with about 2 cc. of a 1% aqueous suspension of potato starch flour,
together with 1 to 3 drops of a 20% aqueous solution of potassium nitrite
and 1 to 3 drops of 5% hydrochloric acid. The starch becomes colored
blue if iodine is present.
Iron. —In manipulating tissues to be tested for iron, take extreme
care to avoid contacts with iron or steel instruments. If a scalpel or knife
isused for cutting sections, clean it very thoroughly; use glass needles for
handling the sections after they have been cut.
Have the sections in water; immerse for 5 minutes in 1% aqueous
hydrochloric acid, to each 25 cc. of which is added 3 drops of a freshly
prepared conc.eiitrated aqueous solution of potassium ferrocyanide (some
workers advise using 8 to 10 drops to each 25 cc. of a 2% solution of the
acid, for 30 minutes to 1 hour). Wash out the acid with distilled water,
and stain the nuclei with alum carmin. Wash out excess dye, mount, and
examine. The sections can be dehydrated and mounted in balsam if

desired. Any iron present should be stained a deep blue, the nuclei red.
Or immerse the sections in a slightly old (yellowish) solution of
ammonium sulphate for 5 to 30 minutes or until the sections become dark
green. Wash out quickly, then transfer to glycerin to which a litth?
ammonium sulphate has been added, and examine. If satisfactory, wash
out with absolute alcohol, pass through xylol, and mount in balsam.
Carmin may be used as a counterstain.
Occurrence, —
The capsules of the iron bacteria are saturated with iron.
This element is also present in many lichens and in the seeds of Sinapis
alba.
Manganese. —Oxalic acid combines with manganese to form man-
ganous oxalate a prompt reaction does not take place, let
crystals. If
the sections dry, then touch a speck of potassium oxalate to them.
Immediate crystal formation should take place.
Occurrence, — Manganese is especially abundant in the cortex and wood
of the Coniferales and in the epidermis of aquatic plants.
198 OENERAL METHODS

Silicon. —Place the sections on a slide, then, without adding any liquids
cover them with a crystal of phenol, and heat until a red color appears
wherever silicon occurs. If it is desired to preserve the sections, clear
with clove oil, and mount in balsam.
Silicon crystals frequently stain with methylene blue and crystal
violet.
Occurrence. — Silicon is especially abundant in diatoms, in the Equi-
setums, Poaceae, and Cyperaceae; and occurs in the form of crystals in
the epidermal cells of the Orchidaceac and Palmaceae.
Nitrates. —Cover the sections with a coverslip, and allow diphenyl-
amine (0.1 g. diphenylamine in 10 cc. 75% sulphuric acid) to run in from
one side of the coverslip. A deep blue color indicates the presence of
nitrates; as the sulphuric acid begins to disintegrate the tissues, the color
changes to a yellowish-brown.
Occurrence. —
Plants growing in waste places and around rubbish
heaps generally are rich in nitrates. Such are to be found among the
following genera: Chenopodium^ Urtica^ Mercurialis^ Solanum, Helianthus.
Zea mays and Cucurbita pepo are also favorable plants.
—
Phosphates. If both magnesium and phosphate are present, the
addition of ammonia causes the formation of crystals of ammonium-
magnesium phosphate. If magnesium is presumably absent, treat the
sections with 1 drop of the following reagent: to 15 cc. water add 25 cc.
of a saturated aqueous solution of magnesium sulphate and 2 cc. of a
saturated solution of ammonium chloride. Ammonium-magnesium
phosphate crystals should form.
Another test is to place the sections on a slide in a drop of a solution
of 1 g. ammonium molybdate in 12 cc. nitric acid. The presence of
phosphates is indicated by the appearance of small yellow black-bordered
drops, which turn into sphaerocrystals, then into cubes and octahedrons.
—Parenchyma
Occurrence. the most favorable being those
of leaves,
of AesculuSy Allium and Ranunculus.
cepa,
Sulphur and Sulphates. — Sulphur occurs in plants principally in the
bound form but is found free in some of the lower plants.
Any of the sulphur bacteria (such as Beggiatoa, or Oscillatoria among
the Cyanophyta) may be mounted in glycerin and the grains of sulphur
are usually visible. This type of sulphur occurs free. Free sulphur is
insoluble in weak acetic acid, weak hydrochloric acid, 1 % chromic acid,
saturated aqueous solutions of picric acid, concentrated sulphuric acid,
and potassium hydroxide; it is soluble in absolute
glycerin, nitric acid,
alcohol, chloroform,and partially so in carbon bisulphide.
Organically bound sulphur occurs in raeristematic tissues. Place
sections in 10% potassium hydroxide, then add 1 or 2 drops of a fresh
 MICROCHEMICAL METHODS 199

10% aqueous solution of sodium nitroprusside; a red color reveals the

presence of sulphur.
Sections to be tested for sulphates may be placed in a 1% solution of
benzidine chloride in 3% hydrochloric acid. Small colorless needles or
glistening scale-like crystals of benzidine sulphate may appear.
Oil- or fat-containing tissues, such as those of cereal seeds, should
first be treated with ether to remove the fats. After the sections have
dried, add 1 drop of 10% hydrochloric acid, then 1 drop of 10% barium
chloride. A granular precipitate of barium sulphate is brought into
being, but it may be somewhat difficult to observe.
—
Carbonates. Add a saturated solution of strontium acetate to the
sections on a slide, then place the latter in a moist chamber for half a
day or longer, since the reaction proceeds slowly.
Chlorides. —
Put several sections, cut with a scrupulously clean scalpel,
in a drop of 5% aqueous silver nitrate on a slide. Examine microscopi-
cally without covering; the precipitate of silver chloride, which seems to be
white to the unaided eye, appears black under the microscope. Transfer
some of the sections, by means of glass needles, to another slide carrying
a drop of 1.5% aqueous nitric acid, add a coverslip, and examine. The
acid clears the sections sufficiently to permit loc^alization of the reactions.
To the remaining sections in the nitrate solution, add ammonia slowly
until the precipitate becomes dissolved. Set aside: in about 1 hour the
precipitate will reappear in the c.rystalline form. The crystals acquire a
deep violet color on reduction.
The silver chloride crystals are stained brilliantly with methylene
blue, Bismarck brown, and eosin.
Thallium sulphate or thallium acetate (1 g. in 15 cc. water and 2 cc.
glycerin) will develop gray or black crystals of thallium chloride in
sections.
Occurrence. —
Roots of Daucus carota and Beta; Solanum; stems of
Primula obconica.

ORGANIC ACIDS
Formic Acid. —Sections may be placed in a few drops of mercuric
chloride solution (1 part of a concentrated solution diluted with 5 parts
water), heated on a water bath for 1 hour, and washed with water
acidified with 1 drop of hydrochloric acid. Then transfer the sections
to 1 drop of 1% potassium hydroxide on a slide. Cells containing
formic acid become blackened.
Occurrence, —^Leaves Abies or Sempervivum;
of cell sap of Urtica.
Oxalic Acid. —Treat sections with a solution of 1 part ferrous phos-
phate in 8 parts of phosphoric acid; an intense yellow color indicates the
presence of oxalic acid. If a considerable quantity of the acid is present.
 —

200 GENERAL METHODS

crystals of ferrous oxalate are formed. If calcium oxalate crystals are

also present, first dissolve them in dilute hydrochloric acid, then add
the ferrous salt.

With manganous nitrate, crystals of manganous oxalate in the form

of six-pointed stars are formed.
Occurrence .
OxaliSy Begoniay Mesembryanthemum, Rheunij RumeXj
SalsoUiy and Salicornia.
Citric Acid. —Sections presumed to containacid may be neutral-
citric

ized with sodium hydroxide and heated in a 5 %

aqueous calcium chloride
solution. A crystalline precipitate of calcium citrate is produced. This
precipitate is soluble in acetic acid, which distinguishes it from calcium
oxalate; it is insoluble in water, thus distinguishing it from the oxalate
and tartrate of calcium.
Calcium citrate crystals can frequently be found in small pieces of
tissue that have been preserved for a long time in alcohol or glycerin.
Malic Acid. —Sublimation of malic acid into maleic acid and maleic
acid anhydride is claimed to be the best reaction for the acid. This
must be carried out at a very high temperature in order to avoid carboni-
zation of the tissues. The crystals will appear in about 24 hours after
sublimation.
If there is much of the acid present, it will crystallize out when the
sections are dried; or if put into 70% alcohol, the crystals appear on the
surface.
—
Tartaric Acid. Sections of tissue treated with a 4% aqueous solution
of any ferrous salt and a few drops of hydrogen peroxide, or 10% potassium
permanganate with the addition of an excess of sodium hydroxide solu-
tion, after a few minutes give a violet color which is produced only by
tartaric acid.
Large crystals of calcium tartrate, which recrystallize from hot solu-
produced when the sections are heated in 20% calcium acetate
tions, are
or 10% calcium chloride.

PECTIC SUBSTANCES
Ruthenium red is the classical dye indicator for pectic substances.

It is a very expensive dye, and its solutions are quite unstable. Make
up only a small quantity of solution as needed: to two or three tiny
ciystals of the dye in a watch glass add distilled water drop by drop
until the solution is a clear reddish-pink in color. Leave the sections
in the ruthenium red solution for 30 minutes, then wash thoroughly, and
mount in glycerin on a slide. All pectic substances acquire a red color.
A 1:10,000 solution of methylene blue likewise stains all pectic
substances, giving a violet color. This color reaction is not so precise
as that given by ruthenium red, since other membrane substances are
 MICROCHEMICAL METHODS 201

also stained. For example, cellulose is usually stained blue, lignin and
some of the hemicelluloses green.
The general procedure of testing sections for pectic substances is as
follow^:
1. Stain with ruthenium red (or methylem^ blue); note localization
of pectic substances. The pectine becomes dissolved in the water of the
reagent.
2. Either heat carefully in 2% aqueous hydrochloric acid for 20 min-
utes, or leave in a solution of 1 part hydrochloric acid to 4 parts alcohol
for 12 hours. transformed into pectine, which dissolves, or
Pectose is

into insoluble pectic acid. (The conditions which determine whether

pectose is to be changed into pectine or pectic acid are not understood.)
Calcium pectate is also broken down, resulting in the formation of calcium
chloride and pectic acid.
3. Wash thoroughly with water. Of all the pectic substances
originally present, pectic; acid is the only one left. Stain the sections
again, and note localization of reaction.
4. Wash in 2% ammonia. This dissolv(\s the pectic acid.
5. After 30 minutes wash in wat(*r, and stain once more. There
should be no reaction for pectic substances.

PIGMENTS
Carotin. —Place fresh pieces of young green leaves in a solution of
20 g.potassium hydroxide in 80 cc. 95% alcohol in a closed vessel kept
in the dark until the chlorophyll is extracted. Remove the sections, wash
for about 12 hours in numerous changes of distilled water, then mount in
glycerin on a slide. Orange-red crystals of carotin and ycdlow crystals of
xanthophyll will appear in a day or two.
If older leaves are used, the carotin probably will not crystallize,
because of the heavier oil content in such leaves; the carotin dissolves
in the oil and appears as yellow droplets.
Carotin and xanthophyll both dissolve and become blue when treated
with concentrated sulphuric acid.
Crystals of carotin are soluble in ether, chloroform, xylol, and benzol
but are insoluble in water and dilute alkalies and acids.
Chlorophyll. —
Put the sections in 1 drop of ether on a slide, and add
a little of a mixture of 20 g. potassium hydroxide in 80 cc. of strong methyl
alcohol. The chlorophyll immediately turns brown, then after a while
changes back to green.
Or place the sections in a very small drop of 25% hydrochloric or
glacial acetic acid on a slide. Large yellowish-green drops ooze out of
the chloroplasts within a few minutes and in about hour long curved
brown crystals of chlorophyllan appear in the pells, Heat the preparation
202 GENERAL METHODS

slowly to about 90®C., whereupon the chlorophyllan is recrystallized

into clusters of straight needles. These crystals are soluble in ether or
chloroform.
—
Xanthophyll. Place fresh sections in 1 drop of chloroform on § slide.
In a few minutes add 1 drop of petroleum ether. The insoluble xantho-
phyll crystallizes out, while the carotin, which is soluble in ether, remains
uncrystallized. ^

PROTEINS
Proteins. — Most of the tests recommended for the detection of
proteins are unsatisfactory because other substances simultaneously
react. With such reactions confirmatory tests should also be made.
A saturated aqueous solution of picric acid is an excellent precipitat-
ing agent for proteins, staining them an intense yellow. Allow the
reagent to react for 24 hours, then mount the sections in glycerin for
examination.
Extremely dilute solutions of eosin, reacting for about 10 minutes,
stain proteins red.
A fair localization reaction is given by potassium ferrocyanide.
Make up the reagent by adding 1 g. of the cyanide to 20 cc. water and
100 cc. glacial acetic acid (sp. gr. 1.063). Leave the sections in this
solution for 1 hour, during which time the proteins will be fixed, then
wash briefly with 60% alcohol, and add a few drops of aqueous ferric^

chloride. A blue color results.

Aleurone. —Aleurone grains are made up of protein crystals and
globules in a protein matrix. Saturate absolute alcohol with picric acid
and nigrosin dye. Place the sections on a slide in a few drops of this
reagent, and observe under the microscope. When the ground substance
of the grains appears blue, stop the reaction by adding absolute alcohol.
The globules should be colorlessand the crystals yellowish-green. The
sections can be cleared with clove oil and mounted in balsam if per-
manent mounts are desired. In place of the nigrosin, eosin may be
employed. With this dye the ground substance becomes dark red, the
globules slightly tinted red, and the crystals yellow.
Occurrence .
—Seeds of the Poaceae.

SECRETIONS
k

Ethereal Oils. —The ethereal oils resemble the fatty oils in that they
stain with the usual fat stains, are blackened by osmic and are acid,
dissolved by fat solvents. The ethereal oils are odorous and volatile,
while the fatty: oils are not. If the sections are placed on a slide and
covered and if; either glacial acetic acid or chloral hydrate solution is run
under the cover, the ethereal oils are easily dissolved, but the fats gener-
 MICROCHEMICAL METHODS 203

ally remain unaffected. Sections, uncovered, may be placed on a slide

and heated over a water bath for about 10 minutes: the ethereal oils
evaporate, and the fatty oils remain.
—
Resins. Resins, found in secretory passages and heartwood, are
divided into seven groups (McNair 1930) which are of only theoretical
interest to the technician.
Thin fresh sections may
be placed in a watch glass, covered with
7% aqueous cupric and allowed to stand for from one to six days
acetate,
until a greenish resin-copper precipitate appears. It would be desirable
first to remove ethereal oils from the sections by distillation. Tests for
fatty oils should also be performed.
—
Aromatic Acids. The aromatic acids, which occur in secretions,
resins, etc., can scarcely be identified by localization reactions. They
are detected mostly by the appearance of the crystals resulting from
sublimation. Benzoic acid sublimes out as feathery clusters of crystals,
soluble in sodium hydroxide. Cinnamic acid gives thin plate crystals.
Ferulic acid crystals are in the form of feathery rosettes and brush-like
clusters at the ends of narrow plates.
—
Gums. Gums, as they occur in plants, are mixtures of various sub-
stances, consequently there are no microchemical tests for gums as such
but only for the constituent substances.
Four distinct classes of gums are recognized (McNair 1930): (1)
those containing arabin or arabic acid, (2) those consisting of mixtures
of arabin and cerasin (cerasic acid), (3) those containing bassorin, and
(4)those containing mixtures of cerasin and bassorin.
—
Mucilages. In some plants, such as the marine algae, mucilage is
very abundant, and in many others it is of constant occurrence. No
satisfactory tests are on record, probably because mucilag(\s include a
number of chemically distinct substances, but the technician when work-
ing with fresh tissues can generally recpgnize mucilage by its slimy
character.
Occurrence .
—Most of the larger Phaeophyta; seeds of Linuin and
Cydonia.

WAXES
Microchemical tests for waxes are the same as those cited for fats.
Waxes are soluble in all ordinary fat solvents, but somewhat less so than
the fats.
Most waxes are to be found on the epidermis or in the cuticles of
leaves and stems. Place sections on a dry slide, cover with a coverslip,
and run in ether from one side of the cover. Allow the ether to evaporate
slowly. Needles, plates, and aggregates of crystals will appear after the
ether has evaporated.
 CHAPTER XVI
SOURCES OF MATERIALS

Many persons have on occasion felt dissuaded from attempting the

preparation of microscopic slides of certain plant materials because of
the presumed difficulty or impossibility of obtaining what might be
wanted. This difficulty is really more apparent than actual, except gen-
erally in the case of tropical plants not in cultivation or of those which
are of quite rare occurrence. There are at present several satisfactory
sources for plant materials, equally available to all interested, and the
cost is usually far less than one would have to stand if the material were
personally collected.
Practically everything in the way of plant life is potential and legiti-
mate game for the botanical technician. The whole problem is simply
one of knowing where, when, and how to look for things, if one must
depend primarily on one^s own efforts. One should become familiar with
local floras, special manuals on the various groups, and other sources
which give detailed information on the occurrence, life histories, and other
facts regarding the special plants in which one might be interested.
Make the acquaintance of local florists and nurserymen, as well as of both
amateur and professional botanists of the vicinity. Establishments
conducted by elderly persons are a more likely source of interesting plant
material than are those run by younger persons primarily concerned in
quick financial returns from commercial crops. However, an acquain-
tance with the latter w^ould be profitable to those interested in plant
pathology. All these people will usually be much interested in what one
is doing; one should be patient and cordial in explaining matters, as it

frequently happens that the most unexpected favors are bestowed. It

makes literally no difference where one might be located: plant materials
of one sort or another are available everywhere.
The modern biological supply concerns, conducted as most of them are
by trained specialists, are veritable lifesavers for the technician. It has
long been the writer^s opinion that too little is generally known of the
services that certain of them are equipped to render. There are, of
course, a number that are in business merely to make money rather than
primarily to serve biologists, but one quickly learns of their unreliability
from the character of the goods which they furnish. Most of the ethical
concerns, unfortunately for botanists, are directed by persons trained
204
 :

SOURCES OF MATERIALS 205

who therefore specialize in animal materials. A

primarily as zoologists,
zoologist who can
turn out superbly injected cats or dogfish simply does
not know a thing about preserving the Chlorophyta without plasmolysis.
For the most part, such concerns deal simply in preserved materials,”
which are not always suitable for slide-making purposes.
There are two concerns which deal exclusively in plant materials and
both are staffed by competent botanists; these are also the only ones which
offer both fixed and embedded materials to technicians who wish to make
their own slides but are unable personally to collect the desired material.
Neither definitely offers such materials in their catalogues, mostly because
of the difficulty of keeping adequate stocks always on hand, but specific
requests will rec^eive careful consideration, and every possible effort will
be made to supply the desired materials. These (*onc(‘rns are:

California Botanical Materials Company, 787 Mtiville Avenue, Palo Alto, Calif.
(Offers the wider variety of all types; the only source for Pacific Coast algal material.)
Dr. Geo. H. Conant, Ripon, Wis. (Best source for the commoner laboratory
types, also for all fungi.)

In addition, there are a few other concerns which, Ix'cause' they are in
excellent collecting regions, can supply certain materials in betU'r quality
than can be obtained elsewhere:

Oregon Biological Supply Company, 4514 S. E. Eighteenlh Avenue, Portland,

Ore. (Excellent for Mniurn^ Polytrichumy and a number of other Bryophyta; also for
Selaginella.)
Carolina Biological Supply Company, Elon College, M. C. (Fine source for
VolvoXj Spirogyray Euglenay Nitellay and other aquatic organisms.)
Supply Department, Marine Biological Laboratory, Woods Hole, Mass. (For
certain east coast algae. Living Fucus can be ordered shipped by air mail during
cold weather, and it usually arrives in good condition for immediate fixation. Preser-
vation excellent on some materials, extremely poor on others. The author has been
unable to obtain unlisted species, and special fixation methods will not bt^ used.)
American Type Culture Collection, George Washington University, Washington,
D. C. (Carries cultures of a great variety of bacteria and fungi, including forms like
AspergilluSy Rhizopus (both plus and minus strains), MucoVy ActinomyceSy Fiisariumy
ZygorhynchuSy and innumerable yeasts. A highly recommended source.)

GLASSWARE, APPARATUS, AND GENERAL SUPPLIES

There are numerous eoneerus scattered over the country which cany
supplies of a general nature, such as staining dishes, dyes, slide boxes,
miscellaneous glassware, chemicals, etc. Some are also the agents for
other concerns which specialize in the manufacture of dyes, reagents, and
similar products. Inspection of their catalogues, obtainable upon
request, will reveal the extent and specific nature of their stocks. Th(‘
following, selected for their location in different regions, large stock list-

ings, and known reliability, may be recommended

 :

206 GENERAL METHODS

Braun Corporation, 2260 East Fifteenth Street, Los Angeles, Calif.

Braun-Knecht-Heimann-Co., 1400 Sixteenth Street, San Francisco, Calif.
Central Scientific Company, 1700 Irving Park Boulevard, Chicago, 111. (Pacific
Coast representative: Redman Scientific Company, 685 Howard Street, San Francisco,
galit)
Chicago Apparatus Company, 1736 North Ashland Avenue, Chicago, 111.
CTi
Standard Scientific Supply Corp., 34 West Fourth Street, New York, N. Y.
A. H. Thomas Company, West Washington Square, Philadelphia, Pa.
W. M. Welch Manufacturing Company, 1515 Sedgwick Street, Chicago, 111.
Will Corporation, Rochester, N. Y.

Slides and Coverslips

Most general supply dealers list slides and coverslips, but the following
supply superior grades of both items:

“^A. H. Th omas Company, West Wa s hington Square^ Philadelphia, Pa.^

Hetlige, Inc., 570!LNnrthem Boulevard, LongHsl and City, N Y.
.

Redili&irScltentilic Company, 58S Howard Street. San Francisco, Calif.

Microtomes
Instruments of American manufacture are preferable:

SpencerLens Company, B u^1q^,N. Y.

Bausch and Lomb Optical Company, ^cheater. JN. Y.

Microscopes
Students in collegiate institutions should already be provided with
suitable microscopes. If one desires to possess his own microscope, Amer-
ican instruments will be found to be as satisfactory as, if not superior to,
those of foreign make. They may be purchased from local optical
dealers or from the optical firms listed above under Microtomes.

Chemicals and Reagents

All ordinary chemicals may be purchased from the general supply
firms. Well-known brands should always be specified, and care should be
taken to see that the materials come in the original containers; this is the
only preventive against adulterations or substitution and deterioration.
The following list includes the manufacturers or sources of special chem-
icals and reagents

Eastman Kodak Company, Chemical Sales Division, Rochester, N. Y. (Special

*

organic chemicals.)
Merck and Company, Rahway, N. J. (Pacific Coast distributor: Griffin Chemical
Company, 1000 Sixteenth Street, San Francisco, Calif.) (For all reagent chemicals.)
General Chemical Company, 40 Rector Street, New York, N. Y. (Baker and
Adamson Reagent Chemicals.) (Pacific Coast distributor: Redman Scientific
Company, 686 Howard Street, San Francisco, Calif.) (Organic and inorganic
chemicals; xylol.)
 SOURCES OF MATERIALS 207

J. T. Baker Chemical Company, Phillipsburg, N. J. (Pacific Coast distributors:

Braun Corporation, Los Angeles, Calif.; Braun-Knecht-Heimann-Co., San Francisco,
Calif.) (General and reagent chemicals.)
Carbide and Carbon Chemicals Corporation, 30 East Forty-second Street, New
York, N. Y. (Pacific Coast distributor: Griffin Chemical Company, 1000 Sixteenth
Street, San Francisco, Calif. ) (Synthetic organic chemicals methyl cellosolve, dioxan,
;

synthetic ethyl alcohol, etc.)

Eimer and Amend, Third Avenue between Eighteenth and Nineteenth Streets,
New York, N. Y. waxes, special chemicals.)
(Essential oils,

California Botanical Materials Company, 787 Melville Avenue, Palo Alto, Calif.
(For special chemicals for botanical microtechnique: tertiary butyl alcohol, hygro-
butol, etc.)
Glyco Products Company, Inc., 148 Lafayette Street, New York, N. Y. (Syn-
thetic chemicals, waxes, resins, and special solvents.)
Baker and Company, Inc., Newark, N. J. (Osinic and chromic acids.)

Dyes and Stains

National Anilin andDye Company, 40 Rector Street, New York, N. Y. (Stocks
are carriedby most general dealers.)
Hartman-Leddon Company, 6010 Haverford Avenue, Philadelphia, Pa.
MacAndrews and Forbes Company, 200 Fifth Avenue, New York, N. Y. (Hema-
toxylin, hematein, and brazilin dyes.)
Akatos, Inc., 65 Van Dam Street, New York, N. Y. (For the tme Griibler dyes.)
Pfaltz and Bauer, Inc., 300 Pearl Street, New York, N. Y. (For Grtibler-Hollborn
dyes and stains.)

Embedding Media
Parowax may be purchased at any large grocery store. The brand
manufactured by the Standard Oil Company of Indiana is far superior to
others in that it does not crystallize readily.
All supply concerns list paraffin in their catalogues, but such paraffins
can be almost anything. It would be advisable to make one^s own
rubber-paraffin embedding medium, as described on page 22, or to
purchase a similar brand, such as Parlax.
Most technicians prefer the Schering-Kahlbaum brand of celloidin.
This is of German manufacture but is obtainable through most dealers or
from Akatos, Inc., 55 Van Dam Street, New York, N.Y. The product
called ^^colloidon” in some catalogues is merely a solution of celloidin and
isintended for medical purposes, not for embedding. There are a number
of types of celloidin on the market, going under such names as Parloidin,
but the author is not prepared to express an opinion on their suitability for
microtechnique purposes.

Mounting Media
Canada balsam of satisfactory quality is difficult to obtain. Most
of the balsam on the market has been highly diluted with xylol or other
208 GENERAL METHODS

solvents, and many samples show the dark brown color caused by exces-
sive heating to accelerate solution. One may, of course, purchase the dry
balsam and dissolve it; but the pure, neutral, nearly colorless, thick solu-
tion, filtered through paper, is best of all. Such a balsam, which can be
diluted with the proper solvents required by special techniques (e.g.y
hygrobutol, dioxan), may be obtained from the following:

Redman Scientific Company, 585 Howard Street, San Francisco, Calif.

Coleman and Bell Company, Norwood, Ohio.
California Botanical Materials Company, 787 Melville Avenue, Palo Alto, Calif.

Diaphane and Euparal are essentially similar products. The first,

which is American in origin, may be obtained from the Will Corporation,
Rochester, New York.
The special synthetic resins used for mounting diatoms are quite
difficult to secure and are expensive. A
high-quality Hyrax may be
purchased from the Braun-Kiiecht-Heimann-Co., 1400 Sixteenth Street,
San Francisco, Calif.
 SECTION II

SPECIAL METHODS FOR THE VARIOUS PHYIA

 CHAPTER XVII
SCHIZOPHYTA
SCHIZOMYCETES
Bacteria, which are to be found everywhere under the most extreme
of conditions, constitute a very low or primitive type of plant life, despite
the high specialization of some kinds. They are extremely minute, sim-
ple, unicellular organisms. A microscope equipped with an immersion
lens is usually required for their examination.
The professional bacteriologist has evolved the study of the bacteria
into a separate science —one so involved and with such a special termi-
nology that the average botanist feels hopelessly bewildered at every
contact with the subject. Botanists are interested in bacteria primarily
for their own sake, not in phagocytosis, hypersensitiveness, or anaphy-
laxis. The teaching of bacteriology is commonly conditioned by medical
school requirements, 'hence the subject is usually viewed with di-staste by
the young botanist, who in most colleges is compelled to study the ba(‘-
teria on his own initiative. The m(^thods of most bacteriologists, more-
over, are more complicated or specialized than th(‘ simple needs of thc^
botanist require. An attempt is therefore being made in the present
chapter to cover the technical aspects of bacteriology as simply and
adequately as possible.
—
Morphology. Bacteria of known dimensions range in size from 0.5 X
0.2 up to 50 to 60 X 4 to 5^.
)li Some of the pathogenic forms are so small
that they are invisible microscopically.
Three basic forms are recognized: the coccus (spherical), bacillus (rod-
like), and spirillum (spiral or segment of a spiral). For a given species
the shape is fairly constant. During multiplication bacteria exhibit more
or less of a tendency to remain attached.
Single coccus forms are nearly spherical, but during multiplication the
form may become elongated or lanceolate. Cells which divide in one
plane only and remain attached occur as pairs, known as “diplococci,” or
in shorter or longer chains, designated as “streptococci.^^ Those which
divide in two directions, one at right angles to the other, form groups
four known as “tetrads.'' Division in three directions to form cubes
produces what are called “sarcinae." Finally, those which divide in any
direction to produce irregular bunches are described as “staphylococci."
Bacilli characteristically are cylinders with a straight axis, of uniform
thickness and flat ends, but there are numerous exceptions. Some types,
211
 .

212 SPECIAL METHODS FOR THE VARIOUS PHYLA

for instance, may have rounded ends; others, more slender in form, are
slightly bent. Spore development causes irregularities in the outline of
the cell. Multiplication of bacilli is solely by division in the plane per-
pendicular to the long axis.
may be either complete spirals or segments of a spiral.
Spirillum types
The spirals may
be large or small, slender or thick. Like the bacilli, the
spirillum types divide in one plane only.
Cell Structure. —
Probably all bacteria possess a capsule, a gelatinous
envelope presumably developed from the outer layer of the cell membrane.
Some produce such a thick capsule that they form slimy
species
growths. Capsules are more easily recognized on forms growing in
animal tissues. In slides prepared from culture media, they are usually
scarcely distinguishable. The capsule is not easily stained by coal-tar
dyes; special methods are required for its detection.

All bacteria possess a cell membrane. In some species it is more

liighly developed than in others. Microchemically, the membrane lacks
cellulose, but in some species the presence of hemicellulos(‘s has been
demonstrated. In still otluT species a substance closely related to chitin
occurs.
Within the membrane the bacterial cell is generally considered to
possess the two chief constituents of most cells, viz.^ cytoplasm and
chromatin. According to the species and also to the stage of growth or
division, the chromatin may exist in the form of definite granules or
may be so finely divided and scattered in the cytoplasm as to be
indi stinguishable
Metachromatic granules, characterized by their great affinity for
stains,occur in many bacteria. Some types of these granules arc sup-
posed to be nuclear in character. Other granules are composed of volutin.
The sulphur bacteria contain granules of sulphur, and the iron bacteria
similarly are filled with particles of ferric compounds.
Very few of the coccus bacteria possess flagella, but the bacillus and
spirillum types frequently show these fine hair-like appendages. The
flagella arc disposed singly or in tufts, over the entire cell or only at one
or both ends of Vjacillus forms. They are the only known means of
locomotion. The have little affinity for stains; consequently they
flagella
are revealed only by special methods.
Vegetative reproduction is by fission. Many bacteria form true
spores, endospores. Each cell produces only one such spore. The spore
may lie in the center of the cell, at one extremity which becomes swollen,
or eccentrically, depending upon the species. Spores are very difficult to
stain. In old cultures of nonspqre-formers a certain proportion of the
cells may become abnormally large with a thicker cell wall; such cells stain

more intensely. These bodies have been described as arthrospores.

 8CHIZ0PHYTA 213

There is a group of organisms intermediate between the true bacteria

and the true fungi which partakes of the characters of both groups. Some
students place the Actinomycetes among the Fungi Imperfecti, where
they perhaps properly belong, but since they are better known to bac-
teriologists and require treatment similar to that demanded by bacteria,
they are included in the present chapter.
—
Sources of Cultures. Cultures are very easily obtained. Bacteria
are always associated with every form of decaying matter. A broth
obtained from meat, potatoes, or other foods, partially exposed to the air
for a few days until an odor is apparent, will yield an abundance of bac-
teria. Scrapings from unbrushed teeth provide others. Water from
sewer outlets, stagnant pools or salt water in which seaweeds are rotting,
all furnish myriads of bacteria. Or a hay infusion, prepared simply by
pouring hot water into a battery jar half full of timothy hay, covering, and
letting the whole stand for a week, is an admirable source for other forms.
For pure cultures of specific forms, it is better to go to a professional
bacteriologist, a commercial bacteriological laboratory, or certain of the
biological supply concerns. There are some concerns which will specially
prepare cultures of highly pathogenic forms, such as B, anthraciSy and
furnish them as a killed suspension in a salt and formalin solution; in such
a condition slides can be prepared at no risk of infection. The Cutter ^

Laboratory, Berkeley, Calif., is such a source. The American Type

Culture Collection is prepared to furnish hundreds of species.
Culturing. —
Each species of bacteria has its own special cultural
requirements. The methods employed for artificial cultivation are
therefore of fundamental importance. Bacteria of medical significance
require complex foodstuffs adjusted to a suitable pH. Such media have a
meat, serum, or blood basis.
A knowledge of hydrogen-ion concentration (pH) and its relation to
the preparation of media on which to grow bacteria is requisite to the
successful cultivation of the organisms. Very few botanical or private
laboratories are equipped for the determination of pH, consequently the
problem of adjusting the reaction is either a difficult matter or impossible.
Fortunately, however, there an easy solution of the matter. This is
is

simply to purchase dehydrated culture media prepared according to

standard formulae, add the required amount of distilled water, and, by
autoclaving at a certain pressure for a specified period, the resulting
medium will have the requisite pH. The ‘‘Difeo'' products, manu-
factured by the Digestive Ferments Company, Detroit, Mich., are
recommended.
Bacteria are usually grown on agar slants. To make the slants, first
prepare the medium and add agar (in the proportion of 1.5 g. to each 100
cc. of medium) if a dehydrated medium already containing agar is not
214 SPECIAL METHODS FOR THE VARIOUS PHYLA

lised. If agar added, dissolve it first by placing the container in a boil-

is

ing water bath. Have standard Pyrex test tubes, scrupulously cleaned,
ready. Fill each tube with about 5 cc. of medium, then plug with a gen-
erous wad of ordinary nonabsorbent cotton, leaving just enough exposed
for the plug to be pulled out without difficulty. Place the tubes in the
autoclave and sterilize for the specified time at the required pressure (this
is usually cited as 15 pounds pressure for 20 or 30 minutes). After
autoclaving, wait for the pressure to go down completely before removing
the tubes. Before the agar has a chance to solidify, place the tubes on
their sides, with the plugged end slightly raised. Take care that the
medium covers the bottom end of the tube, otherwise the slant might
collapse of its own weight after cooling. The bacterium to be cultivated
is streaked over the slanting surface of the solidified medium.

For transferring cultures, a platinum needle or small loop, attached

in a glass or special aluminum holder, is employed. Between every
transfer the wire must be sterilized by heating in a gas flame until white
hot. While making transfers, the tubes must not be kept open any longer
than necessary, otherwise contamination might occur. Hold the tube by
the base in the left hand. The right hand, also holding the platinum wire,
is used for removing the cotton plugs, the plug of one tube being grasped

by the little, finger and that of the second tube between the third and
fourth fingers. After withdrawing the plugs, pass the mouths of the tubes
three or four times through a flame, then transfer the growth, and replace
the plugs. Draw the needle or loop over the growth in the culture tube,
then in the new tube commence at the bottom and streak the needle
diagonally back and forth until the upper limits of the agar are reached.
Sterilize the platinum immediately after using and before placing it down.
Be extremely careful to avoid splattering.
Most bacterial cultures grow best at either of two standard tempera-
tures, 22 or 37°C. For general purposes the first temperature is that of
the average room, but if a more uniform and exact temperature is indi-
cated, an incubator must be employed.
In place of test tubes Petri dishes may be used as culture vessels.
An ordinary Petri dish requires about 10 cc. of medium. It is more
desirable to fill test tubes with that amount of medium in each, then to
plug, and to sterilize. The Petri dishes are cleaned and sterilized. When
one vrishes to make a culture, a tube of medium is melted in hot water and
poured into the Petri dish: the cover is opened just enough to allow the
mouth of the tube to be inserted. As soon as the agar has solidified, the
transfer of culture may be made. As with slants, it should be streaked
diagonally across the surface.
Bacteria are either aerobic or anaerobic, but the average botanist will
scarcely meet with bacteria that can grow only in an atmosphere freed of
oxygen.
 8CHIZ0PHYTA 215

—
Preparation of Smears. The slides or coverslips on which bacteria
.

are to be smeared must be chemically clean. In the center of either, place

a small drop of distilled water. With the platinum needle or loop take up
a very small amount of the culture, place in the drop, and stir the two
together thoroughly but not violently, then spread the drop out some-
what. It is best to place the slides in a warm protected place for 24 hours
to dry, but if in a rush, dry the drop by passing four or five times through a
clean gas flame.
Or the smears, partially dried but still moist, may be fixed in any
standard fixative or over the. fumes of osmic acid.
Dry, fixed, unstained bacterial smears keep indefinitely in arcool place.
If mounted in balsam or other mounting media, stained slides tend
gradually to fade.
—
Fixation of Tissues Containing Bacteria. Plant tissues infected with
bacterial diseases, such as Aplanobacter insidiosum in the roots of Medi-
cago saliva, should be cut into small portions not over 3 mm. square and
2 mm. thick. Fluids containing mercuric chloride are said to be better
than others, but it appears that very little research has been done on this
phase of the problem. It seems probable, in any event, that any fluid
which gives satisfactory preservation of the host tissues will likewise pre-
serve the bacteria, but dehydration should be carried out with tertiary
butyl alcohol as othor fluids are suspected of damaging the bacteria.
Animal tissues should be fixed in Zenker^s fluid:
Potassium V)ichromatc 2.5 g.
Sodium sulphate 1 .
0 K-
Distilled water 100 cc.

Mercuric chloride 5 0 . g.
Glacial acetic acid 5 cc.

Dissolve the bichromate and sublimate in the water with the aid of
heat. Add the acetic acid just before the fluid is to be used. Wash
the fluid out thoroughly with running water, pass through three changes
of pure dioxan over a 24-hour period, transfer to a mixture of equal parts
of dioxan and paraffin oil, then into melted parafin, and embed after
several changes of paraffin have been made.
Stains. —The staining process, so far as bacteria are concerned,
depends upon the solvent condition of the dye. Stains dissolved in
absolute alcohol do not stain well, if at all. Absolute alcohol, further-
more, does not decolorize bacteria, while diluted alcohol is an active
decolorizer. The more completely a dye is dissolved, the weaker its
staining power becomes. The addition of an alkali to a stain renders
the solvent action less complete and intensifies the staining power.
;
The staining process is further dependent upon the nature of the
.

bacterium. Some forms stain easily, others do not. Spores and flagella,
as has already been mentioned, are difficult to stain. Many species
216 SPECIAL METHODS FOR THE VARIOUS PHYLA

possess highly selective staining properties, and this fact is of practical

importance with regard to certain staining schedules. Gram’s stain is
an example, but the differentiation of bacteria into two classes those —
—
which react to Gram’s stain and those which do not is unreliable and
impractical because external circumstances often modify the reaction.
Previous treatment of the bacteria with acids or alkalies frequently
inhibits the reaction. There is, however, no difficulty in finding a
suitable stain for all the different species.
Methyl and crystal violet are the most intense stains which may be
used on bacteria; they usually overstain and must therefore be differenti-
ated. Methylene blue, thionin, basic fuchsin, and safranin give better
differentiation since they do not easily overstain. Bismarck brown and
eosin stain weakly and are commonly used as counterstains.
Mitochondria, in general, are also stained by bacteriological methods
(see page 101 for a staining method to differentiate between the two).
—
General Staining Methods. Bacterial smears are placed directly
into the stain after first having been thoroughly dried. Smears fixed in
killing fluids are washed carefully and then transferred to the stain.
Paraffin sections must, of course, first be deparaffined and brought down
to the appropriate alcohol or to water.
Practically the coal-tar dyes are first made up as saturated alco-
all

holic solutions. Stock solutions are prepared by filling a bottle about

one-fourth full with the dry dye, then nearly filling the bottle with 95%
alcohol. The bottle is then tightly corked, well shaken, and set aside
for 24 hours. If at the end of this time all the dye has dissolved, more

should be added, the bottle shaken again, and set aside for another
24 hours. The process should be repeated until some undissolved dye
remains at the bottom of the bottle.
Methyl {or Crystal) Violet ,
—
The simplest staining method is as
follows:
1. Stain 2 to 5 minutes in crystal or methyl violet (1 part saturated
alcoholic solution to 10 parts water),
2. Rinse quickly in water.
3. Differentiate by dipping into 95% alcohol until most of the color
is gone.
4. Dry as much as possible by blotting with filter paper, then pass

through a flame or put on the slide warming table to complete the drying.
5. The preparation may be examined without mounting, or balsam

and a coverslip may be added.

Anilin Water-Methyl Violet , —
An anilin water solution of either
crystal or methyl violet is considered to give a better stain. First
prepare the anilin water by shaking up 4 cc. anilin oil with 90 cc. distilled
water and filtering the mixture through a wet filter. Add 10 cc. of the
 SCHIZOPHYTA 217

saturated alcoholic solution of either violet to 100 cc. anilin water, and
24 hours to allow the precipitate which soon forms to settle.
set aside for
The stain solution keeps for a week or ten days. Staining occurs within
6 minutes; differentiate as described for the preceding method.
Loeffler^a Methylene Blue. —This is one of the most widely used stains.
Mix 30 cc. of a saturated alcoholic solution of methylene blue with 100 cc.
of 0.05% potassium hydroxide solution. Smears are stained in 2 to 6
minutes; if the solution is heated, the stain is intensified. Sections are
stained for 15 minutes to several hours; decolorized with weak acetic
acid (1:1000) until a faint blue color remains; counterstained with 1%
aqueous eosin, dehydrated with absolute alcohol for 20 seconds, cleared,
and mounted.
Ziehl-N eelson^ s Carbol-Fuchsin. —Add 10 cc. of a saturated alcoholic
5% aqueous carbolic
solution of pararosanilin (basic fuchsin) to 100 cc. of
acid. a good spore stain and is used as described below under
This is

Spore Stains. The solution keeps well.

—
Carhol-Methyl Violet. One part of a saturated alcoholic solution of
methyl (or crystal) violet is added to 10 parts of a 2 to 5% aqueous
solution of carbolic acid.
Carbol- Methylene Blue. —
Add 15 cc. of a saturated alcoholic solution
of methylene blue to 100 cc. of 5% aqueous carbolic acid.
—
CarboUThionin. This is made up of 10 cc. of a saturated alcoholic
rsolution of thionin and 100 cc. of 1% aqueous carbolic acid.
—
Gramms Method. First stain the sections or smears with anilin water-
crystal violet. From the stain transfer to Gram’s iodine solution (1 g.
iodine and 2 g. potassium iodide in 300 cc. distilled water) for 1 to 2 min-
utes. Rinse in 95% alcohol until the violet color is no longer apparent
to the naked eye. If the slides are insufficiently decolorized by the
alcohol, treat again in the iodine solution. If permanent preparations
are wanted, rinse in absolute alcohol, pass through xylol, and mount in
balsam. Otherwise wash smears with water, and dry. With smears the
method works satisfactorily on young, actively growing cultures. Some
bacteria do not stain by the method: such are described as being Gram-
negative, in contrast to the Gram-positive types, which retain the stain.
The above schedule is the classical one used by bacteriologists. A
version which will probably be found more satisfactory by botanists is as
follows.
The anilinwater staining solution should be made up with methyl
violet 6B, or the most highly methylated violet obtainable. Stain the
slides for 5 to 30 minutes, the length of time depending upon the species
and whether the slide holds smears or sections this time will need to be
;

determined by trial. Rinse in anilin water for 30 seconds (the slides

can remain in this solution for some time if necessary), then transfer
218 SPECIAL METHODS FOR THE VARIOUS PHYLA

to the iodine solution for 1 to 2 minutes. Sections become brown-black

in color. Wash in 96% alcohol for 30 seconds, then wash in 96% alcohol
plus 10% hydrochloric acid for 10 seconds, followed by washing in
absolute alcohol, at which juncture no more color should come out of the
sections. Complete clearing in cedar oil (do not use clove oil, as it will

extract the remaining stain), proceed to xylol, and mount in balsam.

The bacteria should be blue or blue-black, tissues nearly or quite color-
less. A weak solution of safranin can be used to stain the nuclei, with
orange G Before one attempts to stain bacteria by
for the cytoplasm.
be ascertained if the bacterium is Gram-positive.
this schedule, it should
The schedule recommended by the Society of American Bacteriologists
(Hucker modification) is as follows. Prepare two separate basic dye
solutions: (1) Dissolve 4 g. crystal violet (85% dye content) in 20 cc.
96% ethyl alcohol; (2) dissolve 0.8 g. ammonium oxalate in 80 cc. dis-
tilled water. Mix the two, and stain smears for 1 minute in the mixture.
Wash in water. Immerse in LugoFs iodine solution (1 g. iodine, 2 g.
potassium iodide, and 300 cc. distilled water) for 1 minute. Wash in
water, and blot dry. Decolorize in 96% alcohol for 30 seconds with
gentle agitation. Cover with safranin counterstain (10 cc. of a 2.5%
solution of safranin in 95% alcohol to 100 cc. distilled water) for 10 sec-
onds. Wash, blot, and dry.
There is available a method for the differential staining of Gram-
positive and Gram-negative bacteria in sections of tissue (Brown and^
Brenn 1931). Stain for about 5 minutes in Harris^ hematoxylin (filter
just before using), wash with acid alcohol (3% hydrochloric acid in
95% alcohol) until the sections are a light purplish-pink, alkalinize the
color to blue with 1 %
ammonia in water, and finally wash thoroughly
with water. Stain for 2 minutes in the following freshly prepared
solution: mix together 5 drops of 5% aqueous sodium bicarbonate to
which has been added 0.5% phenol and 0.75 cc. of 1% aqueous crystal
or methyl violet. Wash off excess stain with water and treat with
Gram\s iodine solution for 1 minute. Wash again with water, then blot
the sections carefully to remove all excess water possible, but do not
allow them to dry. Put the slides in acetone for a few minutes, remove,
hold level with the sections up, and cover with 0.1% aqueous picric acid
until the sections become yellowish-pink. The last step is the critical
one in the schedule; the slides should be held above a white plate or
other white surface. Stop the action by draining off the acid and plung-
ing the slide into a jar of acetone. Pass through a mixture of equal
parts of acetone and then pass through pure xylol, and motint in
xylol,
balsam. The nuclei of the tissue are dark reddish-brown, the cytoplasm
yellowish. Gram-positive bacteria deep violet to almost black. Gram-
negative bacteria bright red.
 8CHIZ0PHYTA 219

Fuelgen’s reaction may be used on bacteria.

also
Staining Methods —
Smears should be made on coverslips.
for Spores.
Fix by passing many times through a flame.
Put the fixed smear in 5% aqueous chromic acid for 5 seconds to
10 minutes (if the spores are not stained at the end of the schedule,
lengthen the time in the chromic solution). Wash in water. Stain with
Ziehl-Neelson’s carbol-fuchsin or anilin water-fuchsin (use pararosanilin
in place of methyl violet as described for anilin water-methyl violet
above) for 1 minute, heating the solution until it steams but does not
boil. Destain 5 seconds in 5% aqueous sulphuric acid. Wash in water.
Counterstain with methylene blue, wash with water, dry in the air, and
finally mount in balsam. The spores should be red, the bacteria blue.
Some workers contend that the use of an acid for differentiation is
undesirable because it damages the organisms and tends to decolorize
the capsules of the spores (Lote 1931). To avoid acids, use may be made
of the fact that some dyes differentiate other stains. Stain smears for
1 and differenti-
to 2 minutes with carbol-fuchsin, rinse with water, dry,
ate with 1 1.5% methylene blue. The time required differs according
to
to the species: some need only 10 minutes, others as long as 1 hour.
Spores, also certain bacteria, are red, other bodies bluish-purple, blue, or
gray. Bismarck brown (0.5 to 0.25%) may be substituted for methylene
blue.
The method gives excellent results, with sharp differentia-
following
tion (Schaeffer and Fulton 1933). Smears are fixed by passing through
a flame three times. Flood the smear with 5% aqueous malachite
green, and heat to steaming several times within 30 seconds. Wash in
running water for about 30 seconds. Cover with 0.5% aqueous safranin
for 30 seconds. Wash, dry, and mount in balsam if desired. The spores
are stained green, cells red.
—
Staining Methods for Capsules. Smears to be stained for capsules
should be made from cultures not over 24 hours old. Dry the smears
in the air, without using heat. Perhaps the simplest method is the
following (Churchman and Emelianoff 1932): flood the smear with
10 drops of Wright^s stain (best purchased made up for use), and leave
until the stain has nearly,but not completely, evaporated. This takes
3 to 4 minutes, and the original blue changes to a pinkish color. Next
wash off as rapidly as possible with a buffer solution of pH 6.4 to 6.5,
and dry without blotting. If the buffer wash is used before the stain
has evaporated suflSciently, the capsule will not be stained. If preferred,
the smears may be left in the stain overnight. The smears are removed
and the stain allowed to evaporate as before, dipped in the buffer solu-
tion and dried. Occasionally there might be a precipitation of the stain
to cause granular deposits.
220 SPECIAL METHODS FOR THE VARIOUS PHYLA

The Hiss capsule stain also gives excellent results and may be employed
if method presents difficulties. Prepare the smears on a
the preceding
very clean coverslip, dry in the air, and fix by passing through a flame.
Flood the coverslip with methyl violet solution (5 cc. saturated aqueous
and steam for a few seconds over a hot
solution to 95 cc. distilled water),
flame. Wash off the stain with 20% aqueous copper sulphate. Do not
wash with water at any time. Blot dry with filter paper (do not dry in
the air or the copper salt will be precipitated out). The capsule is a
faint blue halo around a dark purple cell.
—
Staining Methods for Flagella. Make a thin smear by transferring
a very tiny amount from a culture not over 24 hours old, to 1 drop of
water on a slide. Let dry in the air. Cover with a mordant composed
of 3 parts 5% aqueous tannic acid and 1 part 10% aqueous ferric chloride
for 4 minutes (after Bailey 1930). Pour off the mordant, and cover for
9 minutes with the following freshly prepared solution: 7 drops of the
mordant mixture with 1 drop Ziehl-Neelson^s carbol-fuchsin, with
the successive admixture of 1 drop concentrated hydrochloric acid and
1 drop concentrated formaldehyde. Wash in running water. Flood
with Ziehl-Neelson's carbol-fuchsin, and steam gently for 3 minutes.
Wash in running water, blot, and dry.
There is a second method which affords a slightly better differential
staining (Leifson 1930). Mix the following ingredients in the order
cited:

Ammonium (or potassium ) sulphate, saturated aqueous solution 20 cc.

Tannic acid, 20% aqueous solution 10 cc.
Distilled water 10 cc.
96% ethyl alcohol 16 cc.
Pararosanilin, saturated aqueous solution 3 cc.

Crystal violet may

be substituted for the pararosanilin at the rate of
1.6 cc. instead of 3 cc, of the saturated solution; or methylene blue by
using 5 cc. and reducing the additional 15 cc. of ethyl alcohol to 10 cc.
The stain solution keeps for about a week. Stain for 10 minutes, first
warming the solution to not over 38°C. Wash in water, and dry. To
counterstain: if pararosanilin has been used, 0.1% aqueous methylene
blue plus 1% borax reacts nicely; or if either crystal violet or methylene
blue has been used to stain the flagella, carbol-fuchsin diluted 1:10
counterstains excellently.
Silver staining may be employed (Safford and Fleisher 1931).
also
Make smears Cover with a fixative composed of 100 cc. of
as usual.
one-fourth saturated aqueous solution of picric acid, 5 g. tannic acid and
7.6 g. ferrous sulphate. A few minutes suffices for the mordanting. Wash
with tap water, dry, and cover with the silver stain (to 25 cc. of 2% aque-
 SCHIZOPHYTA 221

0U8 silver nitrate add dilute ammonia until the precipitate which forms
isredissolved, then add more of the silver nitrate until a faint turbidity
results —clear solutions are useless), heat to steaming, and allow to
react for 1 to 2 minutes. Wash in tap water, then dry.
Methods for Metachromatic Granules. Prepare smears
Staining —
as usual. Flood with Gramms iodine for 1 minute, rinse in tap water,
stain with Loeffler^s methylene blue for 20 to 30 seconds, rinse, stain with
1% aqueous safranin for 10 to 15 seconds, rinse, dry, and finally mount in
balsam. Polar bodies are dark bluish-black, while the rest of the bac-
terium is red (Kemp 1931).
Soil Microflora. —The microscopic flora of the soil includes algae
and various fungi and bacteria. Formerly it was considered necessary
to dig a trench in the field soil for inserting slides, but a simpler method is
now vogue (Conn 1933). Merely fill a jelly tumbler with the soil
in
whose be examined, insert^ two or more cleaned slides, cover and
flora is to
set aside for 5 days. Remove one of the slides at the end of the incuba-
tion period, and replace by another. Do the same with a second slide
on the seventh day, and repeat again after the lapse of another 5 days and
7 days for the replaced slides, respectively.
For the miscellaneous collection of organisms that will probably be
found on the slides, only a general stain serves satisfactorily. Rose
bengal is perhaps the most useful. It is prepared in the following pro-
portions: rose bengal, 1 g. calcium chloride, 0.01 g. 5% aqueous phenol,
; ;

100 cc. The slides are preferably laid on a flat surface over a boiling
water bath, the stain solution is poured upon them and allowed to remain
for 1 minute, care being taken that the slides do not become dry; but if
desired, the slides may be stained in beakers of the steaming stain solution.
Determination of Motility.—^When examined under the microscope'
in the actively living condition, many bacteria exhibit motility in fluids.
Some specieshave a rapid movement; others move so slowly that it is
difficult to distinguish between actual movement and pedesis (‘^Brownian
movement^^). Again, not all bacteria which possess flagella exhibit
spontaneous movement under all conditions, since external circum-
stances such as heat, light, electricity, and chemicals may influence the
movements.
To test for motility, make hanging drop cultures from very young
cultures (not over 4 hours old) preferably grown in neutral bouillon. A
hanging drop culture is made by placing a glass ring, 2 to 5 mm. deep, on
a slide; take a circular coverslip of greater diameter than that of the
ring, put a large drop of the culture in the center of the coverslip, care-
fully reverse, and place over the ring so that the drop remains suspended
in the well. To prevent excessive evaporation, both ends of the glass
ring may be coated with a thin layer of vaseline or petrolatum.
 —

222 SPECIAL METHODS FOR THE VARIOUS PHYLA

Special Methods. — It is not possible here to present detailed methods

for all bacteria, consequently manuals which describe all that is known
about each form should be consulted (e.gr., Bergey 1939). Other manuals
should be consult-ed for culture media and cultural methods (Levine and
Schoenlein 1930). However, methods are cited below for some of the
commoner and more interesting bacteria.
>
»

'
<¥ %.

Fig. 24. Rhizohium radicicolum: portion of a nodule from Mdilotua indica with bac-
terial-infected cells. Fixed with chrom-osmo-acetic differentially acidified with HCl and
;

stained with methyl violet.

Rhizobiutn includes the forms which are capable of fixing free nitroge’’..
when growing in nodules on the roots of legumes. Some writers ctll
the association parasitism, others designate it as symbiontism,
and still
others claim it to be a fortituous association. There are two species
generally recognized: R. leguminomm consists of rods
occurring singly
and in Y-shaped formations; B. radicicolum (Fig.
24) occurs singly and
in pairs, often swollen at one end or near
the middle. The bacterial
cells always stain unevenly, perhaps because
they secrete a mucilaginous
 —

SCHIZOPHYTA 223

substance. They are actively motile by means of a polar flagellum, but

some types are peritrichous (Stern and Sarles 1938). Nodules from the
roots of almost any of the Fabaceae or Papilionaceae, or less desirably
from the Mimosaceae, may be fixed in a solution made up of 4 cc. of 2%
aqueous osmic acid and 6 cc. of 1 % aqueous chromic acid, and the speci-
mens may be embedded in paraffin. Sections should be cut not thicker
than 4/x in the lengthwise plam^ of the nodule. Methyl violet, carbol-
fuchsin, or Gram's method may be employed for staining.
If slides of the cause of gonorrhea. Neisseria gonorrheae^ are desired,
take clean slides to a friendly physician, and ask him to make thin

Fuj. 25. Eberthdla typhi: smear from a young culture, dried to slide, fixed with heat, and
stained by Leifson’s method (using crystal violet) for the fiagellae.

smears of urethral discharge from a three- or four-day preferably male

case. Fix by exposure to sunlight for 10 minutes. The Pappenheim-
Saathof stain for phagocytes containing gonococci is specific and strik-
ingly beautiful. Stain smears in the following solution (use specially
certified dyes) for 2 minutes, then wash, dry, and mount in balsam:

Methyl green 0 15
. g.
Pyronin 0 05
. g.
96% alcohol 5.00 cc.
Glycerin 20.00 cc.

2% phenol in distilled water 100.0 cc.

The genera Erwinia and Phytomonas contain practically all bacteria

pathogenic to other plants (Elliott 1930). On artificial media, growth
224 SPECIAL METHODS FOR THE VARIOUS PHYLA

isusually whitish and often slimy. Ermnia includes motile rod-shaped

forms with peritrichous flagella, while the motile forms in Phytomonas
have polar flagella. To show the bacterium in tissues of the host plant,
remove small pieces of the infected tissue, kill and fix preferably in a
strong chrom-osmo-acetic fluid, dehydrate and embed in paraffin, section
at not over Sju, and stain with carbol-fuchsin or methyl violet, counter-
staining with erythrosin.
Eberthella typhi, the cause of typhoid fever, is one of the best forms in
which to demonstrate peritrichous flagella (Fig. 25). Smears should be
obtained from a professional bacteriologist.

Fig. 26 . —
Treponema pallidum: section of fetal lung showing the spirochaetes in abun-
dance. Fixed with 10 % formalin; stained by the Warthin-Starry silver impregnation
method. {From a preparation by Mies Enid Larson,)

There are five genera of so-called ‘4ron bacteria.^^ Most of them

cannot be grown on artificial media. They are typically aquatic, algal-
like filamentous bacteria which may develop conidia but never endo-
spores. The sheath .surrounding them is impregnated with iron. It
may be necessary to remove the iron encrustations with dilute acid in
order to reveal and to stain the cells more clearly.
Rotting marine algae are an excellent source for the so-called ^‘sulphur
bacteria,'^ Thiothrix and Beggiatoa. They are filamentous forms which
show an oscillating motion similar to that of Oscillatoria,
One will have to go to a clinical pathologist for material of Treponema
pallidum the cause of syphilis (Fig. 26).
j
Despite the prevalence of the
disease, it is actually quite difficult to secure suitable infected material;
fetal lung or liver is superior to other tissues. The tissue; cut into very
 SCHIZOPHYTA 225

tiny portions, should be fixed for several days in 10% aqueous neutral
formalin, then washed thoroughly with water, and embedded in paraffin.
Sections should be cut at 5 or 6 m. Clinical technicians and medical
pathologists commonly use the Dieterle silver-impregnation method
(Dieterle 1927), but the Warthin-Starry method (Farrier and Warthin
1930) will give results more to the liking of botanists.

ACTINOMYCETES
The Actinomycetes constitute a group apparently intermediate
between bacteria and molds. They contain innumerable soil organisms
and a few forms pathogenic to man. They consist of extremely delicate
branching threads greatly resembling the mycelium of molds. The ends
of the filaments break up into spores.
The peculiar characteristic of the soil Actinomycetes is their con-
spicuous musty odor. When these organisms are abundant in a given
soil, they give to that soil its characteristic odor. When grown in the
laboratory, the Actinomycetes produce pigments which are frequently
very beautiful and serve for purposes of identification and classification.
The Actinomycetes are better known to medical bacteriologists and
soil scientists than to botanists.

Cultivation. —
The Actinomycetes cannot tolerate an acid medium,
consequently the acid media recommended for yeasts and molds cannot
be used. Most of them will grow on the same media used for bacteria,
but it is too difficult to keep down the growth of contaminating bacteria
on such media. Most Actinomycetes thrive on very weak media which
will scarcely support bacterial growth (Heinrici 1930).
For general isolation of the saprophytic forms soil-extract agar or
Czapek^s agar may be used. The method of growing cultures on slides
as described for the Myxothallophyta may be used for some forms, pro-
vided bacterial growth can be kept down sufficiently. C^oat the slides
with Czapek’s agar.
Pure cultures of an unusually large number of Actinomycetes may bo
secured from the American Type Culture Collection.
—
Preparation of Slides. For general morphological studies, Actino-
mycetes may be treated like yeasts (page 334) or similar fungi, but the
very small size of the former requires more careful methods. An oil
lens must ordinarily be used for examining them.
Permanent preparations of the spore-bearing bodies and the spores
are not difficult to make (Drechsler 1919). Smear a thin film of Mayer’s
adhesive over a coverslip. Carefully drop the coverslip, smeared side
down, on the surface of an actively sporulating colony, and then gently
lift it off again. The spore-bearing filaments will adhere to the coverslip
and will be broken off from the culture but will retain their normal
226 special methods pop the VAmOllS PHYLA

arrangement. The coverslip with the adhering organisms may now be

placed in a dish of fixative and subsequently carried through the staining,
dehydrating, and mounting processes. Any of the basic coal-tar dyes
may be used.
Two staining methods adapted for sections are as follows.
IsraeVs Method. —1. Stain the sections in a concentrated aqueous

solution of orcein, to which has been added a little acetic acid, for several
hours.
2. Wash with water, then dehydrate rapidly.
3. Wash in absolute alcohol for several hours.
4. Wash in xylol, then mount in balsam.
Actinomyeetes are blue, clubs a light red.
Bostroem^s Method. —1. Stain in a 1% solution of crystal violet in

anilin water for 10 to 15 minutes.

2. Transfer directly to picrocarmin (add a strong solution of carmin in

ammonia to a saturated aqueous solution of^ieric acid until a precipitate

is formed; add a trace of phenol to prevent mold growth, then allow to

stand exposed to the air until the solution has evaporated down to a fourth
of its original volume; filter, and allow the filtrate to evaporate to dryness;
make a saturated solution of the resulting crystalline substance in distilled
water for staining) for 5 to 10 minutes.
3. Wash with water, then wash in absolute alcohol until the sections

appear reddish-yellow.
4. Clear in cedar oil or oil of origanum, and mount in balsam.

The Actinomyeetes are dark blue, clubs red, and nuclei reddish-yellow\
 CHAPTER XVIII

CHLOROPHYTA
CHLOROPHYCEAE
The green algae from thedays of microtechnique have been
(earliest

favorite subjects with botanical technicians. Spirogyra^ for example, has

universally been worked upon perhaps more than any other plant. The
reason is that every reaction can be critically observed, and the results,
when different procedures are followed, can be readily compared. Killing
and fixing, dehydrating, staining, and even infiltration with paraffin or
cclloidin may be carried out under direct observation. These algae serve
as superb practice objects for the technician they arc to the botanist what
;

the guinea pig or rabbit is to the animal experimenter. They are superior
to all other experimental types in that what is happening is always plainly
visible. This is quite apart from their own inherent interest.
Occurrence. —Chlorophyta occur everywhere — in pools, lakes,
streams, along rocky shores of the oceans, on damp soils, in ordinary
soils, in salt lakes and salterns, in aerial habitats, and as symbionts or
parasites. Many genera have a specific prefcreiuje for particular habitats
and are to be found nowhere else.

Collection of Material. — Most of the Chlorophyta are easy to collect,

even if the collector has only the haziest idea just what genus or species it

is that is being collected. Simply place the material, if taken from water,
in a bottle of convenient size. The bottle should not be filled more than a
quarter of its capacity with any alga, the remainder of the bottle is to be
filled with the water in which the plants are growing. Lack of air causes
algae to begin rather prompt deterioration. Remove the cork or cap from
the bottle as soon as the return to the laboratory has been made.
Material collected on damp earth can be put in a pasteboard box or in
one of the cellophane wrappers from a pack of cigarettes for transportation
to the laboratory. Do not let such algae dry out.
Examine the immediate neighborhood of aquatic plants for flocculent
masses of algae. Take up some of the plants carefully, hold over the
opening of a wide-mouthed bottle and by running the fingers down the
length of the plant squeeze out the organisms which grow more or less
attached to the plants. Of course, one will scarcely get a pure collection,
but many exceedingly interesting species are to be obtained in this fashion.
227
228 SPECIAL METHODS FOR THE VARIOUS PHYLA

If one finds especially desirable forms, cultures can usually be started from
them.
As soon as the return to the laboratory has been made, the collections
should be examined microscopically. Discard all undesired material,
and preserve the remainder if cultures are not to be made. Watch par-
ticularly for the motile forms while these are still in the living, active
condition. It will probably be impossible to make slides of such organ-
isms, when very few in number and together with other and larger forms,
as they are very easily lost. It would be more practicable to grow a large
quantity of such species in more or less pure (^^unialgaD^) culture and to
make permanent mounts from such a culture. If only a few are available,
they can be mounted directly into glycerin or glycerin jelly and might
retain their color for some time.
Cultivation.— The unicellular and smaller colonial algae are not, as a
rule, difficult to cultivate and to obtain in practically pure culture. Vari-
ous methods following the well known plating procedure as used in
bacteriology have been proposed.
In preparing media on which to cultivate the Chlorophyta, the impor-
tant aspect to keep in mind is that the algae are extremely sensitive to
slight traces of metals.Pyrex glass-distilled water should always be
used, and containers which have previously held solutions of metallic
salts, mineral acids, or formalin should never be used.
If is one that has motile spores, the following method is excel-
the alga
lent. form is sufficiently abundant, platings may be made on a 1
If the %
agar medium prepared with 0.2% Knopfs solution. The alga will grow
rather slowly; it may be three weeks or longer before the colonies can be
observed amongst the numerous bacterial colonies which are rather
certain to appear in the meantime. After a plate showing a good growth
has been secured, allow it to dry slightly for several days to a week, then
transfer small colonies, as aseptically as possible, to vials of sterile dis-
tilled water. Zoospores be liberated promptly. These will
should
generally swim and some can be scooped up
to the surface of the water
with a platinum loop and spread in a drop of nutrient solution over the
surface of sterile nutrient agar in a Petri dish. In this manner pure
cultures are readily obtained and may be transferred repeatedly to both
solid and liquid media. Hanging drop cultures and permanent prepara-
tions can be made of almost any stage in the life history. To embed
material frona an agar culture, cut out small rectangular blocks of the agar
bearing the culture. The algae will adhere to the substrate throughout
the dehydration process (the agar ia nsisily embedded and sectioned).
Sections should be cut at from 2 to bn. To distinguish between the
nucleus and pyrenoids, use a triple combination; for other purposes use
iron hematoxylin.
 : :

CHLOROPHYTA 229

Present-day algologists consider soil-extract media to be far superior

to those composed solely of mineral salts for the cultivation of the green
algae. To prepare the stock solution (Bold 1936), autoclave 500 g. of
good field or garden soil in a flask with 1 liter of glass-distilled water, at
15 pounds pressure for 2 hours. Cool, decant, and filter several times
until the filtrate is clear. The designated as the stock soil
filtrate is
solution.” It may be diluted in various proportions with distilled water
and to each 100 cc. of dilution should be added 1 cc. of 5% aqueous
potassium nitrate. The following dilutions are the most useful (quanti-
ties are in cubic centimeters)

Solution number 1 2 3

Distilled water 94 84 74
Stock soil solution 6 16 25
5% aqueous potassium nitrate 1 1 1

The above dilutions may be solidified by the addition of 1, 2, or 3 g.

agar to each 100 cc. of solution. Since the agar may contain impurities
(unless a specially prepared granulated bacteriological agar is used), it is
wiser to take the precaution to place the agar in a cheesecloth bag, to
wash for 24 hours under running water, and then to soak in several
changes of distilled water.
A large number of purely chemical solutions have been proposed.
The following are the most useful of those which have given excellent
results in the hands of experienced algologists.

Modified Chodat-Grintzesco Medium (Fred and Peterson 1925)

Calcium nitrate 1 0
.
g.
Dibasic potassium phosphate 0 2
. g.
Magnesium sulphate 0 2
. g.
Potassium chloride 0 1
. g.
Ferric sulphate Trace
Distilled water 1 liter
Agar, washed and rinsed 10.0 g.

Autoclave. The reaction should be about 7.3. Pour a large number

of plates of high dilution of the alga to be cultivated. Invert the plates,
place under a bell jar in a northwindow or under artificial illumination
(see below),and allow to incubate for several weeks. From wellrisolated
colonies pick cultures, and streak on the surface of agar slants. To
determine whether contamination is present, make transfers from the
slopes which show a profuse growth into glucose broth and glucose yeast
water. To secure the maximum growth of algae in a short time, add
glucose or sucrose to the Chodat-Grintzesco medium. The carbohy-
 : : :

230 SPECIAL METHODS FOR THE VARIOUS PHYLA

drates are very beneficial, and the sugars are entirely assimilated in about
60 days. Large amounts of alga growth can be obtained by adding 75 cc.
of the medium, without the agar, to tall 16-ounce flat-shaped bottles; keep
in the greenhouse, fiat side down and plugged with cotton.

Modified Detmer^s Solution (Bold 1936)

2 % monobasic potassium phosphate 72 6 . cc.

2% dibasic potassium phosphate 21 .0 cc.

2 % calcium nitrate ]40 0 . cc.

2 % magnesium sulphate 87 5 . cc.

2% potassium chloride 87.5 cc.

0.1 % ferric sulphate 19 5 . cc.

Water 6672.0 cc.

Dissolve each of the chemicals separately in a large portion of the water,

then mix together, adding the calcium nitrate last.

Modified Knopfs Solution (Bold 1936)

Calcium nitrate 2 0 . g.
Monobasic potassium phosphate 0.6g.
Potassium nitrate 0 6 . g.
Ferric chloride (1 % aqueous) drop
1

Water 350.0 cc.

Dissolve the chemicals separately in portions of the water, then mix

together. The solution is considered to be a 1 % Knop solution. For the
Volvocales, dilute to 0.05%.

Modified Benecke's Solution (Bold 1936)

Ammonium nitrate 0.02%

Calcium chloride 0.01%
Dibasic potassium phosphate 0.01%
Magnesium sulphate 0.01%
Ferric chloride (1 % solution) 1 drop per liter

An intense but cold source of illumination is needed for the successful

cultivation of the Chlorophyta. The temperature should never exceed
22®C.; some species require a considerably cooler temperature. Most
people simply keep cultures in a window with a northern exposure, but
uniformly satisfactory results are rarely attained in this manner. A
source of artificial, controlled illumination is superior. The best type is

undoubtedly the new ‘^cold light’' apparatus recently devised by the

General Electric Company, but an equally satisfactory setup can be
arranged by utilizing apparatus found in practically every laboratory.
As depicted in the diagram (Fig. 27), the items consist of a bell jar with a
hole at the top for the insertion of a rubber stopper, a suitable tripod, a
500-watt electric lamp connected to an electric outlet in a suitable man-
 CHLOROPHYTA 231

ner, glass tubing, and rubber tubing for connecting the apparatus to a
water faucet. The diagram illustrates how the whole is put together.
As originally devised, the lamp was placed inside a large beaker placed
inside the bell jar, but experience showed that it was too difficult to keep
the beaker in position. Most 500-watt lamps have long necks, which
makes it possible to immerse the lamp in the water if care is taken not to
allow the metal screw end to get under water. The rate of flow of water
should be regulated so that no heat radiates below the bell jar. The
culture dishes may be arranged on glass shelves placed around the bell
jar, as well as directly below the
latter, and should be illuminated
for about 10 to 12 hours daily.
More detailed directions for
cultivating certain species, espe-
cially where concerned with the
inducing of reproductive phases,
will be given under the species
concerned.
Herbarium Specimens. T h e —
older made a regular
botanists
practice of drying Chlorophyta on
paper for preservation as herbarium
specimens, but not piany follow it
nowadays. Thick cellophane or
sheet mica is often substituted for
the paper.
—
Preservation. All Chlorophyta
can be permanently preserved, after
fixation and washing, in 3% for-
malin, to which should be added 6% Fig. 27.— Diagrammatic section of ap-
,
. . ji
1 i 1 paratus for furnishing artificial light for algal
j. i

glycerin in order that the mate- cultures. {Based upon Bold 1936.)
rial may not become completely

dry in cp^se the water evaporates. Formalin-aceto-alcohol will also

preserve material almost indefinitely.
For preservation in natural colors, use copper-lactophenol:

Phenol, c.p 20 g.
Lactic acid, sp. gr. 1,21 20 g.
Glycerin, sp. gr. 1.26 40 g.

Distilled water 20 cc.

Cupric chloride 0 2 • g.

Cupric acetate 0 2 . g.

Fixation. —Weak chrom-acetic fluids are by far the best for the major-
ity of Chlorophyta, although with some species other fluids are strongly
m special methods for the variovs phyla

indicated. Some technicians claim that the addition of osmic acid is

necessary for the most precise results, but in the writer^s experience this
claim has never been substantiated.
The proportions of chromic and acetic acids to be used vary according
to the species, and even to different lots of the same species. One may
take 1 g. chromic acid and 1 cc. glacial acetic acid to 100 cc. water as the
standard proportions. If fixation appears to be inadequate after these
proportions, reduce the amount of chromic acid, and increase the amount
of acetic acid. Thus 0.7 g. chromic acid and 5 cc. acetic acid to 160 cc.
water may give better results. Fix for 10 to 24 hours, and wash thor-
oughly with water. Some algae, such as certain of the Chlorococcales,
may be fixed satisfactorily for immediate transformation into permanent
mounts, but they cannot well be preserved unmounted for any length
of time.
Chlorophyta are frequently well fixed with formalin-aceto-alcohol
and 5 cc. glacial acetic acid to 100 cc.
in the proportion of 5 cc. formalin
of 50% alcohol. Wash in a change of 50% alcohol after 24 hours, and
proceed to the staining.
For marine Chlorophyta the above fluids (except for the formalin-
aceto-alcohol) may be used, but substitute sea water for distilled water.
Wash out with sea water, then transfer from sea to tap water for subse-
quent treatment.
The flagella and other delicate structures in thf small motile forms
are difficult to fix and preserve, but any sort of attempt may be better
than none. Try mixtures of iodine and formalin as suggested for Volvox.
—
Whole Mounts. The procedures outlined in the chapter on Whole-
mount Methods were designed primarily with the requirements of the
Chlorophyta under constant consideration, consequently reference to
that chapter should be made.
To prepare whole mounts of filamentous forms in nearly natural
color, add 6 parts formalin to 1 part of the copper-lacto-phenol mixture
described above, and fix the alga in this mixture. Then mount in glyc-
erin to which has been added 10 %
copper-lactophenol.
Staining. — A& a rule, iron hematoxylin, with a suitable counterstain,
is the stain par excellence for the Chlorophyta. Since the cell walls are
composed mainly of cellulose, a dye having an affinity for this substance
should be employed if it is desired that the cell walls stand out prom-
inently. Delafield’s or Harris^ hematoxylin can be combined with iron
hematoxylin (for the nuclei and pyrenoids) and fast green or safranin
(for the chloroplasts and cytoplasm, respectively) for this purpose.
Picro-indigocarmin gives a naturalistic green color to many of the colonial
species. Mayer's carmalum (made up with pure carminic acid) may
also be used for the same species.
 CHLOROPHYTA 233

Pyrenoids stain red or reddish after triple combinations; the starch

grains surrounding the pyrenoids should stain violet.

VOLVOCALES

Members of the order are flagellated in the vegetative condition and

may be either solitary or colonial. Typically, the cell is enclosed by a
definite wall, with a cellulose layer next the protoplast. The colonial
species are commonly surrounded by a gelatinous matrix.
—
Polyblepharidaceae. In this primitive family the protoplast is not
surrounded by a cellulose wall but is fairly rigid in the peripheral portion.
Dunaliella^ invariably present in salterns (thus giving them their
characteristic red color), probably the only form which the technician
is

might encounter. It may be mounted directly in glycerin. If found

in a concentrated salt solution, put a drop of the brine containing the
organism on a slide, fix for 1 or 2 minutes by suspending the drop over
the mouth of a bottle containing osmic acid, then let dry, stain in hema-
toxylin, and mount in diaphane. Too much salt may cause trouble in
the foregoing method, consequently a better plan would be to kill the
organisms in formol-iodine and to centrifuge to concentrate the very small
cells, whereupon they may be washed, placed in distilled water, and

subsequently treated for whole mounts.

—
Chlamydomonadaceae. ChlamydomonaSy naturally, is the genus of
greatest significance in the family. It is most likely to be found in
pools rich in ammonium compounds. Once one collects this genus, a
culture should be made; if a small amount is placed over clean white
sand in a Petri dish or similar receptacle and kept just moist, it will
remain in good condition for a long time. If zygotes should be formed,
the culture may be allowed to dry out and can be started into growth
again by simply adding sterile water. If grown on Detmer^s solution
solidified with agar, the colonies remain in a **Palmella^^ condition and
divide actively; if transferred to distilled water or to 0.05% Knop’s solu-
tion, they will return rapidly to* the motile condition. Formol-iodine
fixatives (see below under Volvox) are preferable. Iron hematoxylin or a
carmin stain is satisfactory. To reveal the flagella clearly, special
staining is required; fast green might be tried.
—
Phacotaceae. Treat like Chlamydomonas.
—
Volvocaceae. The most interesting and intensively studied of the
Volvocales belong in this family. The cells are arranged into coenobic
disks or hollow spheres. The genera may all be treated alike in the
manner described below in considerable detail for Volvox. The latter
genus has been selected simply because it is more likely tQ be found and
recognized than are the other genera.
 y :

234 SPECIAL METHODS FOR THE VARIOUS PHYLA

Getting Volvox in a satisfactory condition in nature is mostly a matter

of chance. The best places are deep ponds, small lakes or large quiet
ditches, whose waters are wholly or principally rain water. The plants
are more likely to be found along the edges, but the location varies
according to the species: some grow among grasses and aquatic plants
along the margins, others float in the middle of ponds together with
copepods and other plankton organisms, while still other species prefer
the bottoms. If the colonies arc present in small numbers, they may be
collected in quantity by means of a plankton net of fine bolting silk:
draw it slowly through the water in a likely spot, and if Volvox is present,
the bright green colonies are easily recognized. Let nearly all the water
filter out, then transfer the colonies to a large bottle or battery jar by

means of a giant pipette. The writer once collected more than a quart
of pure Volvox in less than half an hour by this method.
Volvox as well as Goniurriy Pandorinay and Eudorinay can be cultivated
without much difficulty. Luxuriant growtii has been obtained on Nos. 2,
3, and 4 soil solutions (page 229) and in 0.05% Benecke^s and 0.05%
Knop’s solutions (Bold 1936). Cultures may be grown on 1.5% Det-
mer^s agar, on which palmelloid colonies are produced, and may be kept
in this condition for some time. The following solution has also been
recommended
Potassium nitrate 0 25.
g.
Magnesium sulphate 0.26 g.
Calcium nitrate 1 0 g.
.

Monobasic potassium phosphate 0 25 g.

.

Potassium carbonate 0 345 g.

.

Ferric sulphate 0.0125 g.

Distilled water I liter

The pH of the above solution should be about 7.6 at 18 to 20®C.

When ready to use, take 1 part of the solution and 9 parts distilled water,
and pour 100 cc. into flasks of 200-cc. capacity. The flasks should be of
alkalLfree glass if the freedom of the glass from soluble alkalies is ques-
;

tionable,, wash with strong sulphuric acid, and rinse out the acid thor-
oughly with, distilled water. Be sure that the colonies forming the basis
of the culture are reasonably free from other algae^ and use only a very
few colonies for starting each culture. Grow under cool artificial
illumination.
FoZirar does not keep in preserving solutions without gradually
undergoing collapse. Consequently, when one obtains an excellent
culture, whether in nature or by cultivation, it would be well to make it
into slides at once.
In handling Volvox through the killing and subsequent procedures, it
is important to prevent the colonies from becoming clumped together.
 CHLOROPHYTA 235

This can be done by keeping all fluids slightly acidified (with acetic acid
if any particular solution is not already acid). The most satisfactory
killing fluid is composed of the following:

Potassium iodide 2 g.
Iodine 1 g.
Formalin 24 cc.
Glacial acetic acid 4 cc.
Water 400 cc.

Dissolve the iodine and iodide in the water first, then add the othei
ingredients. The colonies may be concentrated, if necessary, by filtering
out the water through coarse paper or bolting silk. The colonies
filter

should be left in the killing solution for about 48 hours, preferably longer.
Protoplasmic connections, flagella, and internal details of reproduction
should all be perfectly preserved. Wash out with water slightly acidified.
Picro-indigocarmin (0.25 g. indigocarmin in 100 cc. of a saturated
aqueous solution of picric acid) is an exceedingly beautiful stain for
VolvoXj provided it can be kept from becoming extracted during the
dehydration. Leave the Volvox in the stain for a week or much longer,
as there is very little chance of overstaining (Fig. 28). Mayer^s car-
malum is also excellent, as is Lynches precipitated carmin, but ^neither is
too precise. The carmin dyes also require a lohg‘ period to take effect,
and dehydration should be by an extremely gradfial process. With
some species, but not all, iron hematoxyluv be used, but the mordant
must be used in very dilute concentration it,tends both to collapse and

to clump the colonies. About 1 drop of ^%tfei?ric ammonium, sulphate

in 250 to 300 cc. of acidified water may be tried. Use for about ^4 hours,
wash with acidified water, then apply the staining solution in high dilu-
tion. Differentiate in a warm saturated aqueous solution of picric
acid, and counterstain with highly diluted fast green.
The problem of getting Volvox through any dehydrating process is
exceedingly difficult. Stages must be extremely gradual, and all diffusion
currents must be avoided. The glycerin evaporation process, with
washing in 95% alcohol, is excellent. The gradual drop method may be
employed with hygrobutol, or dioxan in a very close series of stages
sometimes works beautifully.
Volvox is readily embedded and sectioned. Use the iodine-formalin
fixing fluid, wash out quickly, and dehydrate by a very gradual series.
Almost any desired series may be used. The material is kept in thin
shell vials during all the various processes (the vials in which photo-
graphic developers come are useful); the colonies will sink soon after
each change of fluid, and the liquids may be withdrawn with a pipette.
Leave in the paraffin oven for only a short time, and make changes of
paraffin by means of a pipette warmed to the temperature of the paraffin.
 236 SPECIAL METHODS FOR THE VARIOUS PHYLA

The paraffin may be and the glass broken after

solidified in the vial
cooling, or the material may be poured into
a small paper tray to give a
layer of material 2 to 3 mm. thick. One
will have to sacrifice some
material in trimming down the block for microtoming. Cut sections

28 . whole mount of vegetative colonies. The colonies commonly

Volvoz sp. :

become more or clumped together. Fixed in special formol-iodine fluid; stained with
less
picro-indigooarmin. Dehydrated with hygrobutol and infiltrated with balsam.

at about 4 m. Stain preferably in iron hematoxylin with orange G for

counterstain.
Spondylomoraceaei Sphaerellaceae. —Treat members of these fami-
lies exactly like Chlamydomonas,

Tetrasporales
This order is made up principally of genera which may only occa-
sionally be encountered. Whether unicellular or united to one another in
 CHLOROPHYTA 237

colonies, the cells are surrounded by gelatinous sheaths. These sheaths

are either distinct or confluent.
Cultivation may be in one of the soil solutions, the optimum dilution
needing to be determined by experiment.
The smaller forms may either be dried down on the slide directly
after first killing over the fumes of osmic acid; or they may be killed and
dehydrated as far as 95% alcohol, when a drop of the material may be
placed on a slide smeared with Mayer’s adhesive and then stained with
dyes dissolved in 95% alcohol or clove oil. If the species forms colonies
of a sufficiently large size to be easily handled, they may be killed,
dehydrated very gently, embedded in paraffin, and sectioned. Most
dehydrating fluids are apt to cause excessive hardening of the gelatinous
matrix.

Ulotrichales
The genera in the order possess simple or branched filaments which
make unusually good preparations.
niotrichaceae. —Filaments unbranched.
of all genera are Ulothrix
is the commonest genus, but not so readily found in most regions as
it is

are others. It occurs as light green masses in quiet or running cold water,
attached to stones. Reproductive phases occur only after midnight
and are completed early the next morning; vegetative phases only are
found after about 9 a.m.
Kill and fix in chrom-acetic. Wash, stain with iron hematoxylin and
follow the hygrobutol method, counterstaining with fast green. Fila-
ments of Ulothrix commonly have the annoying peculiarity of clumping
together during the later stages of the dehydration, or at the beginning
of the infiltration, process. However, if the bunched filaments are cut
into portions not over 5 mm. drop of balsam on a
in length, placed in a
slide, and then gently pressed with a needle spatula, they will become
separated. Care should therefore be taken not to place too much
material on each slide.
Ulothrix is easily embedded and sectioned. A large quantity of
filaments showing the formation of zoospores and gametes may be
wrapped in lens paper, carefully tied at the ends with fine thread, and the
whole run up into paraffin. Microtome at 3 to Gm, and stain with iron
hematoxylin and fast green.
Cultivation of vegetative filaments may be in undiluted Detmer’s
solution or in No. 2 soil solution. Zygotes of Ulothrix may be germinated
on an agar medium:
Distilled water 200 cc.

0.06% aqueous potassium nitrate 100 cc.

Dried earth or loam 30 g.

Agar To 1%
238 SPECIAL METHODS FOR THE VARIOUS PHYLA

Autoclave, then pour into Petri dishes or similar containers. Keep the
cultures at 10°C. under artificial light.
Ulothrix is perhaps one of the most desirable forms for demonstrating
the differences between zoospores and gametes. There is an alternation
of generations in the genus (Grosse 1931).
\
—
Microsporaceae. Microsporaj to be expected in early spring in
pools and ditches, has a peculiar wall structure. Treat it as described
for Ulothrix,
Cylindrocapsaceae. —
Treat as for Ulothrix,
—
Chaetophoraceae. Stigeoclonium and Draparnaldia are found in
clear, cool, running water, but the first genus sometimes occurs in quiet
ponds or lakes. In such situations it is frequently found producing
akinetes and is one of the best Chlorophyta in which to demonstrate this
method of reproduction. Kill with 1% chrom-acetic, stain with iron
hematoxylin (paying particular attention to the differentiation of the
akinetes)and fast green, and follow the hygrobutol method. Stigeoclon-
ium usually breaks apart more or less, but Draparnaldia sometimes forms
such large plants that they must be reduced to smaller portions for
mounting.
Genera such as AphanochaetCy ThamniochaetCy and ChaetopeltiSy which
grow upon other filamentous algae, should be worked up together with
the host since it is quite impossible to separate the two.
Protococcaceae. —Protococcus (Pleurococcus) is the only genus occur-
ring in the United States, and should be available almost everywhere.
There is, however, great confusion as to the identification of the Proto-
coccaceae and one should therefore be cautious before concluding that
any unicellular aerial green alga is a Protococcus, Protococcus is easily
isolated and cultivated on Beijerinck^s ammonium nitrate agar:

Distilled water 1000 cc.

Ammonium nitrate 0 6 . g.
Monobasic potassium phosphate 0 2 . g.
Magnesium sulphate 0 2 . g.
Calcium chloride 0 1 . g.

After the chemicals have been dissolved separately in portions of the

water and then mixed together, add 20 g. agar, boil until the latter is
dissolved, then autoclave. Pour into Petri dishes or similar containers.
To inoculate, mix a little of a coating of the alga from tree trunks (prefer-
ably from Tilia)y and pour over the plates. Pour or pipette off any excess
water. The colonies will appear after about three weeks.
Staining of Protococcus is not easy. Many technicians stain with
fast green alone, others with a carmin stain (which obviously gives them
an unnatural color) or with picro-indigocarmin. A really satisfactory
stain has not been described. When iron hematoxylin is used, it is
 CHLOROPHYTA 239

extracted from the very small nuclei before the cytoplasm is sufficiently
differentiated.
—
Coleochaetaceae. Coleochaete is such a small plant that the use of a
high-power microscope is required to detect it. The largest colonies
or thalli probably will not be found to exceed 5 mm. in diameter. The
thalli grow on the stems of various water plants, par-
leaves, culms, or
Typha, and occur from just below
ticularly Sagittaria, Isoetes, Elodea, or
the surface of the water to about 6 inches below (Wesley 1928) and on the
sides of the host that receive the most light.
It is inadvisable toattempt to separate the plants entirely from the
host. Small portions of the host bearing the thalli may be cut away; if
the plants grow on Elodea leaves, whole mounts of the latter are excellent.
Kill and fix with 1 %
chrom-acetic for 1 hour, wash thoroughly, then stain
with either safranin and fast green (the latter in aqueous rather than
alcoholic solution) or Harris^ hematoxylin and fast green, and follow the
hygrobutol method. Beechwood creosote or dioxan might be tried, but
the material will be so hardened after xylol or a similar fluid that it cannot
be mounted. Except for details of zoospore formation, sections are of
little service. Material which is to be embedded should be fixed in a
strong chrom-acetic fluid, dehydrated by a very gradual series, embedded
in bunches, microtomed not thicker than 8 m, and stained with iron hema-
toxylin and fast green.
If zoospores are wanted, arrange slides on the bottom of a large,
shallow dish, fill the latter partially with tap water, place some mature
thalli of Coleochaete in the water, and set the whole in the sun. The
zoospores will attach themselves firmly to the slides, and the latter may
be carried through all the killing, fixing, and staining processes as if they
carried sections. For staining, try acid fuchsin.
Trentepohliaceae. — Trentepohlia is a strictly aerial genus. Filaments
may be scraped off the bark on which they grow and mounted directly
in glycerin jelly without any special treatment. The color is retained
for many years.

Ulvales
Ulvaceae. — Most of the species are marine in habit, but some grow in
brackish, a few in fresh, waters.
Ulvay a marine genus,very common along both coasts and is easily
is

recognized and collected. Fix portions of the thallus, including some

from the marginal region, in 1 %
chrom-acetic in sea water, dehydrate, and
embed in paraffin. C/Zva- tends to become excessively hardened during
the clearing and infiltration if either xylol or chloroform was employed,
consequently, tertiary butyl alcohol, which has little or no hardening
effect, should be used. Since sections cut perpendicular to the flat
240 SPECIAL METHODS FOR THE VARIOUS PHYLA

surface are required if and gamete formation are

details of zoospore
desired, pile several p)ortiohs of thalli on top
one another to a depth of
of
2 mm. or so while the embedding is being done, and press together gently
with a blunt instrument if they do not lie perfectly flat. In this manner
a long series of sections can be cut with a minimum of effort. For repro-
ductive details sections should not be over 3 m; for general structure 10 m
is thin enough. Stain in iron hematoxylin with orange G, or a triple
combination might be substituted.
Even if the thallus of Ulva is only two layers of cells In thickness, whole
mounts are usually unsatisfactory. The presence of considerable iron
in the cell walls inhibits proper staining.
Monostroma, which favors brackish waters, forms a sheet-like thallus
one cell in thickness. Use a weak chrom-acetic, but make the fixation
period very brief. The gelatinous consistency of the thallus causes it to
become readily dissociated. By fixing the entire plant rather than small
portions of the whole, one has a greater chance of avoiding disaster.
Embed as described for Ulva, Material intended for whole mounts
should be dehydrated with hygrobutol, or with glycerin, which may in
turn be washed out with 95% alcohol. Picro-indigocarmin is the pre-
ferred stain.
Enteromorpha has a hollow tubular thallus one cell thick and* favors
more or less saline habitats. It may be treated like Monostroma, but
the thallus would have to be cut open and spread apart for mounting.

ScHIZOGONIALES

Thalli of the Schizogoniales resemble those of the Ulvales. Repro-

duction is by akinetes and aplanospores. It is scarcely to be expected
that any technicians save those specializing on the algae will come into
contact with members Nothing has been described in the
of the order.
literature about their treatment, but Prasiola, which is to be found in the
Rocky Mountains and the Sierra Nevada at isolated stations and at a few
marine localities on both coasts, may be dealt with exactly as if it were
like Ulva (Fig. 29). For whole mounts, stain with picro-indigocarmin.

Cladophorales
The multinucleate cells of the simple or branched filaments have
numerous chloroplasts. Some species are marine, others are fresh-water.
Cladophoraceae. — The thick stratified cell walls are composed of
three layers: an outermost zone consisting chiefly of chitin, a middle
zone rich in pectic substances, and an inner cellulose layer. The absence
of an external pectic layer explains why Cladophoraceae are usually so
heavily encrusted with diatoms and other epiphytic algae.
 CHLOROPHYTA 241

Cladophora is one of the commonest of marine and fresh-water Chloro-

phyta in the United States. It will be found more expedient to select
species, from either fresh or saline waters, that have short branching
filaments not so densely interwoven as to make it difficult or impossible
to untangle them when they have finally been brought into balsam for
whole mounts. Such forms, also, are more likely to be found with
reproductive stages.

Fig. 29 . —Prasiola nevadensis: whole mount of a portion of the thallus, showing charac-
arrangement of the cells.
teristic Fixed with formalin-aceto-alcohol stained with Harris’
;

hematoxylin and fast green.

Kill and fix with a medium chrom-aceticfluid, stain with iron hemato-
xylin anti fast green, and run into balsam preferably by the hygro-
butol method.
Rhizoclonium, Chaetomorpha^ and other common genera of the order
may be treated in the same manner as Cladophora. Some care should
be exercised not to destain the iron hematoxylin too far or to overstain
with the fast green.
—
Sphaeropleaceae. Sphaeropha is of particular interest because of
its method of oogamous sexual reproduction. It develops to maturity
and disappears within a month; consequently it is not often collected. It
is most likely to be found in early or late spring. Fix with chrom-acetic,
stain with iron hematoxylin, but take care not to overdo the counterstain-
ing with either fast green or orange G. Make transfers from one fluid
242 SPECIAL METHODS FOR THE VARIOUS PHYLA

to another very gradual as the unusually large cells of the filaments

collapse readily.

Oedogonialbb
Members unique method
of the order are particularly noted for the
of cell division; manuals should be consulted for a description of th(*
process (G. M. Smith 1933). Very critical staining is required to bring
out the details of the process, and one should employ material killed after
midnight. The presence in certain species of dwarf male filaments bear-
ing antheridia makes the group especially interesting.
All the genera, Oedogoniunif Bulbochaetej and Oedocladium, may be
treated alike technically. Species of all genera may be easily grown in
soil solution No. 3 (see also Mainx 1931). If the material collected is
sterile, it can sometimes be induced to produce antheridia and oogonia

by artificial means. Place not too many of the filaments in a large con-
tainer of any nutrient solution, preferably Knopfs, highly diluted. After
several days transfer to distilled water, and observe from time to tim(‘.

Do not place in direct sunlight.

Fix in a chrom-acetic fluid, with the following proportions: 1 g.
chromic acid, 2 cc. glacial acetic acid, and 100 cc. water; The formol-
iodine fluid recommended for VolvoXj or even formalin-ace^o-alcohol, may
be tried. Iron hematoxylin is the best stain, but the oogonia must ho
very carefully destained to the proper degree. For details of the methods
of cell division and reproduction paraffin sections are deemed necessary
by most workers.
Material may be preserved in a 2% aqueous solution of potassium
acetate to which are added suflficient cupric acetate to color the mixture
and a little phenol to prevent mold growth.

Zygnematales
Members of the order are common, readily distinguished, and easily
.manipulated algae. The cell wall is two-layered, consisting of an exter-
nal pectic layer and an internal cellulose layer. The pectic external
layer becomes so mucilaginous in the filamentous species that they feel
slippery to the touch. In the Desmidiaceae there is a third, intermediate
layer composed impregnated with pectic substances and ferric
of cellulose
salts. Special staining technique is required to differentiate between the
cell layers and their constituent substances (Liitkemiiller 1902).

There are no marine Zygnematales; the species are aU confined to

fresh-water ponds, swamps, creeks, rivers, or ditches.
—
ZygnematacSeae. The most intensively stjudied of all algae, SptVo-
gyra, belongs in this family. The genus is cosmopolitan and should be
 CBLOHOPHYTA 243

readily available to every student commencing microtechnique. Where

Spirogyra too rare, related genera should be easily found in place of that
is

genus. In many localities Zygnemaj as for instance in southern Cali-

fornia, is more common than Spirogyra. As a last resort, well-fixed
material is obtainable from the botanical supply concerns.
It is not an easy matter under average circumstances to obtain Spiro-
gyra and other filamentous Zygnematales in the conjugating condition.
This requires periodic examination of specimens from any given locality.
There is a strong seasonal periodicity in the occurrence of conjugation,
which varies according to the species. Most species conjugate in the
spring, but there are some which do so in summer and others in the
autumn. Contrary to the claims made by a few writers (e.gr., Chamber-
lain 1932), conjugation cannot be induced at will (G. M. Smith 1933) the-
;

writer, despite repeated attempts, has never been able to get any species
to conjugate by artificial inducements. It is probably impossible to
duplicate under artificial conditions the exact combination of circum-
stances which induces conjugation in nature. It is also difficult to keep
Zygnematales in culture vegetatively and is scarcely worth attempting.
Methods for preparing whole mounts of Spirogyra are given in con-
siderable detail in the chapter on Whole-mount Methods, to which
reference should be made. These schedules are applicable to all the
other genera. The combination of Delafield\s or Harris’ hematoxylin
with iron hematoxylin and fast green should particularly yield prepara-
tions of surpassing beauty.
All the genera are easily embedded in parafl5n, but such preparations
are of value only in critical cytological investigations.
—
Mesotaeniaceae. Treat like members of the following family.
There is, however, no impregnation of the cell walls with iron compounds.
—
Desmidiaceae. The unicellular Zygnematales, the desmids, are
among the most beautiful of all unicellular organisms because of their
astonishing diversity in cell structure and especially in ornamentation.
Unfortunately for the technician, who usually does not like to see ‘‘some-
thing of everything’’ in his preparations, too many of the desmids occur
in mixtures of several species or even genera. Sometimes, howeverj the
patient collector may have the good fortune to find some particular form
in unialgal culture in nature. Under such circumstances the most should
be made of these opportunities. Most desmids occur in somewhat acid
waters, or where the pH ranges between 5 and 6. This fact gives a clue
to the method of treatment for these organisms. Follow the suggestions
given for Volvox. Use a centrifuge to get the algae to settle between
changes of fluids. Iron hematoxylin is the best of all nuclear stains,
especially for the larger species, and any desired counterstain may be
used when one seems advisable.
244 SPECIAL METHODS FOB THE VARIOUS PHYLA

It has also been stated that excellent fixation may be obtained with
2 to 3% formalin plus a few drops of acetic acid. Dehydration is by a
6% series of steps of ethyl alcohol to 95% alcohol, allowing 15 minutes in
each percentage. Staining is for 12 to 48 hours in 1% light green in
95% alcohol; wash in clean 95%, then complete dehydration with hygro-
butol, and infiltrate with balsam.

Chlorococcales
The Chlorococcales are mostly unicellular or colonial organisms, of
the greatest diversity in form. Because of the small size of most of the
species, they are difficult to handle during the dehydrating and mounting.
However, if one is fortunate enough to obtain any species in sufficient
abundance, the loss of a considerable proportion of the collection is not
such an exasperating matter.
The proper staining of Chlorococcales is a problem which has scarcely
been attacked. Different lots of the same species react differently to the
same staining procedure. The writer once had the experience of obtain-
ing a beautiful iron hematoxylin stain on one lot of Scenedesmus but
failed completely with material collected from the same locality ten days
later. Others report experiences of the same sort. The difficulty appar-
ently resides in differences in the pH of the water in which the organisms
were growing and which had a direct effect upon the affinity of the cells
for dyes.
For fixation of bulk material use a weak chrom-acetic (with 1 or 2
drops osmic acid solution to each 100 cc. fixative, if desired), or 5 cc.
formalin and 5 cc. glacial acetic acid in 100 cc. distilled water.
A method which has been found to be entirely adequate for all but
the most exacting cytological investigations is to smear a drop of Haupt’s
adhesive over a chemically clean slide, add 1 drop of the suspension
containing the alga, then invert the drop over the mouth of a bottle
containing any osmic acid solution for about 10 seconds. Next put the
slide in a slide box, drop up, and when the box has been filled but before
the drops dry out entirely, place the box, together with a watch glass full
of formalin, in the paraffin oven. The fumes of the formalin will fix the
gelatin of the adhesive and cause the organisms to stick to the slides.
Remove the slides from the oven as soon as the film has dried completely.
Iron hematoxylin staining may When successful, the
be attempted.
results are of surpassing beauty. Counterstaining may be with fast
green. For ordinary purposes, such as class use, a variation of Flem-
ming^s triple combination usually gives an excellent though exceedingly
variable stain. When this combination is used on Scenedesmus, there is
no consistency in the affinity of the different structures for the three dyes
involved. First immerse the slides in 10% alcohol, fqr overnight or
 CHLOROPHYTA 245

longer, to extract the chlorophyll. Place in 1% safranin in 50% alcohol

for 12 hours to 4 days; the time varies with each collection of material, but
there is little danger of overstaining. Wash in running water for 5 min-
utes, then place the slides in 1% aqueous crystal violet for two days.
There is more danger in overstaining with the violet than with safranin.
Rinse off excess stain with water, then place in a saturated aqueous
solution of orange G for not longer than 5 minutes. Rinse* quickly with
water, then with 95% and absolute alcohol for a few seconds in each.
Differentiate with clove oil; the time is variable, but one should leave the
slides in the oil until the nuclei are clearly visible when the preparation
is examined under the microscope. Wash in two changes of xylol, and
mount in balsam.
These algae may, of course, be mounted entire in glycerin without any
special treatment, but they will retain their color for only a short time.
Only the more prominent genera are being mentioned in the following
discussion.
Chlorococcaceae. — Trehouxia is the alga most commonly present in

lichens, though not thft only unicellular one to be found in these plants.
Sections of the lichen thallus {Sticta or Usnea is to be recommended)
should readily reveal the alga. When staining such material for the
algal member alone, disregard the fungal member of the association as it is

impossible to stain both equally well in most lichens. Iron hematoxylin

or a quadruple schedule might be followed.
Protosiphonaceae. —Protosiphon is usually found associated with
Botrydiurn^ and the two are difficult to distinguish in the field. In the
laboratory the iodine reaction for starch may be applied: Protosiphon
reacts for starch, Botrydium does not. It is usually too difficult to sepa-
rate the plants completely from the mud
which they grow. Cultures
in
of Protosiphon are easy to secure (Bold 1936). Remove some of the
plants from the mud with a microneedle, and place in the darkened
portion of a unilaterally illuminated Petri dish containing sterile distilled
water. Gametes which are strongly positively phototaxic are liberated
in 5 or 6 hours. Some of these may
be taken up in a loop and streaked
over the surface of sterile solidified Detmer's agar in Petri dishes. The
germlings will require about two weeks under artificial illumination to
reach a sufficient size. Gamete formation and conjugation may again
be induced as just described. Kill in a weak chrom-acctic fluid, and run
into balsam by a very gradual method, as plasmolysis easily ensues if
sudden changes are made.
— ^

Hydrodictyaceae. Pediastrum is frequently encountered in plankton

collections from pools or large ditches, but it is rarely found in abundance
at most localities where it occurs. Specimens may be isolated and grown
in No. 2, 3, or 4 soil solutions (Bold 1936). This fascinating little alga
246 SPECIAL METHODS FOR THE VARIOUS PHYLA

may be treated as described for Scenedesmus (page 244), or if suflSciently

abundant to be handled in bulk, fix with a weak chrom-acctic or with
formalin-aceto-alcohol, stain with iron hematoxylin, safranin, or picro-
indigocarmin, and run up by the hygrobutol method.
Hydrodictyon widespread and can sometimes be found in enormous
is

quantities in quiet waters. The nets will generally be in the vegetative

condition, but one might be fortunate enough to encounter young nets
within the mother cells of the old nets. Under artificial illumination,
growth in soil solutions Nos. 3 and 4 has been luxuriant. A single young

Fig. 30.— Scenedesmus sp.: whole mount from a pure culture. Fixed with osmic acid
vapor and dried to the slide; stained with safranin, methyl violet, and orange G. Note the
differences in stain retention by the various colonies.

net or a small portion of an older net should be rinsed through several

changes of sterile soil solutions 1 or 2 before being placed in No. 3 or 4
in a large culture vessel (Bold 1936). If vigorously growing nets are
transferred to fresh nutrient solution, zoospore formation will be stimu-
lated (Mainx 19316). Various fixatives have been recommended for
Hydrodictyon. them may cause plasmolysis under circumstances
All of
for which there is no plausible explanation. Formalin-aceto-alcohol
may be used on species which feel rather brittle when handled; for the
others a weak chrom-acetic (say, 0.5 g. chromic acid and 5 cc. acetic acid
to 100 cc. water) be satisfactory. Many of the older technicians
may
found that JuePs gave excellent fixation (96 cc. 60% alcohol, 2 g.
fluid
zinc chloride, 2 cc. glacial acetic acid). Iron hematoxylin with fast green
is a good stain combination, but take care not to overstain with the green.

Run up by a very gradual method into dilute balsam. In mounting, use

scissors fre^ely to cut the nets into suitable portions.
 CHLOROPHYTA 247

Oocystaceae. —Free-living species may be treated as described for

Scenedesmus, Species of Chlorella are found in Hydra, Stentor, and Para-
mecium. Texts on zoological technique should be consulted for methods
of preparing slides of these animals.
Scenedesmaceae. —General methods of ^
dealing with the species
included in this family have been described above. Scenedesmus is an
ubiquitous alga and exceptionally easy to culture (Fig. 30).

SiPHONALES

The genera arc' mostly marine and confined to warmer waters, but a
few are predominantly fresh- water, and three are parasitic.
—
Bryopsidaceae. Bryopsis prefers warmer seas but at least one species
extends into icy waters on both the Atlantic and Pacific Coasts. This
genus usually fruits in spring or early summer and occurs at, or below,
low-tide level. The species are randy f(3und in abundance. Most of the
available forms are sufficiently small for whole mounts. Kill and fix in a
weak chrom-acetic in sea water, transfer to distillcHl water, stain with iron
hematoxylin with a suitable counterstain, and run into balsam by an
extremely gradual schedule. The plants, as usually found, are sterile,
but if brought into the laboratory and kept in cold sea water, the gametes
willappear in three or four days.
Codiaceae. —
The family is a peculiar and interesting one. Tlu'. plant,
body consists of branched coenocytic filamentous cells twisted together to
form spongy thalli of more or less specific form. A few species are
calcified.
C odium was not easy to get into paraffin by the older methods and
usually cracked to pieces during the microtoming. Sections are of
dubious value, except for the reproductive stages. For such studu's kill
in a strong chrom-acetic and get into paraffin by the method described
for the Phaeophyta (page 268). Microtome at not over lOg, preferably
transversely, and stain in iron hematoxylin with either erythrosin or
orange G as counterstain. Whole mounts of the individual cells with
their gametangia attached at one side are of considerable service. Cut
out portions of the thallus under water, and place in 6% hydrochloric
acid until, on gentle stirring, the mass becomes dissociated fragments.
Wash by de(;antation or by pipetting off the w^ater after the mass of cedis
settles down, then place in a solution of 10% formalin and 3% glacial
acetic acid in sea water to complete fixation. Wash out this mixtun'.
thoroughly, stain in iron hematoxylin or Harris^ hematoxylin, counter-
stain lightly with fast green, and proceed to the hygrobutol method.
Halimeda is a tropical genus and may be found in Florida. The
plants are calcified but are easily sectioned after fixation in a fluid con-
248 SPECIAL METHODS FOR THE VARIOUS PHYLA

taining at least 5% acetic acid. Microtome perpendicular to one flat

surface, and stain with iron hematoxylin and fast green.
—
Derbesiaceae. Derhesia somewhat resembles Bryopsis in habit, also
prefers warmer seas and has about the same geographical range. Treat
like Bryopsis,
Vaucheriaceae. — Vaucheria is the most conspicuous fresh-water
representative of the order since it is to be found almost everywhere.
One may on pots in the greenhouse, on damp soil, in quiet fresh
find it
waters, in brackish and saline waters. It usually forms felt-like masses,
(jasily distinguishable by their glistening appearance when examined in
sunlight. Sterile material is worthless and uninteresting; only material
showing at least antheridia and oogonia is worth preserving. Vaucheria
is reputed to be difficult to kill and fix satisfactorily, and this reputation

is often found to be sustained. Formalin-propiono-alcohol gives good

I'esults with most species; the weak and medium chrom-acetic mixtures

too often cause considerable plasmolysis and, as a rule, are not to be

recommended. The amount of formalin in the formalin-aceto-alcohol
should be increased to 10 cc. with the usual 5 cc. acetic acid to 100 cc. of
50% alcohol. After this fluid very satisfactory results have been
obtained with even a short balsam infiltration schedule, since Vaitcheria,
if properly fixed, is not so prone to become plasmolyzed during the later

processes as are many other filamentous Chlorophyta. Iron hematoxylin

is by far the best basic stain, but great care must bo taken to differentiates

the oOgonia properly, as they commonly tend to remain overstained.

Use fast green for counterstain.
If the only available material is
sterile, it can sometimes but not
often be induced to produce the
reproductive structures. The fol-
lowing methods have been sug-
Fiq. 31
.
—
Vaucheria terreatrie: whole
gested. If placed in a 4% aqueous
mount of a germinating aplanospore. Fixed
with formalin-aceto-alcohol; stained with solution of sucrose, aplanospores
iron hematoxylin and fast green.
may be obtained in a few days
and then germinated in a nutrient solution (Fig. 31). In the same
solution, if pl^ed in bright sunlight, antheridia and oogonia may also
appear. To induce the formation of zoospores, place the material in
Knopfs solution diluted to 0.2% strength for a week or longer, then place
in tap water, and put in a cool dark place. Signs of zoospore develop-
ment may be seen within two days. If such signs are not forthcoming,
strengthen the nutrient solution, pflace the culture in the sunlight for
several days, and then put back in the dark. Production of zoospores
under such conditions may continue for a fortnight. In place of Knopfs
diluted solution, one might try a 2% sucrose solution.
For bubit studies preserve the material in 3% formalin,
 CHLOROPHYTA 249

SiPHONOCLABIALES

The genera are all subtropical or tropical marine plants.

Valoniaceae. — Valonia is common in Florida. Portions of the thallus
may be cut out and fixed in Bouin^s fluid,which appears to be as good as
any (Schechner-Fries 1934). Rather thin sections (at about should
be cut, and these may be stained with iron hematoxylin.
—
Dasycladaceae. Most of the species are calcareous, which explains
why exceedingly about the technical manipulation of these forms is
little

available. However, since the lime-encrusted Rhodophyta have been

shown to offer practically no difficulty, there is no reason why the similar
forms in the Chlorophyta should not be equally amenable to treatment.
Species of Acetahularia and Neomeris from Florida are less than 10 mm. in
height and should be easy enough to decalcify and mount entire. Other
species of much larger size are also to be found in Florida.

CHAROPHYCEAE
The Charophyceae are placed by most recent writers with the Chloro-
phyceae (G. M. Smith 1938). Chara and Nitella are the principal genera;
both are aquatics inhabiting ponds and lagoons and to a lesser extent
ditchevs. Chara generally grows in warmer waters; the most luxuriant
growth the writer has ever seen occurred in small pools on the floor of
Death Valley.
Chara is not mentioned in most botanical texts, but it nevertheless is
a most interesting and instructive plant and incidentally is one in which
the fixation and staining will try tKe skill of the technician. This is
because of its fragile structure, the calcium deposits, and its tendency to
become exceedingly brittle if proper care in the dehydration is not taken.
It is as difficult as with the aquatic Angiosperms to stain sections sharply.
Cultivation. — Chara is easily grown in a large aquarium or in a jar of
at least 2-gallon capacity. Only a few plants should be put in the con-
tainer, and they should be rooted in a layer of pond mud and sand about
I inch deep.
Germinating stages can be secured by the culture method only. It
has been claimed (Chamberlain 1932), but not substantiated by the
experience of others, that old plants of Chara can be collected from a
dried-up pond, stored for at least a month, and the zygotes removed and
germinated in a shallow container containing tap water. Filamentous
growths are .supposed to appear shortly and lateral buds give rise to the
typical Chara plant. These germlings, if available, are easily mounted
entire by the general methods for the Chlorophyta.
—
Whole Mounts. There are two general methods of preparing whole
mounts of the sexual organs and thallus of the Charophyceae. In one
the (^ustomaiy killing, fixing, and staining are carried out as desired; in
260 SPECIAL METHODS FOR THE VARIOUS PHYLA

the other the plants are killed and fixed so as to retain the natural colors
of the different organs.
In preparing mounts by the first general method, kill and fix in a
strong chrom-acetic fluid or in formalin-aceto-alcohol. Wash out the
killing fluid, which should have dissolved most of the encrustations, and
stain by any desired in toto combination. Mayer^s carmalum, Harris^
hematoxylin with fast green, methyl green acidified with acetic acid, or
even safranin and fast green, may be tried. It will scarcely be possible
to obtain excellent internal staining of older antheridia or oogonia, but
the development of the younger sex organs may be easily demonstrated.
The apical cell will b('. too hidden by the enveloping leaves to be seen in
whole mounts, although there may be species of Nitella in which this
would be possible. Older antheridia may be run up entire, then crushed,
teased apart carefully in a drop of balsam, and a coverslip added; thus the
filaments, shield, manubrium, and capitula may be observed.
Permanent mounts of the sexual organs and thallus, in whi(‘h all the
natural colors arc retained, are most instructive and appanmtly not too
difficult to prepare (Woods 1929).
1. Soak the desired portions in cool tap water for two days to remove
part of the encrusting lime.
2. Remove air by pumping or by immersion in cool boiled tap water.
3. Fix in the following solution for 4 hours:

Water 200 oc.

Glacial acetic acid 8 cc.

Cupric acetate, c.p 1 g.

(Cupric sulphate may

be used in place of the acetate.) The volume of
solution should be fifty times that of the material. Agitate occasionally
to dislodge the bubMes of carbon dioxide.
4. Pour off the fixing fluid, and add to it just sufficient concentrated

ammonia to change the blue color to a decidedly purplish tint. Pour

back over the material, and let it remain for about 1 hour or until the
thallus has turned a markedly blue-green color.
5. Wash in running tap water for 10 minutes, then soak in several
changes of distilled water for 5 minutes. The thallus should now be a
bluish-green, the antheridia orange, and the oogonia brown and green.
Transfer to 5% glycerin in distilled water. If plasmolysis occurs here,
omit the washing in tap water, and go directly to the 5% glycerin.
6. This step must be carefully followed if the orange and brown pig-

ments are to be preserved. Place portions of the material on chemically

clean slides, add sufficient of the 5% glycerin, then cover with No. 2
coverslips, and begin artificial evaporation at once. In warm and dry
regions this may be done outdoors. Otherwise place the slides on an
 CHLOROPHYTA 251

asbestos board, and adjust in front of an electric heater in such a manner

that the water evaporates steadily, but not suddenly, from under the
coverslips. Add 5 or 10% glycerin as needed, but use solutions of higher
concentrations toward the end of the process, which should not require
more than 10 to 12 hours. Examine oc(;asioually to avoid plasmolysis
or swelling. A little practice is needed to perform this step properly.
7. When the solution has reached the consistency of pure glycerin,
remove the coverslips, transfer the material to new chemically clean slides,
and mount in glycerin jelly. Put aside for a week to harden, then seal
carefully with balsam.
Fixation. —All calcified species require a fluid containing acetic acfid
in order that the calcium encrustations may be dissolved. For the apical
cell and younger development, a mc'diurn to strong (‘hrorri-
stag(*s of sexual
acetic fluid is satisfactory. It would also be us('ful to fix some of the
material in a fluid giving a basic fixation image to nveal the vacuolar
system mon', clearly. Since the nuclei in the internodal regions incrc'ast'
in nunib('r amitotically, special atbuition should be devoted to the fixation
and subsequent staining if it is desired that this feature be emphasizc'd.
Formalin-aceto-alcohol has beem found to fix the older reproductiv(‘
organs satisfactorily, but it is useless for the apical cells.
The apical portions of the branches arc easily removed; it is not ne(!es“
sary to dissect away the side ^Teaves^^ or smaller branches sin(‘e it is not
difficult to oriemt the pieces so that longitudinal sections through th(‘
apical cell are secured. The side branches bearing the developing s('x
organs should b(‘ cut off separately, and as the organs ar(> approaching
maturity, they should be removed individually or in groups of two or
three. Orientation otherwise will be difficult since the internodes have a
tendency to curl during dehydration. Embed the branches on one side,
and section parallel in this plane at lOg for the younger reproductive*,
organs and at 12g for the older stages. Apical cells should be cut at Sg.
Staining. —The quality of the staining is largely de'pc'ndent upon the^

fixation, and with some speci(^s it is rather difficult to obtain a sharp differ-
entiation. Frequently everything else must be sacrificed for some;
particular structure, such as the antheridia. A triple combination is

good for the apical cell and For the earlier stages
for anatomical details.
in the development of the sex organs iron hematoxylin is satisfactory, but
it is usually rather unsatisfactory for the later stages. For these, safranin
and fast green have given sharp staining. In staining the developing
antheridia, care must be taken not to overstain, and all other structures
should be ignored. One might attempt Feulgen^s reaction. The devel-
oping oogonium is so gorged with foodstuffs that it is not easy to give
it a satisfactory stain. During the metamorphosis of the antherozoids,
the blepharoplast may be revealed with iron hematoxylin.
 CHAPTER XIX
EUGLENOPHYTA
The euglenoid flagellates may not be considered as belonging among
plants, but as they contain pigments identical with those in the Chloro-
phyceae, one might as well be somewhat arbitrary with classificatory ideas
and treat them as plants (G. M. Smith 1933, 1938).

Euglenales
Occurrence. —These organisms are most commonly found in small
pools rich in organic matter; the pigmented species are far more abundant
than the colorless types. Forms which are not free-floating occur on
algae, plant debris, or upon small crustaceans. One genus, Ev^glenanior-
phaj is endozoic in habit, occurring in the intestinal tract of tadpoles of
Rana.
Structure. —All free-swimming species possess a
periplast, which may
be flexible or In two genera the protoplast is surrounded by a
rigid.
lorica with an opening at the anterior end through which the flagella
project. The lorica, composed of a firm gelatinous substance, is without
any trace of cellulose but usually is heavily impregnated with iron com-
pounds. Paramylum, an insoluble carbohydrate with a chemical formula
similar to that of starch, is the chief product of photosynthesis it does not
;

react to the ordinary chemical tests for starch. Cysts with thick walls are
common in many genera.
—
and Manipulation. Since only one genus, Euglena^ is of
Cultivation
common occurrence and has been cultivated artificially with its technical
handling worked out, the discussion of the various genera will be limited
to this genus. Most of the free-floating species can be treated similarly
as far as fixation and staining are concerned. Etiglenamorpha must be
treated as smears.
Euglena occurs in pools or ponds rich in organic matter, forming a
greenish scum. Other organisms will probably be found in company with
it, but a fair degree of separation may be obtained by placing a small

amount of the scum in a large covered dish containing 1 liter of pure tap
water and^^l g. of malted milk. In a few days there should be an abun-
dance of Euglena, and subcultures can be prepared with this culture as a
base.
252
 EVGLENOPHYTA 253

A
1. great many methods have been proposed for cultivating Euglerm
(see also Mainx 1927). A choice may therefore be made between the
following:
Although not all species will grow equally well on this particular
medium, perhaps the most useful one is split pea infusion, prepared by

Fig. 32 . —
Euglena viridia: whole mount of pure culture. Fixed with ScHaudinn’s fluid;
stained with Lynch’s precipitated carmin and indulin. The flagella are faintly visible in
some specimens. (From a preparcUion by Miss Enid A. Larson,)

boiling 40 split peas in 1 liter of tap water (Baker 1926). Add enough
citric acid to prevent excessive growth of bacteria.
2. Anautoclaved mixture containing 0.1% yeast extract and 2 g.
sodium acetate per liter of distilled water has given excellent results.
3. Quince-seed jelly may be used for keeping
cultures in an inactive

condition: boil 20 g. dry quince seeds for 30 minutes in 1.5 liters distilled
264 SPECIAL METHODS FOR THE VARIOUS PHYLA

water, then pass the thick exudate, together with the water, through a
fine wire (80-mesh) sieve. Increase the volume to 2.5 liters with distilled
water, and place in a stoppered bottle, where it will keep for many

months. Put some of this medium in a test tube or small flask, and inocu-
late. Such cultures will last almost indefinitely. Molds are likely to put
in an appearance; they may be prevented by rendering the medium
slightly alkaline. Place the cultures in moderate light, and look for the
Euglena on the side of the vessel toward the light. If a split pea infusion
be inoculated with material from a quince-seed jelly culture, abundant
active forms will be produced within a few days.
4. Small cubes of coagulated egg albumen added to sterilized tap

water constitute^a good medium.

5. Soil dilutions No. 2 or 3 as recommended for the Chlorophyceae

are reported to support luxuriant growth (Bold 1936).

The most satisfactory killing fluid appears to be Schaudinn^s heated
to 56°C. For best results when studying nuclear details, the organisms
should be killed late in the evening; the largest number of mitoses are
to be found at 10 p.m. Material should be stained in toto. For staining,
a long iron hematoxylin method or Lynches precipitated carmin (Fig. 32)
is preferable. A counterstain is required to reveal the motor apparatus;
eosin or Bordeaux red, or even light green, may be used. If eosin or
Bordeaux red is selected, which seems rccommendable, apply it before
using the ferric iViordant in hematoxylin staining. Other stains that may
be employed include Harris’ hematoxylin, safranin and fast green, or a
triple combination.
Get the Euglena into dilute balsam by the hygrobutol or beechwood
creosote method.
 CHAPTER XX
PYRROPHYTA
DINOPHYCEAE
The Dinophyceae are mostly marine planktonic organisms restricted
to the warmer parts of the ocean. The fresh-water species, with a strictly
algal organization, are not known in the United States. Several species
occur as parasites in protozoa and metazoa. The majority of the species
are unicellular (G. M. Smith 1933, 1938).
A fewspecies have naked protoplasts, but the majority have cellulose',
wallswhich usually consist of a definite number of articulated plates.
The unicellular motile forms, as well as the zoospores of immobile forms,
have a distinctive structure. There is always a transverse or a spiral
groove completely or partially encircling the cell. All motile forms have
two flagella inserted in the groove. The general color of the organisms
is yellow-brown, but others may be blue-green. The principal food
reserve of the fresh-water species, as a rule, is starch, that of the marine
species is an oil.

Some investigators claim that the Dinophyceae are best studied in

the living condition, but this appears to have come about because they
paid more attention to the plates on the external walls than to internal
details. For permanent preparations, fix with 1% chrom-aeetic, stain
with iron hematoxylin, Harris^ hematoxylin, Mayer’s carmalum, or
safranin, and counterstain with a transparent dye such as erythrosin or
orange G. Dehydrate rather slowly with hygrobutol, and infiltrate with
very highly dilute balsam.

m
 CHAPTER XXI
CHRYSOPHYTA
With the exception of the diatoms, members of the Chrysophyta are
rarely collected in the United States, simply because so few searches art^

made for them. Most of them are plankton organisms existing below tlu;

surface of the water, but others are to be found in pure waters of pools
and ditches. The diatoms, on the other hand, are to be found in all

kinds of waters and are decidedly cosmopolitan.

Chrysophyta have cell walls that are usually composed of two ov(t-
All
lapping halves and are frequently more or less silicificd; there is an
absence of starch formation, the reserve foodstuffs consisting of oils and
leucosin (Pascher 1924, 1937; G. M. Smith 1938).
Most of the Chrysophyta degenerate very rapidly after removal from
the habitat, consequently fixation should be effected as promptly as
possible.

XANTHOPHYCEAE (HETEROKONTAE)
The Xanthophyceae are so rarely collected and so little known to the
average American botanist that an extended discussion appears to be
unnecessary. Two genera, however, are common and may be encoun-
tered. Trihonemaj a filamentous genus, may be dealt with as if it were
one of the filamentous Chlorophyta. Botrydium is the only siphonaceous
genus in the class and may, when conditions are favorable, form an exten-
sive growth. A portion of the damp substrate may be dug up and the
organisms carefully washed out under running water. Kill and fix in a
medium chrom-acetic with iron hematoxylin, and bring up to
fluid, stain

85% ethyl alcohol extremely gradually over a period of a week or longer.

Counterstain with fast green in 95% alcohol plus a little methyl cellosolve.
Then wash with a change of 95% alcohol, and add hygrobutol at the rate
of 1 drop per hour or longer; days or even two weeks should be allowed
for the dehydration process. Infiltrate just as gradually with highly
dilute balsam. Mount in depression slides.

CHRYSOPHYCEAE
Chrysophyceae are as rare and as seldom collected as are the
Xanthophyceae.
JHnohryon is said to be widespread in standing fresh watel^, and
ocoMlionally tq pccur in abundance. Mp^t illuetratiQU^ depict merely
•
 CHRYSOPHYTA 257

the more or less silicified lorica. There is no record of dependable meth-

ods for fixing and staining material.
Hydrurus is occasionally very abundant in the cold waters of montane
streams in mid- or late spring. The plants have a feather-like appearance
but are tough in consistency. As the name of the sole species, H. foetidus,
indicates, it generally has a disagreeable odor. Because of the yellowish-
brown color, it may easily be ignored on the assumption that it is merely
a mass of disintegrating green algae. Kill and fix with formalin-aceto-
alcohol or a 1% chrom-acetic fluid; stain with iron hematoxylin and fast
green. If dehydration and infiltration with balsam are attempted, it
should be done very slowly because of the danger of collapsing the gelati-
nous portion of the thallus. Direct mounting in Karo would probably
be better.

BACILLARIOPHYCEAE
Diatoms are almost ubiquitous organisms wherever there are aquatic
or moist situations exposed to sunlight (Boyer 1927a, 19275). Some
genera prefer fresh waters and others marine habitats; again certain
species are strictly planktonic, and others grow attached to rocks, to other
algae, or to aquatic Angiosperms. Most species prefer cooler waters or
at least are more abundant during the spring and autumn, but that
others can survive in hot waters is evidenced by their abundant occur-
rence in Death Valley streams.
Collection and Preservation. —Diatoms may for convenience be
divided into two groups: fossil and living.
Among the living diatoms most collectors will find the fresh- water
forms more easily obtainable, although under average conditions the
marine species can be secured in greater abundance and in purer cultures.
At most times of year, but particularly under any given set of circum-
stances, certain species will be found to predominate over others.
In fresh- and brackish-water situations, diatoms may be free floating
on or below the surface. One method of securing such species is to obtain
a jar of at least 1-gallon capacity, with not too wide a mouth, whose sides
are painted black or covered with black paper, and to fill with the water
in which the diatoms are living. Place in bright sunlight within a day or
;

so the living diatoms will congregate on the top and around the edge,
from which they can be removed by means of a giant pipette. Mud from
the bottoms of ponds, lakes, shallow bays, and sluggish streams may be
treated in the same manner.
Diatoms grow very abundantly in the form of slimy masses on rocks
and on other algae and aquatic Angiosperms that have been standing in
the water for some time. Such slimy masses are easily scraped off, but
if the host is old and decaying, it is a better plan to place the entire mass
 ;

258 SPECIAL METHODS FOB THE VARIOUS PHYLA

in the collecting bottle and[ later to get rid of the host tissues by chemical
maceration and dissolution.
Marine plankton forms are most easily collected by towing a plankton
net, made of fine bolting vsilk, through the water. In order to collect a
suflSciently large volume of material, a large net should be towed behind a
motorboat. The net should float near the surface. About 3^ hour^s
towing will be required. Many marine species possess long hair-like
processes (as in Chaetoceros) consequently the materials should not be
handled roughly at any stage of treatment. Marine species growing
attached to various large algae frequently occur as ^^unialgal cultures,^'
either as single specimens in large numbers or grouped in fasciae by the
millions. Those species which develop fasciae exhibit marked preferences
as to their hosts and also in many cases produce the fasciae in such definite
macroscopic forms that they are commonly mistaken for members of the
Phaeophyta.
All living diatoms are easily fixed and stained if it is desired that
internal structural details be revealed. A weak chrom-acetic fluid will
give the best fixation it should be based on sea water in the case of marine
;

species. The standard stains may be employed (iron hematoxylin is

recommended), but differentiation will be somewhat difficult because of
the siliceous envelopes, and some unevenness must be expected. Dehy-
drate by the hygrobutol or dioxan method; infiltrate with highly dilute
balsam, and evaporate the solvent very gradually.
Plants of Polysiphonia, Vaucheriay Cladophora, and Chara made into
whole mounts, according to methods applicable to those genera, fre-
quently have excellently fixed and stained diatoms attached to their
thalli.
‘^Diatomaceous earth’’ consists mainly of fossil diatoms, which may
be either fresh- water or marine in origin, and is found in various parts of
the world, as in Ireland, New Zealand, Nevada, and particularly in Cali-
fornia. If samples cannot be obtained by personal collection, it is a
simple matter to purchase a small quantity from certain of the supply
concerns or from diatom specialists.
—
Preparation of Diatoms for Taxonomic Study. Most diatomologists
are not interested in internal structure, their attention being primarily
concentrated on the surface sculpturing of the cell walls. Their methods
of preservation are, therefore, crude in the extreme, since the siliceous
walls are usually not damaged by ordinary reagents. Most of them
simply add from 5 to 10% formalin to the water to act as a preservative.
The formalin should be added as soon after collection as possible because
degeneration changes set in promptly.
To determine some genera and most species accurately, everything
except the cell walls must be removed: diatomologists call this the clean-
 CHRYSOPHYTA 259

ing’^ of diatoms. The mass containing the diatoms is placed in a large

test tube or small beaker, and a small quantity of hydrochloric acid is
added. The acid serves a twofold purpose. If an effervescence is noted,
it indicates the presence of chalk, limestone, or other calcium Compounds,

and these substances must be entirely removed by the addition of more

acid until all effervescence has ceased. The other purpose of the acid
is to loosen those which adhere to other objects, consequently the diatom

material should be left in the acid for a day or two. Shake the contents
well, add considerable water, and strain into a small beaker through coarse
muslin (to remove the larger pieces of debris, portions of other plants,
sand, etc.). If calcium compounds appear to be present, the material
must be washed thoroughly with water, otherwise the next step in treat-
ment will leave irremovable precipitates of calcium sulphate. For mak-
ing rapid washings, a centrifuge may be employed. The tubes should
have round bottoms (like those of test tubes) rather than the narrowly
attenuated ones with which most centrifuges are equipped. The centri-
fuge should be revolved slowly; since the diatoms will settle quickly, not
many turnings will be needed. If no calcium compounds appear to be
present, two thorough washings suffice. Pour off most of the water, add
a small quantity of sulphuric acid, and warm gently over a flame. The
acid will char or dissolve most of the organic matter present. Next add
a small crystal of potassium bichromate. Considerable chemical activity
is usually generated, and chromic acid is liberated. The liquid turns
greenish. If the mass not completely freed of debris and the diatoms
is

well cleaned, wash with water, and repeat the sulphuric acid-bichromate
process. When finally clean, the diatoms arc well washed with water. If
the diatoms are to be mounted as strews in balsam, simply wash with
two or three changes of hygrobutol or dioxan, and infiltrate with balsam.
Mounting of individual specimens may be carried out as directed below.
The manipulation diatomaceous earths depends on whether the
of
mass is composed almost exclusively of empty shells or mixed with more or
less debris. The former condition rarely occurs, but in such cases the
material requires no further cleaning. Most samples of diatomaceous
earth must be subjected to a laborious cleaning process before the diatoms
will be in a condition suitable for critical study (Shropshire 1931).
A 1-inch cube of the earth is broken up into portions about the size
of peas. These are placed in a small Erlenmeyer flask together with three
times the bulk of sodium acetate and enough water to moisten the entire
mass thoroughly. The flask is boiled in a water bath for 15 minutes, then
set aside to cool quite undisturbed. When cold, a small crystal of sodium
acetate is dropped in; the forces of crystallization thus set up serve to
break up the masses of earth. Warm water is added in excess and the
mass brought to a boil. The flocculent material is poured into a beaker,
260 SPECIAL METHODS FOR THE VARIOUS PHYLA

then the boiling and crystallizing process is repeated with the unbroken
part until all the material is broken up. The sodium acetate must now
be removed by repeated washings. Water is added to the material, and
the container is allowed to stand undisturbed until the diatoms have
settled; the water is then cautiously decanted, and more clean water is
added. The process should be repeated several times, until it is certain
that all traces of the acetate have been removed. If only small quantities
are handled at a time and proper care is taken, a centrifuge may be
employed to hasten the washing process. The washing completed, all
possible water is poured off, and concentrated sulphuric acid is added in an
amount equal to about three times the bulk of the material. The con-
tainer is placed in a sand bath, brought to a boil, and the boiling permitted
to continue for at least 15 minutes. The sand bath should be placed
under a hood or out in the open air because of the dangerous fumes
evolved during the next operation. Remove the source of heat. Add
carefully drop by drop (by means of a pipette) a concentrated aqueous
solution of potassium permanganate until the solution becomes bleached.
The mass of material is then cautiously poured into a large flask contain-
ing at least 500 cc. of cold water. The material is next washed thoroughly
with several changes of clean water until all acid is removed (the centri-
fuge may again be utilized). The material is then poured into a 500-cc.
beaker, sufl[icient water and a teaspoon of any good soap powder are
added, and the solution is boiled for about 20 minutes. The material is
thereupon again washed thoroughly. Most diatomaceous earths have
been completely cleaned after this laborious treatment, but if small
particles of dirt still adhere to the diatoms, the soap treatment may be
repeated. If microscopic examination shows the material not to be clean
enough for mounting, the only recourse is to repeat the entire process,
beginning with the sodium acetate. If the material is quite clean, it is
ready for mounting.
—
Mounting of Diatoms. For most practical purposes, mounting in
balsam is as good as in any other medium. The larger diatoms have
their charateristic markings revealed clearly enough, but for the very
small species, which require examination under an immersion lens,
mounting must be in Hyrax, Styrax, or some other synthetic resin with a
high index of refraction. It should be borne in mind that the diatoms
are entirely without color (they cannot be stained after having been
cleaned); consequently a mounting medium with a different refractive
index is required. It is, however, not an easy matter to obtain satis-
factory samples of synthetic mounting media, and the aid of a specialist
on the group may have to be enlisted.
The elaborate groupings of diatoms which give the cranks on the
subject so much delight are of no particular scientific value; sometimes
 CHRYSOPHYTA 261

even the alleged aesthetic value is questionable. However, if one wants

to go to the trouble of designing a rosette or what not with different
diatoms, he will gain the skill and fine touch necessary for the prepara-
tion of type slides of diatoms for taxonomic purposes.
The simplest method of preparing strew diatom slides, especially
where quantities of such are required, is to drain off all possible water
from the cleaned material, to wash with three changes of hygrobutol,
and to transfer to diluted balsam. Instead of evaporating the solvent
until the balsam has become sufficiently thickened, as is ordinarily done,
mounting should be begun as soon as the diatoms have settled down.
Place a small drop of thin balsam on the slide, pick up a tiny amount of
diatom material with a needle spatula, and place in the drop of balsam.
Pass a cleaned 15 or 18 mm. No. 1 circular coverslip through an alcohol
flame, then place on the drop of balsam. Accurate judgment is needed
both in estimating the size of the drop of balsam, which should be as small
as possible yet enough to spread to the periphery of the coverslip, and in
picking up just enough of the diatom material so that it becomes spread
as evenly as possible throughout the balsam without excjessive over-
lapping of individual diatoms.
Some diatomists merely place a drop of the watery solution of diatoms
on a on a copper plate and, while still
coverslip, let it dry, then heat
warm, reverse over a drop of mounting medium on a slide. By this
method too many diatoms tend to collect at the periphery of the drop of
water; the difficulty can be remedied by smearing the slip with a thin
film ofMayer^s adhesive. The diatoms must be thoroughly dried before
being mounted. Air is frequently trapped within them, but most of it
can be caused to disappear if the slide is heated.
In making designs or special-type slides, the desired diatoms are first
picked out of the general mass by means of fine pipettes (perform this
operation under a binocular microscope with suitable magnifications)
and set aside to dry. Diatoms are more easily removed from alcoholic
than from aqueous solutions. Small circular coverslips are smeared
’

with a very thin film of acetic gelatin (liquid gelatin thinned with an
equal volume of 60% acetic acid) and this allowed almost to dry. Many
mounters prefer to make their adhesive by adding 2 parts clove oil to 3
parts acetone-soluble celloidin, which is spread as thinly as possible on
the chemically clean coverslip. The diatoms are picked up by means of
a bristle (a cat^s whisker will do) and arranged as desired under the micro-
scope. Place the completed coverslip on a warming plate until thor-
oughly dry. A drop of Hyrax (or similar medium) is placed on the
coverslip and the latter inverted and gently pressed down on a warmed
slide. If the Hyrax is too thick, it may be thinned with benzene.
 CHAPTER XXII
PHAEOPHYTA
PHAEOPHYCEAE (MELANOPHYCEAE)
In North America the bro\ra algae are exclusively marine plants.
Elsewhere only three fresh-water species are known. The brown algae
are far more complex in both structure and reproductive processes than
are the Chlorophyta or the Cyanophyta, but less so in both respects
than the Rhodophyta.
In size the thallus of the Phaeophyta varies from simple or slightly
branched filaments of a single row of cells and simple membranaceous
forms, varying from a single layer to several layers of cells in thickness,
up to solid plant bodies of various forms. Many of the smaller forms
are epiphytic upon or endophytic within other algae. An alternation of
generations occurs in most if not all of the Phaeophyta outside of the
Fucales. The two generations are either similar or dissimilar in size and
vegetative structure. The sporophyte may be smaller or larger than
the gametophyte. In some genera the gametophyte is an annual and
the sporophyte a perennial, but in other genera both generations are
annuals and in still other genera both are perennials.
—
Occurrence. Phaeophyta are predominantly cold-water plants, but
there are a few which prefer warm seas. Some, such as Pelvetiopsis and
an occasional Fucus, are found at the high-tide line, but the majority
prefer the middle littoral and sublittoral zones. In a few particularly
favorable locations species normally occurring at deeper levels may be
collected without much difficulty during extremely low tides. Under
such conditions on the Pacific Coast it is easy to obtain small plants of the
giant kelps, Macrocystis and Nereocystis.
There is not so great a variation in the colors pf the various Phaeo-
phyta as exists among the Rhodophyta. Some are a light yellowish-
brown, others appear darker because of a greater thickness of the tissues,
while many crustose species become so dark brown as to appear black
when more or less dried out during low tides. There are a few Rhodo-
phyta which might be mistaken by the inexperienced collector for Phaeo-
phyta, but such can readily be distin^ished if the chemical tests noted
under the former phylum are applied.
There is an excellent manual for the Phaeophyta of the north Atlantic
Coast (Taylor 1937) and apother for the Pacific Coast species (Setchell
and Gardner 1925).
262
 PHAEOPHYTA 263

Collecting. —Nearly
all Phaeophyta which are not crustose, endo-
phytic, or epiphytic grow firmly attached to rocks and frequently where
they enjoy the full force of the surf. It is generally difficult to get them
loose; at times one may have to work quickly between waves, when
extreme caution is necessary. The upper part of the plants may be cut
off with a large sharp knife, but one usually wants to get the entire plant.
The easiest method of prying the plants loose is to use an old chisel with
a blade about 1 inch wide or a stout geologist’s pick, and to cut against
the rocky substrate. The rocks are usually in a disintegrating condition,
and it is and quicker to chip pieces of rock loose than to chop
easier
through the mass of stout hapteres.
Although some collecting can be done at all times, the best specimens
are always obtainable during the lowest tides. At such periods the
larger forms, which grow below mean low-tide levels, can be secured.
A tide table for the locality should be available; it may be found on the
maritime or waterfront” pages of local scaix)rt newspapers, or a tide
may be obtained, usually gratis, at sporting
table for the entire year
goods stores that cater to fishermen. Tide levels and times differ
enormously for a few miles apart; consequently the tables of
localities
mean and times, which accompany the tide tables, should
differences
be consulted and the proper allowances made.
Masses of algae cast ashore should, as a rule, be ignored, since they
are either too dry or in too advanced a stage of decomposition to be
worth examining. At certain parts of every rocky beach, determined
by a combination of currents and winds, masses of floating algae, cut
adrift from deeper regions, occur. One may cautiously wade out in such
areas and select the better, fully intact and nondiscolored specimens.
Material from such sources is fully satisfactory for slide-making purposes,
but one runs a certain danger of criticism if locality herbarium specimens
are made therefrom. If the material should be used merely
is dried, it

for purposes of identification and the label should always bear the nota-
tion ^‘cast ashore.”
In clambering over rocks in search of material, one should proceed
with great caution, as it is very easy to slip and receive bruises or abra-
sions. If one slips against jagged rocks, barnacles or mussels, open cuts
on legs readily result, and when the salt water gets into the wounds, they
can become very painful. Concentrate on collecting algae and forget
about surf bathing or getting a tan or other extraneous matters ^except —
for keeping an eye open for waves and the incoming tide. Wear stout
hip boots even if the water is warm; these are mostly for protection
against slips and consequent abrasions. If the rocks are densely covered
,

with algae, place one foot down firmly, twisting it sidewise to get a good
hold, before lifting the other foot, and do not attempt to take too long
^4 SPECIAL METHODS POR THE VARIOUS PHYLA

steps or to jump from one projecting rock to another. In wading

through walk
pools, very slowly, with short steps and the foot not lifted
too high, and in a direction so as to avoid reflections from the surface.
Search under and around large masses of algae for smaller forms. Exam-
ine shady banks and overhanging ledges; the plants in such situations
are apt to be different from those growing on the sunny sides. In
removing plants, grasp the entire mass firmly as close to the base as pos-
sible and pull up with a twisting motion, or use the chisel or pick to
assist in loosening them.
The plants, as removed,may be placed in a galvanized or enamel-
ware bucket of about 2-gallon capacity. Do not put any water in the
bucket, but if the algae show signs of drying out, simply immerse momen-
tarily in cold sea water, and replace. If the algae are placed in water,
most of the spores, gametes, oogonia, etc., may be liberated. Do not
cut the smaller epiphytic species from their hosts since it is frequently
necessary to identity of the host when classifying the epiphytes.
know the
If cultures are to be started, put only one species in a bucket; otherwise
contamination may result. In a general collection there is little danger
of spoiling if the different sorts are mixed, but Polysiphonia, Pterosi-
phoniaj and Desmarestia (particularly D. herbacea) should never be per-
mitted to come into contact with other algae.
As soon as the return to the laboratory has been made, sort out the
material. The species which cannot withstand much drying should be
worked up first. If an immediate identification is not possible, place
some of the material in a tank of running sea water for later study. If
any material appears to have suffered from drying, place in cold sea
water for a short time to restore turgidity.
—
Preservation of Material. 1. In Liquid, In general, all Phaeophyta
may be preserved in 8 to 10% formalin in sea water, plus the addition
of enough borax to render the solution distinctly alkaline. Unfortu-
nately, this solution corrodes all except all-glass receptacles. After
remaining in the formalin solution in sea, water for about a week, this
fluid may be washed out with two or three graduated mixtures of sea
water and tap water and finally stored in 3 to 5 %
formalin in tap water
plus sufficient borax to prevent the fluid from becoming acid and about
5% glycerin.
2. —
Rough Drying, Old newspapers may be spread in some place
where the air is as dry as possible and not in full sun. The material may
then be spread apart on the newspapers, then turned over from time to
time so that the drying may be m6fe uniform and molding is avoided.
The specimens should be taken up once in a while and shaken gently so
that the branches will not adhere but will dry separately. If the atmos-
phere is damp or if the specimens are unusually large, it may be necessary
 PHAEOPHYTA 265

to utilize gentle artificial heat in order to avoid rotting or molding.

The specimens might be placed in a warm room or near a radiator,
stove, or other source of heat. On
the other hand, the specimens must
not be dried too rapidly, or they will become too brittle. When the
specimens have become fairly dry, but before they have become too
brittle to handle without damage, they should be rolled into a ball or
roll, tied with twine, and allowed to continue drying. They may finally
be wrapped up in newspapers or placed in suitable boxes.
The plants should not have been washed in other than sea water;
the salt left in them during the drying process serves to keep them
flexible. After drying, even if they have remained in that condition for
years, they may be soaked out in either fresh or salt water and studied
as if they were freshly collected specimens.
To prepare dried specimens for mounting as herbarium specimens,
soak them in sea water until they are restored to approximately the
original condition, then transfer to a mixture of equal parts of water,
ethyl alcohol, and glycerin plus 10% phenol until thoroughly penetrated,
then they may be dried on paper. Or they may be dried without placing
on paper and will retain their flexibility indefinitely if stored in tin or
airtight boxes.
3. Herbarium Specimens —
Practically all forms (^an be dried on paper.
,

The more slimy forms, such as FucuSy Pelmtia, and HesperophycuSy will
shrink badly by the time they are thoroughly dry. Most small sped-
mens will adhere readily to the paper but species belonging in the Fucales,
Dictyotales, and Laminariales will need to be glued to the paper with
waterproof glue after they have dried completely. Most of the Lami-
nariales are too large for the ordinary herbarium sheet; one must either
search for small specimens, cut the specimens into suitable lengths, or
mount selected portions of mature plants.
To prepare herbarium specimens, obtain a flat ti*ay of a size to
receive easily a standard herbarium sheet. Of course, one does not have
to use the regulation-size herbarium mounting paper: almost any con-
venient size of paper that will not shrink badly from wetting may be
used. Ectocarpus and similar small forms may, for example, be mounted
on plain standard 3 X 5-inch index cards. Students have mounted
specimens on thick 8}4 X 11-inch notebook paper and incorporated the
finished mounts in their notebooks. Fill the tray somewhat less than
half full with sea water, slip the sheet of paper in and float the specimen
above the paper. Hold the stipe or thicker end of the specimen at Pne
end of the sheet and partially raise this end out of the water, ^
small brush or needle arrange the rest of the speqinxen the papei^ grad-
ually lifted out of the water. Drain off excess water, lay the paper on a
drying blpttpr, mi cover the speeimeu with ^ piece or two of cheesecloth
266 SPECIAL METHODS FOB THE VARIOUS PHYLA

or part of an old linen bed sheet. Place a couple of driers over the cloth,
followed by a piece of corrugated board. The next specimen may now
be added. If the specimens are being mounted on small pieces of paper,
several such pieces may be arranged on a single drier. After 2 hours
remove and replace with dry ones; do not try to remove the
all driers,

cloths. If the atmosphere is too damp for drying to take place, some
form of gentle artificial heat must be arranged. The driers should be
replaced at least twice a day with dry ones until the specimens are
thoroughly dry. Between changes, keep the mass under moderate
pressure.
Cultivation. —Methods have never been devised for growing to
maturity other than the smaller filamentous species; the larger species
with massive mature structures have been grown to the older germling
stage only. Cultivation methods have, in fact, been directed mainly
toward obtaining the gametophytes in genera in which these structures
are microscopic in size and cannot be found in nature.
No matter what the means of reproduction, cultural methods and
solutions are essentially identical. The following solution has given
excellent results (Schreiber 1931):

Sodium nitrate 0.1 g.

Dibasic sodium phosphate 0 02 g.
.

Distilled water. 50.0 cc.

Sea water To 1 liter

Autoclave at 15 pounds pressure for about 20 minutes. The solution

may be solidified with 13^^% agar if it is desired to raise the germlings
on such a medium. If the cultures are to be carried for longer than
three months and slides are not to be made of stages younger than well-
developed sporelings, it is preferable to use a solid substrate. Cultures
have been carried along for 3 to 5 years on such media.
If zoospores are to be germinated, obtain a small battery jar about
inches in diameter and 3 inches deep. Place two clean slides at
opposite sides on the bottom, then lay two more slides across the ends
of these slides and continue stacking slides until the pile is about
or 2 inches deep. Pour in the culture solution, using more than enough
to cover the stack. If gametophytes with sex organs are to be raised,
use a similar jar of wider diameter, and arrange the slides in one layer on
the bottom and stacked in one row around the periphery, then fill with
nutrient solution. It may be more satisfactory to use large coverslips
(24 X 50 mm.. No. 2) in place of slides for gametophyte growth.
Place small pieces of fruiting material in the culture dishes. Portions
of the laminae of the Laminariales bearing sori, or receptacles of the
Fucales, should have been collected some time previously and kept in
the refrigerator for several hours or overnight/ then rinsed thoroughly
 PHAEOPHYTA 267

but quickly in many

changes of sterile sea water before being placed in
the culture. The aim
of this procedure is to get rid of any foreign spores
and diatoms that might be present, but it is nevertheless very difficult
to keep down diatom growth in cultures. Most of the spores will have
been discharged by the next morning, whereupon the pieces of fruiting
material should then be removed. The spores of most species will have
germinated by the second or third 'day; growth at first is usually rapid
so that within a week there is a brown layer over the bottom and sides
of the culture vessel and on the slides placed therein. Keep the cultures
in a cool location in moderate light.
Slides may be removed at any desired stage of growth of the germlings
and plunged into 1% chrom-acetic in sea water. Fix for about 2 hours.
Wash thoroughly in sea water, then transfer gradually to distilled water.
Staining of both germlings and gametophytes may be with iron hema-
toxylin; but if details are obscured, treat for 1 hour with cold 1/N
hydrochloric acid, rinse with distilled water, and apply Harris’ hema-
toxylin. After either hematoxylin, counterstaining may be with orange
G. Before proceeding with the staining, determine on which side of the
slide the best or desired plants occur, mark this side so that the growths
will not be accidentally wiped off, then clean the other side of the slide
and the edges.
Two methods are available for dehydrating and mounting the slides.
The germlings and gametophytes are easily plasmolyzed by too violent
changes of fluids.
1. Place the slides in a large, shallow flat-bottomed dish containing

an ample amount of 7% glycerin to which a trace of formaldehyde has

been added (G. J. Hollenberg, unpublished). The reason for the for-
maldehyde is that fungal growths occasionally appear in the glycerin.
Place the container in a warm place, protected from dust, for the glycerin
to concentrate. Remove the glycerin with 95% and absolute alcohol,
as in the glycerin method (p. 119), and follow with a graded series of
absolute alcohol and xylol mixtures, allowing 30 minutes in each, until
the material is in pure xylol. Return the slides again to a large flat-
bottomed dish containing a little xylol. This time place the slides with
the attached gametophytes on the lower side, and support ea(;h end of
the slides by means of glass rods or thin slides. Carefully add an ample
quantity of 3 %
balsam in xylol, and allow the solvent to evaporate in a
warm dust-free place. Just before the balsam becomes of a mounting
consistency, remove a slide, quickly wipe the balsam from the back and
other exposed portions of the slide with a cloth moistened with xylol,
then add a coverslip.
2. A fasten and equally good procedure is to dehydrate with hygro-
butol after the staining has been completed. Place the slides in a suitable
 2d8 SPECIAL METHODS FOR THE VARIOUS PHYLA

container in water or in alcohol, if they have already been partially

dehydrated, and adc^ small quantities of hygrobutol at a time. Do not

add too much hygrobutol at a time, and allow at least 30 minutes between
additions. Pour off some
mixture from time to time as the pro-
of the
portion of hygrobutol increases. Finally give two changes of pure
hygrobutol, and mount in thin balsam. However, if plasmolysis results
upon mounting, which is not likely since the hygrobutol will harden the
specimens somewhat, place the slides in 5% balsam in hygrobutol, then
evaporate the solvent, and mount as described in the first procedure
outlined above.
Whole Mounts. —Filamentous forms such as PylaieUa, Ectocarpus^
Streblonemay and Sphacelaria, frond tips of Desmarestia, Myriogloia and
similar forms, gametophytes of the Laminariales if loose rather than

attached to slides, etc., are perfectly fixed in 10% formalin in sea water.
If a chroin-acetic fixatioq image is desired, use the standard formula
given below, but for the more delicate forms, dilute it considerably with
sea water.
Whether fixed with formalin or chrom-acetic, wash out the fixative
thoroughly with sea water, and transfer the material through (1) a
mixture of 75 parts sea water and 25 parts distilled water, (2) equal
portions of sea and distilled water, (3) 25 parts sea water and 75 parts
distilled water, allowing the material to remain in each mixture for at
least 1 hour. The transfer to plain water may be completed by washing
in several changes of distilled water, whereupon the material is ready for
staining. Iron hematoxylin may be recommended for most species (but
not for those with thick with a counterstain of orange G. Dif-
thalli),
ferentiation of the hematoxylin more satisfactory with a 1% solution
is

of ferric chloride. Or one may use Harris^ hematoxylin, especially for

reproductive phases, particularly when a sharp, transparent stain is
required for revealing internal details of reproductive phases. For
example, whole mounts of the thalli of Myriogloia with all stages in the
development of the zoosporangia are very satisfactory after this stain.
A counterstain should be used on filamentous species, but it is usually
better not to employ one on thick thalli. Dehydrate with hygrobutol,
and transfer to highly dilute balsam. Plenty of material of the fila-
mentous species should be mounted on each slid^ as stages in the develop-
ment of the reproductive bodies are usually none too abundant.
—
Fixation and Embedding. With the exception of species belonging
among the Fucaceae, the Phaeophyta are exceptionally easy subjects
technically, this statement embracing all forms from those that are tiny
filaments up to the giant kelps of the Pacific Coast. The Fucaceae are
very troublesome to manipulate because of the excessive slime content
of the thalli.
 :

PHAEOPHYTA 269

Two standard fixing fluids are applicable to the majority of the

Phaeophyta and will be found to meet practically all needs. The
simplest is a 10% solution of formalin in sea water, in which the material
should be allowed to remain for at least several days before being sub-
jected to further treatment. The second fluid is a 1% chrom-acetic in
the following proportions:

Sea water (filtered) 100 cc.

Chromic acid 1 g.
Glacial acetic acid 1 cc.

Saponin 0 5
. g.

If a quantity of the stock solution is to be made up, add the saponin

only as needed. Material should be allowed to remain just sufficiently
long to become properly fixed; wash out thoroughly with copious quanti-
ties of sea water. For many softer forms and
which are deli-
for those
cately filamentous, the fluid should be diluted up
volumeto one-half its
with sea water. Such forms are sufficiently fixed in about 4 hours, but
portions of the thick fruiting laminae of the Laminariales need to remain
in the fluid for about 24 hours. The transfer from sea water to a paraffin
solvent is readily effected by a graduated series of fluids, as noted in the
following table (the volumes are in cubic centimeters)

Sea water 90 80 65 50 35 20
Distilled water ^ 10 20 30 35 40
I

96% ethyl alcohol 1

5 10 15 20 30 40

From the last mixture proceed to the 50% solution of the tertiary butyl
alcohol method. Since the tissues are readily penetrated by the fluids,
changes may be a little more rapid than enough. Only
usual: 1 hour is

sufficient fluid to cover the material it should then

completely is required ;

be discarded and not used over again. Dioxan has proved to be very
unsatisfactory for the Phaeophyta, as it is nearly impossible to remove it
completely during infiltration. Xylol and similar fluids will harden the
tissues excessively. The time in the paraffin oven needs to be as short as
possible since the heat of the oven has a deleterious effect upon the
tissues. The paraffin penetrates rapidly; consequently the time in the
oven may be half that ordinarily required for vascular plants.
—
Microtoming. All phaeophytean tissues are quite easily sectioned.
Very few are so hard that they cause trouble; such tissues might better
be embedded in celloidin. Among
these are the hard stipes of Cystoseira
and old stipes of the larger Laminariales. Sections of embedded material
as thin as 2 and 3m are readily cut, provided a paraffin of extremely fine
consistency (such as Parlax) is employed for the embedding.
270 SPECIAL METHODS FOR THE VARIOUS PHYLA

Staining. —The Phaeophyta, because of the nature of their cell-wall

structure, are not easy to stain adequately and sharply by ordinary
procedures. The cell walls are composed principally of cellulose, and the
gelatinous portion is a pec tic compound know as ‘‘algin.^^ The pro-
portion of the two substances varies greatly both according to the
speciesand to the tissues. The walls and cytoplasm as a rule both stain
deeply and quickly and are very hard to differentiate if overstained.
Care must also be taken not to allow the tissues to remain too long in
killing fluids containing chromic acid since the tissues are readily over-
chromated and bleaching is useless. Overchromated tissues are notori-
ously difficult to retain on the slides, and this is particularly true of the
brown algae. Except for sections of blades with zoosporangia, it is
always advisable to avoid going below 70% alcohol during the staining
process, but a momentary rinsing in water to remove excess stain usually
does not cause the sections to float off.

Combinations of stains commonly used on stems, leaves, roots, etc.,

of the vascular plants are entirely useless on the Phaeophyta. There is,

for example, no xylem in these plants; consequently the use of safranin

would serve no purpose. Other combinations are therefore necessary.
A combination that gives excellent results and rather good differentia-
tion on anatomical material is Bismarck brown and fast green. Deparaf-
fin the slides and bring down to 70% ethyl alcohol. Place in a 1%
solution of Bismarck brown in 70% alcohol for 20 minutes (if left in th('-
stain for longer than 30 minutes, it becomes impossible to extract the
stain). Remove slides individually and wash for 5 to 10 seconds in each
of two jars of 95% alcohol, (After the first jar becomes too saturated
with excess stain, discard this alcohol, replace with the alcohol from the
second jar, refilling the latter with fresh alcohol.) Remove the slide
from the alcohol, quickly wipe the underside dry with a clean cloth, then
apply the fast green from a dropping bottle (the dye should be a methyl
cellosolve-clove oil-alcphol solution), and allow to remain for about 5 to 8
seconds. Differentiate for a few seconds in a mixture of 2 parts clove oil
and 1 part each of absolute alcohol and xylob pass through a xylol wash,
thence into pure xylol, and mount in balsam. In sections of the stipe
of Macracystis the sieve tubes and plates take the green and all other
structures the brown in shades varying from pale straw to dark brown.
Zoosporangia are best stained with iron hematoxylin and orange G.
Mordant for 20 minutes, and leave in the hematoxylin for not longer
than 2 hours; it is very easy to overdo the staining. If it appears to be
impossible to obtain a clear differentiation with the hematoxylin, advan-
tage may be taken of the aflinity of zoospores for acid fuchsin. The
zoosporangia of Postelsia, which do not take a good hematoxylin stain,
 PHAEOPHYTA 271

are clearly stainedby the fuchsin. Use a 1 % solution of acid fuchsin in

70% and
alcoholstain for not longer than 20 minutes. Rinse the slide
by plunging two or three times in tap water, then pass through two jars of
95% alcohol (strong alcohol has little effect on the stain, hence the neces-
sity for rinsing in water). Then counterstain with fast green as described
in the preceding paragraph.
Antheridia and oogonia, mi(;rospores and macrospores, and similar
reproductive bodies should be stained with iron hematoxylin, although
the male elements occasionally take a better stain with Harris^ hema-
toxylin. If a counterstain is desired, use orange G.
Taxonomy. —The classification of the Phaeophyta is now based upon
the life cycle (Kylin 1933, G. M. Smith 1938, Taylor 1937), and this
scheme has been adhered to in the following discussion.

ISOGENERATAE
Two similar generations alternate; they are identical in vegetative
structure.

Ectocarpales
A large number of families were formerly included in the order, but
as the life histories of the various species became better known, they were
transferred to other orders. This has incidentally made possibki a
greater uniformity in technical procedures.
Ectocarpaceae. —The species in the family are all filamentous forms,
commonly epiphytic on other algae. If large enough and sufficiently
abundant, remove from the host or other attachment. If too small,
work up both host and epiphyte together, and scrape the latter off with
needles when ready to mount; or the two may be brought into paraffin
and sectioned together. For whole mounts fixation is superior in a
weak chrom-acetic fluid. Ascocyclus, Streblonema and some species of
Ectocarpus bearing sporangia on the prostrate filaments are better
sectioned than mounted entire. Iron hematoxylin with a light counter-
stain of orange G is the most pleasing stain combination, but care should
be taken not to leave plurilocular sporangia overstained. Iron hema-
toxyliQ, forsome reason, does not always stain some species sharply; in
such cases, resort to differential acidification, and stain with Harris'
hematoxylin and orange G. This combination is especially good for
critical cytological investigations (Fig. 33).
In Pylaiella the sporangia are mostly catenate, usually intercalary
in position. Some species have a strong tendency to become brittle
during dehydration for whole mounts; consequently the process should
be rather gradual.
272 SPECIAL METHODS FOB THE VARIOUS PHYLA

Ralfsiaceae* —The flat, crustaceous thalli may be scraped off the

rocks on which they grow, all the debris possible washed away, and the
specimens embedded and sectioned.

Fig. 33.— Ectocarpua acutua: whole mount of portion of mature plant with gametangia.
Fixed in 10 % formalin in sea water, ionized with HCl, stained with Harris’ hematoxylin and
orange G, dehydrated with hygrobutol and mounted in balsam.

—
Heterochordaxiaceae. Heterochordaria is one of the commonest
brown algae on the Pacific Coast. The plants are too thick for whole
mounts but are easily embedded and sectioned. Microtome transverse
sections of the ramuli for the gametangia at not over 6m and stain sharply
with iron hemat/oxylin and orange G.
 PHAEOPHYTA 273

Sphacelariales

The family Sphacelariaceae is the only one, members of which are

likely to be found in the United States. The plants occur rather rarely.
Most members of the order are filamentous plants ranging from 4 mm.
to 5 cm. in height, all species being characterized by prominent apical
cells. The plants become polysiphonous in the older portions. In
some species of Sphacelaria lateral branches are converted into pro-
pagulae. Permanent whole mounts are best made by the hygrobutol
method, after staining with iron hematoxylin. The counterstain should
be applied in very dilute solution since the tissues have an unusually
strong affinity for acid stains. In Sphacelaria and Stypocaulon, the
latter occurring only on the north Atlantic Coast, mitoses are conspicuous.
At midnight or shortly thereafter, fix portions of the tips about 6 mm.
long in 1 % chrom-acetic, embed, and section longitudinally, perpendic-
ular to the flat surface, at 3 m- Stain critically with iron hematoxylin,
and counterstain very lightly with orange G.

ClJTLERIALES
Cutleria is said to occur in Florida; otherwise the order is not repre-
sented in the United States. (For technical methods, consult Yama-
nouchi 1909, 1912.)

Dictyotales
The Dictyotales differ from the other orders in the presence of aplano-
spores and in a heterogamous method of reproduction. Members of
the one family, Dictyotaceae, prefer the warmer southern waters, and
representatives occMir on both coasts.
In Dictyota growth is by means an apical cell; in the other genera
of
it is by division of marginal cells. The apical end of the thallus of Dictyota
should be sectioned parallel to the flat surface, the thalli of the other
genera should be cut perpendicular to the flat surface. Sections should be
cut at about 12m; staining is superior with iron hematoxylin with or with-
out a counterstain of orange G or erythrosin. Many people make whole
mounts of the apices of the thalli.
The aplanospores, oogonia, and antheridia are arranged differently in
the various genera; a knowledge of their location or the nature of their
distribiftion is therefore necessary when fruiting material is desired.
The antheridia and oogonia, however, are usually in dense sori and always
project beyond the surface. The sporangia are usually scattered, but
in NeurocarpuSy for example, they are arranged in sori along either side

of the midrib. The oogonia and antheridia may be on the same frond
or plant or on different individuals and disposed in some definite arrange-
 :

274 SPECIAL METHODS FOR THE VARIOUS PHYLA

ment or scattered. In many species one is more likely to find aplano-

spores than antheridia or oogonia. In order to obtain the best fixation
of the critical stages in the development of the antheridium and oogonium
of the forms with thick thalli, a chrom-osmo-acetic fluid is preferable.
The following formula has proved to be satisfactory with Dictyota,
Padina, and Zonaria:

1% chromic acid in sea water 10 cc.

2% osmic acid in 2% chromic acid 0.06 cc.

Glacial acetic acid 0 5 cc.
.

The centrosomes and radiations should show up clearly after staining

with iron hematoxylin and no counterstain.
In Zonaria, which is common on the southern California coast, an
astonishingly large number of apical cells are readily obtained, and
developmental stages are easy to follow out. The following formula
has been used

Chromic acid 1 g.
Glacial acetic acid 3 cc.

1% aqueous osmic acid. 1 cc.

Sea water 96 cc.

HETEROGENERATAE
Two dissimilar generations alternate. The sporophytic is usually
macroscopic, the gametophytic microscopic.

Haplostichineae

Growth is trichothallic, the thallus being composed of one or more

^
and
filaments their branches.

Chordariales
Practically all material of the Chordariales that one collects is the
sporophytic generation; the gametophytes are known only from cultural
studies. The methods cited in the introductory paragraphs to
cultural
the Phaeophyta may
be followed ;to x)btain the gametophytic phases.
—
Chordariaceae. Chordaria, the more prominent one of the several
genera, grows farther south on the Atlantic Coast than on the Pacific.
Sections of the cylindrical fronds should readily reveal the zoosporangia.
—
Coliodesmaceae. Coliodesrne is common on the Pacific Coast. It
usually grows on large specimens of Cystoseira or Cystophyllum. Portions
of fronds of various devdopmental stages may be cut out, embedded,
and sectioned in the vertical plane to show the unilocular zoosporangia.
Elachisteaceae. —The species are pulvinate, microscopic in size, and
epiphytic on various larger algae. Work up portions of the host bearing
 PHAEOPHYTA 275

the epiphytes by the hygrobutol method, finally scraping off the filaments
carefully, and mount in balsam.
—
Myrionemataceae. The thallus consists of a prostrate disk, com-
posed of radiating filaments more or less closely united and not more than
two cells in thickness, and erect filaments, which may be free or united
in a common jelly. All the genera grow upon other algae or on marine
Angiosperms; the pneumatocysts of Macrocystis arc particularly favored
by species of Compsonenia. Cut out portions of the host bearing the
epiphyte, embed, and section in the transverse plane of the host tissues
(Fig. 34).
Leathesiaceae. —Most of the species grow on rocks, a few on other
plants. The thallus is thick, cariiose, and composed of filaments held

Fig. ’64:.—Myrio7iema strangulans: section of a young colony on Ulva lohala, with two
young zoosporangia in the center. Fixed with chroni-acetic in sea water; stained with iron
hematoxylin and orange G.

together in a thick jelly. Cut portions of the thallus into small pieces,,
embed, and section.

SPOROCHNALES
Sporochnaceae. —Two species of Sporochnus occur rarely on the
Atlantic Coast from South Carolina southward. Whole mounts may be
made of portions of the branches with terminal filaments; otherwise
sections are indicated.

Desmarestiales
M3rriogloiaceae. —Sections of embedded portions of the filaments
should show the unilocular zoosporangia.Prepare whole mounts of the
tips of the branches to show their interesting structure; stain with
Harris' hematoxylin (Fig. 35).
276 SPECIAL METHODS FOR THE VARIOUS PHYLA

—
Desmarestiaceae. Members of the family occur on both coasts,
Desmarestia being th0 most widespread genus, and certain of the species
have the characteristic peculiarity of turning a verdigris green on drying
and of bleaching other algae when coming into contact with them.
Decomposition commences almost as soon as the plants are taken from the
ocean. For this reason, when preserving material of the genus, the
plants should be placed in a quiet pool, and the desired tissues removed
and immediately immersed in tjie killing fluid. Portions of the stipes

Fig. 36
. —
Myriogloia amieraonii: portion of a whole mount of thallus with radiating
filaments and zoosporangia. Fixed with 1 %chrom-acetic in sea water; stained with
Harris’ hematoxylin, dehydrated with hygrobutol and infiltrated with balsam.

and frond are valuable for morphological study; whole mounts of the
apices of the fronds to demonstrate the trichothallic apical growth are
easilymade. The zoosporangia are very difficult to locate, and one may
have to examine a large number of fronds of fully mature plants before
finding them; they probably appear only in the autumn.

Polystichineae

Growth not trichothallic ; thallus parenchymatous.

PUNCTARIALES
Stilophoraceae. —A single representative, Stilophora rhizoides, occurs
from North Carolina to Massachusetts and fruits at the end of the sum-
mer. Whole mounts of the apices are possible, but sections should be
out for the reproductive structures.
 PHAEOPHYTA 277

Asperococcaceae. —The fronds are sometimes ligulate, but the mon^

common species are saccate. a widespread Pacific Coast
Soranthera is

epiphyte (Fig. 36), occurring generally on Rhodomela, Plants in all

stages of development are usually to be found on a single host plant; conse-
quently it is not difficult to prepare sections showing all stages in the
growth of the sori and zoosporangia. Section the thallus transversely

Fia. 36 . —Sorarithera ulvoidea: whole mount of zoospores just ))eforo germination.

Diatoms are also present. Fixed with 10% formalin in sea water; stained with iron
hematoxylin.

at about 7/x. Asyerococcusy equally common on the Atlantic Coast, is

also excellent and may be dealt with similarly.

Punctariaceae. —The simplest forms in the family are monosiphonous;
the more complex species are either filamentous and solid, saccate, or

membranous. The zoosporangia and gametangia form definite soi’i.

Whole mounts of portions of the thallus are easily prepared, but as they
are useless for detailed studies, sections are advisable.
—
Scytosiphonaceae. Scytosiphon lomentariay the best-known species,
occurs on both coasts and is easily found. It usually produces game-
tangia in the autumn. Cut the long frond into pieces about 5 mm. in

length, embed, and section transversely at about 8 m- The frond is

likely to be very densely covered with diatoms and other small organisms.
 :

278 SPECIAL METHODS FOR THE VARIOUS PHYLA

Laminariales
The order includes the giant kelps, largest of all known marine
plants. The plants, as ordinarily seen, represent the sporophytic genera-
tion: the gametophyte is microscopic and in most of the genera is very
much reduced. The fronds are usually differentiated into three regions
(1) a holdfast, varying from discoidal in some species to clusters of simple
or branched hapteres in others; (2) a stipe, cylindrical or more or less
flattened and simple to dichotomously or irregularly branched, and (3)
one or more flattened blades. The zoosporangia may be borne either
on the typical blades or on specialized sporophylls and are generally
grouped into sori of considerable extent. Growth takes place in most
species in a meristematic tissue intercalated between the blade and
stipe; in N
ereocystis growth occurs throughout the entire plant.
There is no necessity for dealing with the families or genera separately
since the same technique serves for the entire group with respect to the
various structures.
Holdfast .
—
Cut out small portions of the main part; or in the case
of branching hapteres, remove pieces of both the older parts and the
apical end. Embed, and cut both longitudinal and transverse sections
at 10^1. There is little or no tissue differentiation. A single stain
suffices: a 1 %
solution of Bismarck brown in 70% alcohol or a 0.2% solu-
tion of fast green in 95% alcohol will stain deeply enough in a few min-
utes. Wash quickly in 95% alcohol, clear, and mount.
Stipe .
—
In some species the stipe may attain a diameter of more than
15 cm., thus becoming so large that portions must be cut out for easier
manipulation. It is easy to cut such material in the fresh condition
with a sliding microtome, exactly as in the case of stems. However, it
will be better to embed since the cells are so small that rather thin
sections are indicated. The solid portions of the hollow stipes of Nereo-
cystis and Postelsia average 1.2 cm. in thickness, but it is not in the least
difficult to embed and section these parts. The stipes of the other
genera are decidedly variable in their internal structure.
Mucilage ducts are present in some,, absent in others. When present,
there may be some difficulty in fixation; the remedy is to increase the
percentage of acetic acid in the fixative. As the sections are retained
on the slide only with great care, it is advisable to cover them with a thin
film of celloidin before removing the paraffin with carbol-xylol.
The stipe of Macrocystis is peculiarly interesting because of the
presence of large sieve tubes and sieve plates. Embed, and cut trans-
verse sections at 14)Lt and longitudinal sections at 12jw. On account of
the more or less accentuated twisting of the stipe, the longitudinal sections
will rarely show entire sieve tubes and on the whole are usually disap-
pointing. Stain with Bismarck brown and fast green.
 PHAEOPHYTA 279

The and those of some species of Alaria exhibit

stipes of Pterygophora
concentric rings. In a few species the stipe becomes too cariilaginous
to be sectioned readily in paraffin. Resort might be had to celloidin.
The stipes of the algae cannot be soaked in water in order to soften them
previous to microtoming, as is frequently done with woody stems,
because they will promptly swell up and disintegrate.
—
Blade and Sporophylls. Despite their rather thick and tough,
leathery character the blades and sporophylls of all members of the order
present no difficulty whatever.

Fig. 37
.
—
Poatelsia palmarformis: Portion of cross section of lamina with zooaporangia
in the depressions. Fixed with 1% chrom-acetic in sea water; stained with acid fuchsin
and fast green.

When borne on tlie blade, the areas containing the sori appear a
darker brown against the light; but in some forms such as Postelsia
the presence of zoosporangia can be determined only by microscopic
examination of small pieces of the blade. In the Alariaceae and in
Lessoniopsis of the Laminariaceae the zoosporangia are borne not on
the blade but on specialized sporophylls. The latter appear as a rule at
certain seasons only and a careful watch must be kept for their appear-
ance. Cut but small portions of the blade or sporophyll, taking care to
obtain a series of stages from the youngest to the oldest, and embed in
paraffin (Fig. 37). In Laminaria especially, the tissues tend to become
very hard, but nevertheless they are surprisingly easy to microtome.
To show the origin and development of the zoosporangia and
paraphyses, the sections should not be thicker than 3^. Staining is
preferably with iron hematoxylin alone. On thicker sections, for general
morphology, acid fuchsin and fast green, or Bismarck brown and fast
green, should be employed.
Gametophytes, —
These are obtainable only by special culture methods,
which can be readily carried out only at a properly equipped marine
station. Methods are described in the introductory paragraphs to the
Phaeophyta.
 :

280 SPECIAL METHODS FOR THE VARIOUS PHYLA

Cyclosporeae

A diploid generation only is present.

Fu GALES
The order occurs on both coasts of North America. It contains two
families: Fucaceae, in which the fronds are flattened in two ranks in one

Fig. 38. —
Fucus vesiculosis: portion of a cross section of a receptacle with a micro-
sporangial conceptacle. Fixed with chrom-acetic in sea water; stained with iron hema-
toxylin and orange G.

plane without differentiation into axial and lateral branches, and Sar-
gassaceae, in which the branches arise on all sides of the main axis.
Fucaceae.—Fwcw.s is unquestionably the most widely studied of all
Phaeophyta but is exasperatingly troublesome to the technician because
of the chemical nature of the mucilage. The pieces of frond are usually
lefthard and “dry” by the usual fixation and dehydration methods; the
macrosporangia are too often completely disrupted.
Fixation may be in the following fluid

Chromic acid 1 g_
Glacial acetic acid 3 cc.
Glycerin 10 co.
Seawater 90 co.
 PHAEOPHYTA 281

Wash with 10% glycerin in sea water, then place in 10% glycerin, and
set aside for the water to evaporate. Wash out the concentrated glycerin
with a mixture of equal parts of 95% alcohol and tertiary butyl alcohol,
then with a short series of mixtures of these two alcohols in which the
butyl alcohol is gradually increased. Finally give two changes of pure
tertiary butyl alcohol, and infiltrate with j^araffin. Or one might, after
washing out the glycerin thoroughly with the mixture of equal parts of

Fig. 39
, —
Fucns vesiculoms: portion of a cross section of a receptacle with a macro-
sporangial conceptacle. Fixed with 1 % chroiii-acetic in sea water; stained with iron
hematoxylin and orange G.

the two alcohols, go to a mixture of equal parts of absolute alcohol and

tertiary butyl alcohol (giving at least three changes to make ct^rtain that
all glycerin has been removed), thence to one of equal parts of absolute

alcohol, tertiary butyl alcohol and and infiltrate with celloidin.

ether,
For the younger stages of micro- and macrospore development, the
very tips of the fronds should be selected for fixation. On the Atlantic
Coast, F. a dioecious species, predominates. On the Pacific
vesiculosiiSy

Coast, F. furcatus is a widespread monoecious species. Botanists on

the east coast prefer the first-named species, whereas those on the west
coast use the monoecious species in the classroom. From the technical
standpoint, F. furcatus is the more difficult of the two. For the first
282 SPECIAL METHODS FOR THE VARIOUS PHYLA

8 to 10 mm. be microtomed in the longi-

of the tips, the sections should
tudinal plane, perpendicular to one flat face, at 10/i. Older portions
should be cut transversely at about the same thickness. Stain with iron
liematoxylin and orange G (Figs. 38, 39).
For demonstrating the apical cell of the Fucaceae with its five cutting
faces (four lateral and one posterior) and for the origin and development
of the conceptacles, nothing is better than Pelvetia fastigiata. The con-
ceptacles originate quite close to the apical cell (many tips will have two
adjoining apical cells) ;
the one-celled to several-celled stages are usually
found in preparations showing the apical cell or cut for this structure.
Remove the anterior 2 mm. growing young
of the apices of vigorously
fronds, taken preferably from plants which show definite indications of
being in the fruiting condition. Fix with 1% chrom-acetic, and embed
by the standard tertiary butyl alcohol method. Section the tips trans-
versely, parallel to, and perpendicular to the flat surface in order that
the apical cell may be observed in different views. This method ought
also to show several stages in the development of the conceptacles; as
these are scattered irregularly over the thallus, they can be obtained
only in a hit-or-miss fashion. Sections should be at 8 m, and staining is
precise with iron hematoxylin and orange G.
If one is located in the Middle West or far from the coast, that need
be no deterrent to working with living material of Fucus or of many
other Phaeophyta. During cold weather material will travel safely from
either coast,
—
Sargassaceae. Probably all the genera within the confines of the
United States possess fronds provided with vesicles, which constitute an
easy character for distinguishing the genera. In Sargassum the vesicles
occur singly; in the other genera they are seriate. Sargassum for the
most part prefers the warmer seas, but at least one species ranges as far
north on the Atlantic Coast as New England. Cystoseira is to be found
farther north on the Pacific side than on the Atlantic (Florida) Standard .

methods are applicable to these two genera. In Cystoseira the conceptacles

are formed in the more or less metamorphosed terminal branchlets;in Sar-
gassum they are borne in specialized axillary branches, which are slender
and more or less forked. Cystoseira is scarcely less favorable than
Pelvetia for illustrating the origin and development of the conceptacles.
In Sargassum it is known that the development of the oogonia in general
is periodicand simultaneous. Desired stages can be found only at cer-
tain hours on definite days. Meiosis can be found in the oogonium in
the daytime as well as at night.
In most of the genera, but particularly in Cystoseira^ the macrospores
contain numerous large chromatophores which take a deep stain with
iron hematoxylin and are diflScult to differentiate clearly.
 CHAPTER XXIII
CYANOPHYTA
MYXOPHYCEAE (CYANOPHYCEAE)
The Myxophyceae are of widespread occurrence (Geitler 1930-1932,
G. M. Smith 1933) and one cannot fail sooner or later to come into
contact with them. There are only three things about them to annoy
the technician: (1) to get any particular free-living specit\s, with certain
exceptions, in a sufficiently pure condition; (2) to get enough material of
such forms; and (3) the primitivity of the nuclear organization, wffiich
frequently makes exact stain differentiation difficult. If these do not

present too great annoyances to the technician, the group will be found
to be decidedly interesting, and preparations of any type will be most
valuable acquisitions to one’s collection of microscope slides.

Before starting to work up any species, it should be carefully studied

in the living c.ondition. Generic and specific determinations arc, as a
rule, best made on living material, supplemented w^here necessary by
simple chemical tests.

A few Cyanophyta are unicellular, but most of them form filamentous

or other types of colonies. In filamentous colonies, a single row’ of cells

is called a ^Hrichome^^; the trichome together with the gelatinous sheath

is called a filament.” Some filaments may contain more than one
trichome.
The cell wall is composed of cellulose or hemicellulose. Earlier
reports that the wall is chitinous have been disproved by recent investi-
gations. The gelatinous sheath is composed mostly of pectic compounds.
A definite nucleus or nucleolus is absent, but the central body, which
takes up most chromatin stains, contains nucleic acid and therefore may
be considered as being nuclear in nature. The reserve foodstuffs are
mostly glycoproteins but include other proteins and also oils.
—
Occurrence. Cyanophyta are to be found everywhere in an astonish-
ing diversity of habitats, but they are principally fresh-w^ater organisms.
Pure growths, however, are rarely found; these are usually species of either
Anabaena^ NostoCf or Oscillatoria, The habitats of the more prominent
forms will be described in the discussions under the orders.
Cultivation. —Many Cyanophyta are easy to culture. Knop’s,
Benecke^s, or Detmer^s solutions, diluted to about 0.2/N strength, are
excellent culture media. Other fluids, such as artificial sea w^ater,
Wettstein's fluid (Wettstein 1921), or the following may be used:
283
284 SPECIAL METHODS FOR THE VARIOUS PHYLA

Tap water 100 cc.

Ammonium nitrate 0.02 g.
Dibasic potassium phosphate 0 05 g.
.

To obtain soil Myxophyceae, inoculate a medium consisting of 100 cc.

water and 0.02 g. dibasic potassium phosphate (in a 3-liter flask) with 1
or 2 g. garden earth. Place in partial sunlight at 16 to 20°C. Or make
the following solution and inoculate in the same manner:

Water 1 liter

Neutral potassium phosphate 0.2 g.

Magnesium sulphate 0 2 . g.
Potassium sulphate 0 2 .
g.
Calcium carbonate 0 1 .
g.
Ferric chloride Trace

Preservation of Specimens. —
Herbarium specimens are easily pre-
pared. Simply spread and dry the specimens on pieces of mica or thick
cellophane. They may be wetted when it is desired to examine them
microscopically, and then allowed to dry again. The process may be
repeated indefinitely. Habit material may be preserved in 3 or 4%
formalin.
—
Fixation, in general, is easily accomplished.
Fixation. The simplest
fluid a 3 to 5% formalin solution^ in which the algae should
is

remain for a week or longer (Haupt 1923). Bouin^s fluid is said to work
well. The material may be dehydrated by any desired method and
embedded in paraffin. Sections should be cut at 2^ for detailed cytologi-
cal investigations.
Mitochondrial methods are wholly unsatisfactory (Guillierrnond
1926).
Staining. — Staining may be for one of three purposes: (1) general, (2)
for identification of various cell contents, or (3) to outline the gelatinous
structure. It is rarely possible to accomplish all three simultaneously.
General staining, in turn, may be vital or on killed material. For
vital staining very dilute aqueous solutions of either neutral red, cresyl
blue, or toluidin blue are most useful. Preserved or microtomed material
will be more or less stained by almost any of the coal-tar dyes; methylene
blue is often used.
Volutin may be identified by placing the material in 0.1% aqueous
methylene blue and, after the stain has reacted, adding a little 1 sulphu- %
ric acid; a deep blue or black color results.

Plasmodesma, if present, may be demonstrated with a mixture of

and 100 cc. of
6 cc. of a concentrated alcoholic solution of basic fuchsin
3% aqueous phenol. Place some of the filaments on a slide in a large
drop of the solution, and heat gently over a flame. The protoplast will
 CYANOPHYTA 285

shrink and stain an intense red, but the plasmodesma will be clearly
revealed.
For internal details iron hematoxylin surpasses other stains in the
all

quality of the results obtained. Counterstaining may

be with erythrosin,
fast green, or anilin blue. It would also be productive of valuable
results to trj^ Feulgen^s reaction. Picro-indigocannin gives a superb and
occasionally quite life-like stain to many species.
The gelatinous envelopes may
be demonstrated by using moderate
concentrations of the counterstains mentioned above, or of crystal
violet, ruthenium red, toluidin blue, or methylene blue.
—
Whole Mounts. All the Cyanophyta, with the exception of those
which form huge gelatinous colonies (such as Aphanothece, Aphanocapsa,
and Coelosphaerium) and those which invade other plants, are easily
mounted entire. Stain as desired, dehydrate by a gradual hygrobutol
method or glycerin concentration procedure, and infiltrate with very
highly diluted balsam. A very short schedule, satisfactory for all but
critical studies, consists simply of placing a drop of formalin-fixed
material to dry on a clean slide and proceeding directly to the staining,
mounting in balsam from xylol. Of course, one can mount directly in
glycerin or glycerin jelly without staining.

Chroococcales
Chroococcaceae. —Gloeocapsa and Gloeothece may be treated as dried
mounts, as they whether or not Mayer’s
will stick readily to the slip,
adhesive is used.Merismopedia is frequently found; the flat colonies
are easily stained and mounted. Stain some colonies with iron hema-
toxylin, others with an alcoholic carmin stain or with picro-indigocarmin,
and mount some of the different colorations under the same cover.
Aphanocapsaj Aphanothexe, Microcystis, and Coelosphaerium are
difficult subjects; quite apart from this fact, many algologists claim that
these genera are not worth mounting. The writer has found Microcystis
ichthyloblobe to be comparatively easy to manipulate; stain with Mayer’s
carmalum. The others can probably be mounted in glycerin only, as
all attempts to get them into balsam or paraffin have met with failure

because of the complete collapse of the gelatinous matrix. Dissect the

larger colonies into fragments before proceeding with the staining or
dehydration.

Chamaesiphonales
Dermocarpaceae. —Dermocarpa, a marine genus, is common on both
the Atlantic and Pacific coasts. On the west coast Rhodomela is almost
certain to be liberally encrusted with D. fucicola (Fig. 40). Embed not
too old portions of the host, cut transversely at 8 to lOp, and stain with
286 SPECIAL METHODS FOR THE VARIOUS PHYLA

iron hematoxylin and erythrosin. Reproduction is by means of endo-

spores, whose development is easily followed out, since a series of stages
are usually found in each preparation. (Some writers describe the
endospores as gonidia, produced in an enlarged cell which functions as a
gonidangium.) Ceramium is frequently found covered with. Dermocarpa
on the older filaments; host and epiphyte may be mounted together as
whole mounts. On the Atlantic Coast, D. prassina is abundant on
different algae and may be treated like the west coast species.

Fig. AQ.— ’-Dermocarpa fucicola: section of two colonies on Rhodomela larix, showing
the formation of endospores. Fixed with 1 % chrom-acetic in sea water for 10 minutes;
stained with iron hematoxylin and erythrosin.

Xenococcus grows, usually in profusion, on other marine algae, which

are small enough to permit the epiphyte and its host to be worked up

together and mounted entire. If the staining is critical enough, such

preparations are just as good as sections.

Hormogonales
Oscillatoriaceae. —The best-known genus is Oscillatoriay although
there are others which are perhaps rather more interesting. Spirulina,
Arthrospira, Lynghya, Phormidiurriy and Microcoleus, all of which have
fresh-water, brackish-water, and marine species, are of the greatest
value both for technique and for instructional purposes.
These forms may all be treated technically according to the general
methods. The cells of the larger species, especially in Oscillatoria,
are rather liable to collapse if too violent changes of fluids are made.
Those growing in warm salt water should also be handled cautiously;
it would be well to warm the killing fluid to the temperature of the water

in which they are growing, and let it cool slowly. It would be interesting
 CYANOPHYTA 287

to compare the fresh-water and marine species when prepared by identical

procedures.
—
Nostocaceae. Anahaena and Nostoc are cosmopolitan forms and are
readily obtained in abundance and generally also in a pure condition.
Nostocy which sometimes is found in enormous gelatinous balls in the
Rocky Mountain region and in British Columbia, seems to prefer cooler
and more protected habitats than does Anahaena. The latter is usually

I'lG. 41. —Nostoc colony in thallus of Anthoceros carolinianus. Fixed with fonnalin-
propio no-alcohol; stained with iron hematoxylin aiid fast green.

a free-living aquatic alga, but Nostoc is sometimes found on damp soil.

Two species of Anahaena are parasitic. A. azollae infests Azolla; material
of Azolla fixed and stained as described for that genus will afford excellent
preparations of the alga. Section the younger and vigorously growing
branches in the vertical-longitudinal plane at 11^; the alga will be found
at even the very apex of the host and is easily identified if staining is in

safranin and fast green. A. cycadeae inhabits the cortical tissues of the
roots of various species of Cycas. Section portions of the embedded
rootiS transversely at 10/x. The parasite is ndt eavsy to locate in most
 288 SPECIAL METHODS FOR THE VARIOUS PHYLA

roots. For whole mounts of free-living species, stain with iron hema-
toxylin or picro-indigocarmin. Nostoc may be terrestrial, aquatic, or
and occurs in the form of gelatinous nodules of various sizes,
parasitic,
some becoming as large as 50 cm. acro.ss. The species found free floating
or attached to various substrata in swiftly flowing streams are more

Fio. i2.—Anabaena, Microcyatia and OacUlatoria: whole mount of the so-called “

Was-
serbltlte.” Fixed with formalin-aoeto-alcohol; stained with iron hematoxylin and fast
green.

easily manipulated technically than those growing on damp ground.

Kill and fix in weak chrom-acetic, 6% formalin, or in formalin-aceto-
^cohol. Small colonies may be run up entire and the colony crushed
just before the coverslip is applied. Larger colonies are very easily
embedded and sectioned for detailed investigations. Cut at any thick-
ness up to 8/i. Stain critically in iron hematoxylin. A oounterstain of
ftcid fuchsin (1% in 70% alcohol) will differentiate the sheaths enclosing
 CYANOPHYTA 289

the individual trichomes from the common gelatinous matrix; anilin blue
is also excellent for the purpose and perhaps less gaudy. Anthoceros
is

and its allies are parasitized by Nostoc, which can be easily recognized in
sections of the host thallus (Fig. 41).
Akinetes are most easily found in Cylindrospermum: they are developed
next to the heterocysts at one end of each trichome and are sometimes
found in a catenate series. Treat like free-living species of Anabaena.
Scytonemataceae. —Members of
this family are easily recognized by
their false branching. This character is shared by the Rivulariaceae,
but in this family the trichomes are conspicuously attenuated and possess
terminal hairs. Most of the genera are cosmopolitan in distribution.
In Scytonema the filaments grow over damp soil or on dripping cliffs
and are usually so interwoven as to form a felt-like mass of considerable
extent. Tolypothrix and the remaining genera arc aquatic.
All species may be treated alike. The hygrobutol method has been
the most successful one. The basic stain is preferably iron hematoxylin.
A counterstain is necessary to reveal the sheaths and the nature of the
false branching. For this purpose orange G, fast green, acid fuchsin,
or anilin blue may be employed. These algae are easily embedded and
sectioned, but whole mounts are ordinarily adequate.
Stigonemataceae. —Genera belonging to the family are characterized
by true branching. Heterocysts are always present, but akinetes are of
rare occurrence. Siigonema is most likely to be collected. Treat as in
the preceding family.
Rivulariaceae. —
Cyanophyta which have trichomes conspicuously
attenuated either from base to apex or from the center toward both
extremities belong in this family. Each trichome usually terminates in
a hair. There are both fresh-water and marine species, the latter pre-
dominating. Submerged species usually grow on the stems of other
plants, within the gelatinous envelopes of other algae, or on rocks or
wood.
Rivularia and Gloeotrichia are perhaps the best known of the genera,
but others, sUch as Calothrix and Dichothrix, are quite as interesting
and useful. The nodule-forming species may be treated like similar
species of Nostoc, other species by the general methods for the phylum.
The soft nodules of Gloeotrichia are very apt to become dissociated in
the killing fluid. A drop containing dissociated filaments may be spread
on a slide smeared with a thin layer of adhesive and fixed in 95% alcohol
for a few minutes. Stain for 24 hours or longer in safranin, extract the
stain with acidulated water until only the internal details remain clear,
then counterstain with fast green to bring out the sheaths; or iron hema-
toxylin with any suitable counterstain may be used. Complete dehy-
dration and mount in balsam.
290 ' SPECIAL METHODS FOR THE VARIOUS PHYLA

Colonies of Rimlana may be fixed with 1% chrom-acetie or with

formalin-aceto-alcohol and the fluid washed out; then the trichomes may
be critically stained with iron hematoxylin, dehydrated to 85% alcohol,
and counterstained with fast green, and dehydration may be completed
with hygrobutol, and infiltrated with balsam; finally a single colony may
be crushed on a slide and the coverslip applied.
 CHAPTER XXIV
RHODOPHYTA
RHODOPHYCEAE
In structure and particularly in methods of reproduction the Rhodo-
phyta are the most diverse and complicated of all algae. From the
technical standpoint very little indeed has previously been done on the
phylum as a whole.
In size the Rhodophyta generally are smaller than most of the Phaeo-
phyta, but larger than the Chlorophyta. The designation ^^red algae
is somewhat of a misnomer, for some appear to be greenish
s})ecies
{Iridaeay H
alosaccion) and others have a brownish color {N emalioriy
Gracilariay Ceramium). It has l)f>en claimed that sterile Rhodophyta
may be distinguished from Phaeophyta by the slower rate at which they
turn green when dipped in water heated to a temperature between 60
and 70°C. Some known member of the Phaeophyta, such as Fucus or
Laminaria^ should be used as a guide in making this test. A simpler
method may be found in the fact that when the larger Rhodophyta are
placed in the standard chrom-acetic fixing fluid, they lose all color very
rapidly, whereas most Phaeophyta are scarcely affected.
—
Occurrence. Although the Rhodophyta are predominantly marine
plants, there is a considerable number of fresh-water species (Skuja 1938).
These prefer shallow running waters and belong mostly to the primitive
groups. Batrachospermurriy so commonly employed in elementary botany
courses as a representative of the red algae, is the most widespread genus
as well as the one with the largest number of species. Very few of the
fresh-water species are red in color; they are mostly bluish, green, grayish,
or brownish.
The Rhodophyta prefer warmer waters, but a few representatives
may be found in northern latitudes. In the United States the greatest
range in forms is to be found in Florida and along the Pacific Coast from
Puget Sound southward. Like the brown algae, the red algae grow
mostly on rocks or where the shores are rocky. On sandy beaches few,
if any, red algae can be found, except on pilings, stone jetties or similar

structures. The coralline types are practically the only red algae occur-
ring in tide pools, which are otherwise well filled with representatives of
other algae and aquatic Angiosperms. The Rhodophyta prefer the mid-
dle and lower littoral regions: the more deeply the plants grow, the
291
 ,

292 SPECIAL METHODS FOR THE VARIOUS PHYLA

brighter red their color becomes. Those growing near the upper tide
line become more brownish darker {Chondrus and some
(Agardhiella)
species of Gigartina), olivaceous (Gastroclonium, Lomentaria) or even ^

blackish {Endocladia^ Rhodomela). One should therefore beware of being

deceived by color differences when collecting Rhodophyta.
There is no satisfactory manual available for the Pacific Coast
Rhodophyta, outside of one regional manual (Kylin 1925), but an excel-
lent manual for the Atlantic Coast species was recently published
(Taylor 1937). A comprehensive review of the anatomical features of
the phylum (Kylin 1937 see the bibliography for references to the litera-
;

ture) has recently been published.

Collection and Preservation. —These
procedures are identically as
described for the Phaeophyta making herbarium specimens,
(p. 263). In
most Rhodophyta will adhere firmly to the paper and need no gluing or
taping. They dry quickly but are susceptible to mold infection.
Fixation.—The two standard fixing fluids described as being satis-
factory for the Phaeophyta serve equally well for most Rhodophyta;
viz,j8 to 10% formalin (neutral) in sea water, and a 1% chrom-acetic
fluid. Certain groups, however, notably the Bangiales, Ceramiales, and
Gigartinales, present exceptional diflBculties, and special methods will
occasionally be cited.
It must be emphasized in the strongest possible terms that Rhodo-
phyta should not be left in any of the various chrom-acetic mixtures any
longer than it takes to fix them properly. In some cases this is a matter
of seconds. The filamentous forms and some thalloid genera, such as
Porphyra, will break up quickly, and others will literally become dis-
solved into a gelatinous mass if left in the killing fluid for even 1 minute
too long. Such Rhodophyta are good illustrations of the fact that
killing and fixing are two separate processes, even if they are usually
obtained with the same fluid; all Rhodophyta are promptly killed, but
few ever become sufficiently fixed for further treatment. As a safe rule,
it may be stated that fixation is completed in about half again the length

of time that it takes for the natural color to disappear. For example, if
the color disappears in 2 minutes, fixation (or what passes for fixation)
is completed 1 minute later, hence the washing out of the fixative should

be begun immediately. In neutral 10% fornaalin in sea water, material

may be left for several months without appreciable harm, provided it is
kept out of the light.
Embedding. —This may be carried out exactly as described for the
Phaeophyta. Difficulty isbe encountered with jnany species.
likely to
Even if the material appears to have been killed satisfactorily, there will
be many occasions when it will be discovered that fixation actually was
not Effected; the material either becomes excessively hardened or shrinks
 RHODOPHYTA 293

completely after it has been carried beyond 80% alcohol. The Rhodo-
phyta have a tendency to become brittle if left too long in the paraffin
oven, consequently the time devoted to this process should be as brief
as possible. In any event, infiltration takes place very quickly with
practically all species.
Whole Mounts. —The best fixing medium is an 8 to 10% solution of
formalin in sea water, but as it is necessary to get rid of the natural pig-
ments before staining can be effected in certain genera, a 1% chrom-
acetic should be used on these. Even the fronds of large forms like
Ceramium and Ptilota are perfectly preserved by the formalin solution
and are easily stained and mounted entire. Fresh- water forms may be
fixed in the standard chrom-acetic or formalin-aceto-alcohol fluids com-
monly used on the fresh-water Chlorophyta.
On no account should iron hematoxylin be used on whole-mount
material of marine Rhodophyta: it is practically a certainty that the
material will become completely dissociated when the mordant is poured
on or when the stain solution is applied after washing out the mordant.
The reason is that the acid of the mordant dissolves the gelatinous matrix
and allows the cells to become separated. By far the most satisfactory
results, as well as exceedingly beautiful preparations, may be secured with
Harris^ hematoxylin and an erythrosin counterstain.
Species that have a loose filamentous structure held together in a
gelatinous matrix are adapted for the making of temporary mounts which
are exceedingly serviceable for investigating the structure and develop-
ment of complicated carpogonial branch systems. Place the fresh mate-
rial in 6% hydrochloric acid in sea water, and let remain overnight or at

least for several hours, whereupon it will be found to have become more
or less dissociated. Put a small portion on a slide, cover with a No. 2
coverslip, and crush and spread gently. The filaments will become
separated, and the entire reproductive structure with all its ramifications
may be located and studied. This is impossible in the case of even very
thick paraffin sections. If it would be possible to do so without too
much handling of the material, the dissociated mass might be stained,
dehydrated with hygrobutol, and mounted in balsam.

Bangioideae

Bangiales

The majority of the species are marine, but a few fresh-water species
are found in each of the families.
Erythrotrichiaceae. —
Most of the genera are marine, and when grow-
ing epiphytically are inseparable from the host. Erythrotrichia is com-
mon on both coasts and is an instructive form because of the formation
294 SPECIAL METHODS FOR THE VARIOUS PHYLA

of monospores by oblique walls in vegetative cells. Fix in 8% formalin

in sea water, stain carefully with Harris^ hematoxylin, counterstain with
erythrosin,and run up by a gradual hygrobutol method (Fig. 43). Dis-
regard fixation and staining of the host, which is usually in a bad
condition.
Bangiaceae. — Bangia is fairly common on both coasts. Fixation is
difficult, whether for whole mounts or embedding, because of excessive

Fig. 43 . —
Erythrotrichia carnea epiphytic on old filaments of Cladophora: this prepara-
tion well illustrates the utter impossibility of separating host and epiphyte and why they
must be run up and mounted together. Fixed with 1 % chrom-aeotic in sea water; stained
with Harris’ hematoxylin and erythrosin.

plasmolysis. As the thick gelatinous envelope will x^revent the cells from
breaking apart, iron hematoxylin may be used on material intended for
whole mounts. The standard chrom-acetic fluid diluted in half with
sea water sometimes gives good results on material to be embedded. A
group of thalli should be embedded and sectioned together transversely
at about S/x.
Porphyra is very common and abundant on both coasts. It is a
diflScult alga to fix adequately, but in this respect it is not so difficult as
Bangia. The thallus in most species is too thick for clear staining for

whole mounts. Picro-indigocarmin is the only stain which, in the writer's

experience, does not hopelessly overstain. The reproductive regions may
usually be recognized as differently colored areas along the margins of
 :

RHODOPHYTA 295

the thallus. The fluid which has given the best fixation for embedding
and sectioning is composed of
Saturated aqueous picric acid 100 cc.
Chromic acid 1 g.
Hydrochloric acid, c.p 6 cc.

While observing the color changes, watch the vegetative areas of small
portions of tlie thallus, not the already discolored reproductive regions,

and stop fixation as soon as all color has disappeared. Microtome^ trans-

Fig. 44 . —
Porphyra perforata: cross section of thallus with both vegetative cells and
reproductive regions. Fixed with 1 % chrom-acetic in sea water; stained with iron hema-
toxylin and fast green.

versely at about 7 m; staining is generally very good in iron hematoxylin

(Fig. 44).

Florideae

The great diversity in thallus structure and methods of reproduction

lead to technical difficulties of one sort or another. Even species in the
same genus will not react equally wcdl to an idt^ntical method of treatment.
Laurencia spectabilis and L. virgata afford an example of this sort; the
latter is far more amenable to treatment than the former, although the
two are scarcely different structurally.
The following discussions will not go into any great detail, except in
the case of such common laboratory forms as Polysiphonia. This, how-
ever, should emphatically not deter one from trying the admittedly
difficult Florideae, for these plants, when properly worked up, constitute
some of the most beautiful and instructive preparations imaginable.

Njcmalionales
The order includes a few fresh-water and innumerable marine species.
It differs from the other Florideae in that a tetrasporic generation is
absent. The general impression seems to be that Nemalion is the only
representative of the order worth studying. Such is not the case:
Nemalion is r^^ther unsatisfactory if one wishes to secure an abundance
of all stages in the development of the carpogonia, gonimoblasts, and
carpospores. Cumagloiay which unfortunately is restricted to the Pacific
Coast, is a more satisfactory genus.
296 SPECIAL METHODS FOR THE VARIOUS PHYLA

Chantransiaceae. —Acrochaetium^ a filamentous marine alga, which

is more abundant on the Atlantic than on the Pacific Coast, is espe-
far
cially desirable for illustrating reproduction by means of monospores.
The plants are said to be not always in a good condition for study. Sper-
matia and carpogonia occur in some species. Pieces of the host bearing
the epiphyte may be fixed for about 10 minutes in 1% chrom-acetic,
stained with Harris^ hematoxylin and erythrosin, and then dehydrated
by a gradual hygrobutol method; the epiphyte may be carefully scraped
offwith needles and mounted alone. Some species are endophytic and
others endozoic sections apparently can be made without difficulty.
;

—
Batrachospermaceae. One of the commonest fresh-water Rhodo-
phyta is Batrachospermumj found in cool small streams, ponds) and pools.
It is easily recognized by the whorls of lateral branches on the conspicuous
central axis. Most species are a pronounced bluish or olive-green in
color when fresh and growing in the light; when found in shady places or
deep water, the color changes from reddish to purple. Adult fruiting
plants may be found only during the spring season. Fix in formalin-
aceto-alcohol or in a weak chrom-acetic, stain in iron or Harris^ hematox-
ylin, counterstain with erythrosin and make whole mounts. When
ready to mount, cut the thalli into suitable small portions.
—
Lemaneaceae. Small plants of Lemanea, which grows on rOcks in
turbulent fresh waters, are readily mounted whole, but longitudinal and
transverse sections of the maturing thallus are required if stages in the
development of spermatia and carpogonia are desired. Sections should
be cut at 4pt for spermatia and 7 m for carpogonia. In staining sections,
iron hematoxylin is preferable, but it is not always possible to get the
spermatia and carpogonia differentiated to an optimum point for both
on the same slide. The carpogonia do not retain the stain well, whereas
a prolonged treatment is required by the spermatia. Counterstain with
erythrosin.
—
Helminthocladiaceae. The more interesting species are grouped in
this family. A given species may be either monoecious, dioecious or
rarely both. The spermatia are borne on the ends of short, and often
densely crowded, branches. The carpogonia originate on short carpo-
gonial branches; the terminal cell elongates into a trichogyne. The
fertilized egg develops into gonimoblasts whose terminal cells produce
carpospores.
Curmgloia andersoniiy as previously noted, is one of the best species
to use (Fig. 45). Helminthora, which does not occur in the United States,
and Liagora, to be expected in Florida and perhaps farther north, are the
next best, but Nemalion can, of course, be used if none of the other genera
is available. NemoMon^ which grows in scattered colonies on rocks in
the )ipper or middle littoral zone and is not common, is readily recognized
 RHOmPHYTA 297

in the fieldby the very slippery feeling of its dull brownish-red, terete
thalli. In Liagora some species are slimy, but in others the gelatinous
matrix is more or less calcified if a fluid containing acetic acid is used on
;

the calcified forms, no trouble should be encountered.

In order to obtain the youngest stages in the development of both
spermatia and carpogonia, the very tips of the thalli must be used. The
younger plants are better. The terminal 1.5 or 2 mm. of the thallus
should be placed in one vial containing the killing fluid, the next 2 mm. in

Fig. 4^b.—Cuinagloia andersonii: median longitudinal section of portion of a carpogonial

plant, showing the essentially filamentous structure of the thallus. Fixed with chrom-
acetic in sea water; microtomed at 16/x; stained with iron hematoxylin and erythrosin.

a second vial, and the third 2 mm. in still another vial; portions of the
thallus over 5 or 6 mm. from the tip will ordinarily show only mature
structures. The standard chrom-acetic fluid may be used, or a few drops
of a 1 % aqueous solution of osmic acid may be added if desired, as well
as 1 g. of urea or saponin.
To show the filamentous structure of the thallus, longitudinal sections
of the apex should be microtomed at about 18/x, transverse sections a
few microns thinner. For the development of the spermatia, longitudinal
sections are best, cut at 8/i. To show the characteristic downward growth
of the carposporophyte in Nemalion, which is rarely diagrammed accu-
rately in botanical texts, longitudinal sections may be cut at 12iL£. For
studies in greater detail of either the spermatia or carpospores, the sec-
tions should be much thinner; 6/* is about right, Such thin sections
 —

298 SPECIAL METHODS FOR THE VARIOUS PHYLA

naturally do not show the general topography of the thallus as well as

do the thicker sections.
Staining exceedingly difficult with some genera, but iron hematox-
is

ylin may be tried. The destaining must be rather carefully controlled

A j),
'

Fiu. 46. Gloiophlom confuaa: longitudinal section of thallus with throe carpogonia
(one above the other, slightly to the left of the center). The very small size of the carpo-
gonia and their burial within the thallus indicate the careful search that must be made to
locate them. Fixed for 10 minutes in 1 % chroin-acotic in sea water; stained with iron
hematoxylin and fast green.

aKSthe sex organs lose the stain long before the vegetative portions are
properly differentiated. Erythrosin differentiates the carpogonia and
spermatia unusually well, but the staining period with this dye should be
twice as long as when used on other plants.
It has lately been found that Johansen^s quadruple stain affords
clear and sharp results not obtainable with other stain combinations;
the material should hav^ been fixed in a chrom-aoetio fluid as formalin-
Preserved material does not take a good stain.
 RHODOPHYTA 299

All the genera in the family lend themselves to the preparation

of whole mounts, but only fresh material should be used for this purpose.
Preserved material becomes too acid to take and retain stains. The
thalli of Nemalion and Cumagloia arc so slippery that they can hardly be
kept in glass containers; small strainers, obtainable at any five- and ten-
cent store, should be used to hold them. Pieces of thallus about 2 inches
in length should be fixed in 1 %
chrom-acetic in sea water to remove the
pigment; wash thoroughly, transfer gradually to distilled water, then

Fia. 47 .
—
Gloiophloea confusa: longitudinal section of thallus with two young carposporo-
phytes, the lower one being in median section. Fixation and staining as in Fig. 4(3.

pass through 10, 20, and30% ethyl alcohol. Stain with Harris^ hematox-
ylin,wash, and differentiate. Dehydrate somewhat slowly by a gradual
hygrobutol method, and infiltrate with balsam. When ready to mount,
cut the thallus into portions about 1 cm. in kmgth with scissors. Jt will
be necessary to apply considerable pressure on the coverslip to spread the
thallus sufficiently; it would be better to crush the piece of thallus with
the flat side of a scalpel before adding the coverslip.
—
Chaetangiaceae. Scinaia, Gloiophloea, and Whidbeyella are found on
the Pacific Coast, Galaxaura on the south Atlantic. All are equally
interesting, but a little difficult to manipulate. The first three are soft
plants, but this is a deceptive quality, as they occasionally become so
300 SPECIAL METHODS FOR THE VARIOUS PHYLA

cartilaginous after passing through the higher alcohols that they can
be neither embedded nor sectioned. Gloiophloea is exceptionally fine for
showing the development of the gonimoblasts from the carpogonium
(Figs. 46, 47). Galaxaura is more or less calcareous, but the hints given
as to the treatment of Liagora also apply here.
The apical tips of the plants should be collected if one is studying the
reproductive phases, as suggested under the Helminthocladiaceae. The
cells composing filaments, antheridia, and carpogonia are so small that
rather thin sections, at least 7 to 8 m, are indicated. For general topog-
raphy the sections may be cut a little thicker. Staining is very beautiful
with iron hematoxylin, which is comparatively easy to differentiate in
this family.

Gelidiales
The order differs from the haplobiont Nemalionales in being diplo-
biont; the carposporophyte develops directly from the carpogonium.
The plants are mostly slender or wiry and rather tough.
Gelidiaceae. —Gelidium is a widespread genus, and some species are
of considerable economic importance as the source of agar. (?. car-
tilagineum is common on the Pacific Coast, G. crinale on the Atlantic, and
other species are to be found on both coasts. The mentioned
species first
is excellent for illustrating the development carpogonium peculiar
of the
to the family, but the trouble is to get the material into paraffin without
its shrinking or becoming too cartilaginous. Satisfactory methods have
not yet been devised. The standard ehrom-acetic fluid succeeds only
with the apices of the fronds or with the very youngest plants. Section
the material in the longitudinal plane.

Cryptonemiales
Dumontiaceae. —
In this family the carpogonial branches and auxiliary
cell branches are distinctly separated from each other. Several genera
are available for demonstrating this condition. Cryptosiphonia woodii
is supposed to be one of the best, but the technique for this plant has not

yet been sufficiently refined; the material is very apt to shrink completely
during dehydration. Fresh or well-preserved material may be treated
with hydrochloric acid and the complicated carpogonial filament and the
carposporophyte observed in their entirety.
—
Squamariaceae. The anatomy of Peyssonnelia is rather interesting;
the genus occurs from Florida to Maine. There are several other Atlantic
Coast genera. The usual methods should prove satisfactory.
—
Grateloupiaceae. In this family the carpogonia and auxiliary cells
are formed on inner branches of the cortical filaments; the gonimoblast
arises from a large basal stalk cell. On both coasts Grateloupia and
 :

RHODOPHYTA 301

Halymenia are the genera most be found, and on the west coast
likely to
Prionitis is very favorable material. not at all difficult to secure
It is
series of stages by following the general methods.
Corallinaceae. —
The coralline red algae superficially form a decidedly
forbidding group of plants because of the heavy incrustation of calcium
salts on their fronds. There is little real basis for the claim that the
group is technically impossible since preparations of even the larger and
more heavily encrusted species are easily made. One need only make
(certain that the lime has been completely removed during fixation. The
compact nature of the fronds permits exposure of the pieces of material
to the softening action of acids for long periods.
The larger crustaceous forms, which grow on rocks, stones, or wood-
work, should be carefully scraped off in small portions. The small
crustose forms, which usually grow on other algae, are best removed
together with part of the host. For example, leaves of Phyllospadix or
Zostera or the main axis of Laurencia encrusted with Epilithon or Melo-
besia may be cut into short portions, dropped into the killing fluid,
embedded, and sectioned together. It is of no avail to attempt to sepa-
rate them. All the erect-branched species should be cut into convenient
short lengths. It is better to cut across the middle of an internode (or
segment) of the larger species than to break the joints apart at a node.
The nodes are not calcified.
All the smaller species included in Melobesia and Epilithon are per-
fectly fixed and sufficiently softened within 10 minutes or slightly longer
in the standard chrom-acetic fluid. In Epilithon (which is placed in
Lithotharnnion by some authors and in Melobesia by others), particularly,
a fine series of developmental stages of all the reproductive phases are
easily obtained. The larger forms may be placed in the following solu-
tion until soft (a month or longer may sometimes be required)

Distilled water 100 cc.

Sodium chloride 1 g.

Formalin 10 cc.
Glacial acetic acid 8 cc.
96% alcohol 50 cc.

Dissolve the salt in the distilled water first, and add the alcohol last,
pouring it in slowly while stirring the mixture there should not be a white
;

turbidity. Wash out with 50% alcohol, and begin the dehydration proc-
ess with the 50% step of the tertiary butyl alcohol method. The
material usually microtomes with surprising ease. Staining is excellent
with either iron or Harris^ hematoxylin and erythrosin.
In genera with such broad joints as those of Amphiroa there may be
some difficulty in locating the sex organs, although tetrasporangia are
easily located.
302 SPECIAL METHODS FOR THE VARIOUS PHYLA

—
Gloiosiphoniaceae. One species of Gloiosiphonia occurs on the
Atlantic Coast, two on the Pacific. Whole mounts of portions of the
plant bearing tetrasporangia are possible, but since the material responds
nicely to treatment, sections should be made.
Callymeniaceae. —The
two most important genera are CallophylliSj
with several species on the Pacific Coast, and Callymeniaj with one species
on the west and two on the Atlantic coasts. Plants of the former are
easily collected at very low tides, but Callymenia grows in such deep
water that only floating or dredged plants can be picked up.

Fiq. 48. —
Callophyllis furcata: longitudinal section of thallus perpendicular to flat
surface, with two young carposporophytes. Fixed with 1% chrom-acetic in sea water;
stained with iron hematoxylin and erythrosin.

Callophyllis probably the easiest of all Rhodophyta with thin, flat

is

fronds with which to work. Fixation with chrom-acetic is perfect; there

is no shrinkage or hardening during the embedding process, staining is

precise with iron hematoxylin, and it is a comparatively simple matter

to secure a complete series of stages from the two-celled procarp to mature
cystocarps (Fig. 48). Cut off the tips of the plants about 6 mm. back
from the apex, and section these in a vertical longitudinal direction at
lOju. The remainder of the thallus may be cut up into portions about
4 mm. long and microtoraed transversely. Most of the plants collected
will be cystocarpic; finding the spermatia and tetrasporangia is a matter
of sectioning and examining material from a large number of plants. For
this reason only material from a given plant should be placed in each vial.

Gigartinalbs

For a discussion of this order, one

cannot do better than to study
Kylin^s papers (Kylin 1928, 1932) and to follow the bibliographies listed
 EHODOPHYTA 303

therein. A large number of the genera mentioned in these papers are to

be found on either coast of the United States (see also Taylor 1937).
Practically all of the species are perfectly fixed with the standard
chrom-acetic fluid, but the species in the Gigartinaceae are exceedingly
troublesome. The writer has never obtained really good fixation of
either Chondrus or Iridaea after innumerable trials; success was had with
two or three species of Gigartina but not at all with others. Species with
the cells closely packed together are easier to manipulate than those
with a rather loose cellular organization. Firmness to the touch does not
always connote solidity of stru(;ture; for instance, thalli of species of
Gigartina which have a rigid feeling when pressed with the fingers never-
theless become reduced to formless masses either during fixation or wash-
ing. Since the gelatinous substance permeating the large intercellular
spaces is of a carbohydrate nature, there is no known method of pre-

cipitating or coagulating it, as can be done with proteinaceous substances.

Sometimes treatment with hydrochloric acid, either during or immedi-
ately after fixation, prevents excessive dissociation. Very young portions
of the thallus are generally more solid than the older parts; since these
bear the spermatangia and carpogonia, good fixation can usually be
obtained. Small portions of the thallus cortex with mature cystocarps
can be removed and fixed separately.
Agardhiella and Gracilaria are particularly to be recommended; they
arc easy to fix, to section, and to stain with iron hematoxylin. If care
is taken! to obtain the youngest fruiting tips, a fine series of stages in the

development of the carpogonial branches and carpospores is readily

secured.
In the following discussion, those families which have no easily col-
lected representatives on cither the Atlantic or Pacific Coast are omitted.
Solieriaceae. —Agardhiella tenera
is found all along the Atlantic Coast

and A. coulteri on the Pacific side. They are exceptionally easy algae to
work with. Sarcodiotheca, occurring in Washington, is likewise easy.
The plants are heterothallic, large, bushy, with cylindrical, tapering
branches that have a peculiarly fleshy, firm texture. The tetrasporangia
and cystocarps are scattered, the spermatia are formed in patches on
young branches. For the origin of all stages, remove 2 to 3 mm. lengths
of the tips of the branches, and microtome longitudinally at 7 to ll/i.
For developing gonimoblasts and tetraspores, use the next few millimeters
of the tips, but section transversely at 12 m. The mature cystocarps are
easily recognized as darker red spots in the thalli ;
cut out short portions
bearing such spots, and microtome them transversely at 10m. Stain all

stages with iron hematoxylin and fast green.

—
RhodophylUdaceae. Two genera occur on the north Atlantic Coast.
RhodophyUis has flat, compact membranous thalli which appear to be
304 SPECIAL METHODS FOR THE VARIOUS PHYLA

unlikely to give difficulty during fixation and dehydration. Fix short

portions of the apices for the younger developmental stages; otherwise
treat as in the preceding family.
Plocaxniaceae.—Two species of Plocamium are to be found on the
Pacific Coast. The thalli are richly branched, somewhat flattened, and
ifear the reproductive bodies on the small side branches. Remove a
group of these branches, embed, and microtome in the horizontal longi-
tudinal plane at lO/n. Mount as serial sections, since neither sperma-
tangia nor carpogonia are abundant. Stain with iron hematoxylin, but
avoid overdoing the counterstain.
Gracilariaceae. —On the Pacific Coast Gracilaria sjostedtii is very
abundant in and its manipulation gives absolutely no trouble,
late spring,
although spermatangia and carpogonia are difficult to find. The devel-
oping and maturing cystocarps are recognizable immediately as “ warts
scattered along the long, cylindrical thallus; section these both longi-
tudinally and transversely, mounting only sections going through th(^
pore. There are two species on the Atlantic Coast; one is cylindrical and
the other flattened. All these species are found in shallow, quiet, and
warm bays or coves.
Gigartinaceae. —
Four genera which superficially do not appear to
be closely related are placed in the family: Chondrus, Iridaea {Iridophy-
cu8)f Rhodoglossurriy and Gigartina. The exceedingly variable Irish
moss,^^ Chondrus crispus, occurs on both coasts. Spermatia and carpo-
spores are produced during the summer and tetraspores during the
autumn. The plants become extremely brittle during dehydration, but
if the process is made very gradual, efforts to get young tips into paraffin

may be successful. Section these tips in the vertical longitudinal plane

at 10/i.

Only one very small species of Gigartina grows on the north Atlantic
Coast, whereas the genus has about 10 species on the Pacific side, some
of them being among the largest of all Rhodophyta. The taxonomy of
these species is still chaotic despite recent attempts to straighten it out.
The cystocarps of Gigartina are borne in special short branches which
densely cover the thallus in most species. As all stages in development
save the very youngest are readily recognized externally, a series of stages
is easy to collect. G. canaliculatay a slender plant which feels soft when
handled in the fresh condition, is really cartilaginous and becomes so
hardened and desiccated during dehydration that it cannot be micro-
tomed. If paraffin blocks of this and other species of the Gigartinales
are placed in water, the portions promptly “blow out.’^ In fact the
sections can hardly be placed in water to straighten them out, as they
invariably twist themselves out of the paraffin. Celloidin embedding is
not much better. G. hinghamiae has proved to be the species most amen-
 RHODOPHYTA 306

able to treatment, but it must be said that a satisfactory fixing fluid and
dehydration method for the entire family has yet to be devised.
Iridaea is one of the commonest red algae during the summer and
autumn along the Pacific Coast. All reproductive phases are buried
within the thallus. A long series of killing fluids and various dehydration
methods were utilized in an attempt to manipulate this genus, but no
conclusive results were obtained. The most satisfactory fixation was
obtained with the following:

l/N hydrochloric acid in sea water 90 cc.

Formalin 6 cc.
Glacial acetic acid 6 cc.
Chromic acid 1 g.

Stenogramme interrupta, which may be found at, or below, the lowest

tide levels along the central California coast, is the only species in the
family with which the writer has had consistently good fixation and
staining results. Remove 4 mm. of the apices of the thalli, then fix
separately the next, the third, and the fourth 4-mm. portions. The apices
should be microtomed in the vertical longitudinal plane, the others
transversely, at not over 8/u, In the apical portions the origin and devel-
opment of the procarps or spermatia will be found; young stages of tetra-
spore formation will be found in similar portions from tetrasporangial
plants.

Ceramiales
Included in the order are innumerable forms which are exceedingly
beautifulwhen observed under the microscope, whether living or stained
and mounted. For the most part these species are filamentous, but some
are polysiphonous, others frondrose, while still others are more or less
saccate and solitary or colonial. Few technique difficulties are likely to
be encountered with any of the species.
—
Ceramiaceae. Material of the species belonging to this family is
abundant and easily collected. The difficulty is to obtain the sexual
reproductive phases, as the bulk of the material usually collected tends to
show only tetraspores, if fruiting at all.

All the species are readily transformed into permanent mounts. As

the family is intended for whole
strictly marine, fixation of material
mounts is excellent in 10% formalin in sea water, or the usual 1 % chrom-
acetic fluid may be employed. The change from sea water to distilled
water should be more gradual than usual as there is sometimes a tendency
for the tetraspores to collapse either during this change or during the
dehydration process. Staining is very good in Harris' hematoxylin with
counterstaining in erythrosin.
 306 SPECIAL METHODS FOR THE VARIOUS PHYLA

Material intended for embedding may be fixed in the standard chrom-

acetic solution, which should be diluted with an equal portion of sea
water. the material must be transferred
If division figures are desired,
directly from ocean into the killing fluid, and the only time when
the^
mitoses are likely to be caught is around midnight. The time of fixation

Fig. 49.—Ceramium californicum: whole mount of a portion of a vegetative plant.

The banded appearance characteristic of the genus, and the protoplasmic connections
between adjoining cells, are clearly shown. Fixed in 1 % ohrom-acetio in sea water for
4 minutes; stained with Harris’ hematoxylin and erythrosin.

is extremely short, even with the highly diluted fluids, hence its progress
must be carefully watched. The material should remain in the paraffin
oven for the briefest possible period. It would save much time and work
later on if the pieces of material were embedded in bunches. Staining
seems to be best with iron hematoxylin and erythrosin.
Ardiitiamnionis one of the most widespread of the various genera. It
grows on the piles of wharves and docks, on the bottoms of sm^ll boats,
 RHODOPHYTA 307

and on the laminae and stipes of the larger Laminariales. The plants
generally contain tetraspores; plants with gonimoblasts and spermatia
are not often encountered, but when found a fine series of developmental
stages is often present. Whole mounts are preferable; critical attention
needs to be paid to the staining.
Callithamnion and Ceramium are also common, but it is sometimes
difficult to collect them, especially the latter, when in fruit. Ceramium^
however, has such an interesting thallus structure that even sterile mate-
rial is worth mounting entire (Fig. 49). Compared to the other
well
genera, Ptilota may appear to have too thick a frond for whole mounts to
be made, but such is not the case since a transparent stain is readily
obtained with Harris’ hematoxylin and erythrosin. Griffithsia is easily
mounted whole, but as the reproductive regions may be obscured if the
pieces of frond are not carefully examined before the coverslip is applied,
sections may
be more useful. Fix in diluted 1% (ffirom-acetic, section at
3 to 5g, and stain with iron hematoxylin.
Spermothamnion is known from the Atlantic Coast, but it is said to
be difficult to find in the fruiting condition, and spermatangia have not
been ^Found.
Delesseriaceae. — Most of the species in this family possess mem-
Inanous fronds, and all are easily fixed and microtomed. On the Pacific
Coast one may select Nitophylluyn, which attains a considerable size and
is (‘asily collected. There are a number of other genera, specie's of which
may be found on either or both coasts, which are so closely related to, or
otherwise so resemble, Nitophyllum that they may be treated similarly.
These include: Erythroglossurn^ Polyneura, Heteronema, Delesseria^ Crypto-
pleura Memhranoptera^ and others.
^

Remove the very tips of the fronds from the plants, and microtome
these longitudinally both perpendicular and parallel to the flat surface at
about lOg. Stain with iron hematoxylin and counterstain cautiously to
avoid overstaining. The procarps are rather difficult to locate, not
being any too numerous. Older portions of the fronds may be sectioned
transversely.
Rhodomelaceae. —Some of the commonest and most interesting of
all red algae are included in the Rhodomelaceae. It contains Polysi-
phoniay which has doubtless been studied more extensively than any other
red alga. This perhaps is more because of its general availability than
for any other reasons, since there are other genera easier to handle and
of simpler structure but with the same methods of forming the reproduc-
tive bodies. Pterosiphonia is an example.
Despite their wide distribution many of the Rhodomelaceae are diffi-

cult to collect at most localities, either for lack of abundance or of a

healthy and vigorously growing or fruiting condition. The various
308 SPECIAL METHODS FOR THE VARIOUS PHYLA

speciesgrow on rocks, in sandy areas between protecting rocks {Poly-

dphonia especially), or on piles or other larger algae. One will find
oneself compelled to study the habitat preferences of the different genera
rather carefully and to collect material frequently over a long period in
order to secure all the stages in reproduction. If not sterile, one too
frequently finds only tetrasporangia in most of the material collected.
Some species have been found to complete development and disappear
within 40 to 60 days.
A word of caution concerning Polysiphonia and related genera is in
order: the plants rapidly begin to putrefy after being removed from th(‘
water and evolve a characteristic odor resembling that of hydrogen
sulphide; consequently they should be placed in a 6% formalin solution
in sea water while being collected and later transferred to some other
fixing fluid.
All the polysiphonous forms ‘may be treated alike. This statement
genem: Polysiphonia Pterosiphonia^ Heierosiphoniu
includes the following ^ ,

Herposiphonia, and Lophosiphoniay most of which are Pacific Coast

genera. For whole mounts 10% formalin in sea water is satisfactory; for
embedding and sectioning, the regular chrom-acetic or the following fluid
may be used:
1% chromic acid in sea water 25 cc.

1 % glacial acetic acid 10 cc.

Sea water (filtered) 66 cc.

Fix 5 to 40 minutes, depending upon the ^Houghness^^ of the species, and

wash out thoroughly with sea water. Cut longitudinally at 5 to 10/li and
stain with iron hematoxylin or a triple combination. If the species is

one that shows a tendency to shoot to pieces when placed in a chrom-

acetic fluid, the following alcoholic formula will overcome the trouble:

70% ethyl alcohol 100 cc.

Formalin 6 cc.

Wash with 60% ethyl alcohol, and avoid going into water.
With material intended for whole mounts it is necessary to get rid of
the natural pigments before a satisfactory stain can be obtained. The
pigments inhibit staining. The process of extracting them is simple:
wash out the formalin killing fluid, place the material in 1 chromic acid %
in sea water, watch the process of color extraction (which should take
place rapidly if the material is exposed to sunlight), and stop as soon as
completed by washing with sea water. Harris^ hematoxylin gives a
superb differentiation to material intended for whole mounts counterstain ;

with erythrosin. Dehydration is preferably with hygrobutol.

The nonpolysiphonous forms, which for the most part possess thalli
top thick for whole mounts, present no diflBiculties in the way of fixation
 RHODOPHYTA 309

and staining. Portions of the older thalli of the polysiphonous forms may
also be sectioned for anatomical details.
Rhodomela larix is very common on rocks in the middle littoral zone
on the Pacific Coast, and there are three species along the Atlantic.
Embed portions of the thallus, and make both transverse and longitudinal
sections.
Ricardia saccata is an unusually good type in which to demonstrate
the origin of the procarps and the development of the cystocarps (Fig.
50). The plants form brownish grape-like clusters on Rhodomela larix.
Hmall specimens may be fixed entire, but portions should be cut out of the
older inflated sacs. Microtonu* transversely at 6 to

Fig. 50 .
—
Ricardia saccaia: cross section of portion of saccate thallus with cystocarps
containing, at loft, a young carpogoniuiu and, at right, carpospores. Fixed with chrom-
acetic in sea water; stained with iron hematoxylin and erythrosin.

Laurencia includes two or three species on the Pacific Coast and

stn^eral in Florida. L. virgata is an unusually good form with which to
work, as it is easily embedded and all developmental stages can be fol-
lowed out (Figs. 51, 52, 53).

Rhodymeniales
There are far more tropical and subtropical representatives of the
order than there are species occurring in the United States, and there
also are more on the Pacific than on the Atlantic Coast. The order is
made up of plants somewhat diverse in habit, but all give little or no
difficulty from this standpoint, with the exception of Halosaccion.
Rhod3mieniaceae. —Halosaccion forms obovate^ hollow plants which
are difficult to section and stain and are not worth the trouble that they
entail.
310 SPECIAL METHODS FOR THE VARIOUS PHYLA

Rhodymenia has one species which extends from New Jersey into the
Arctic regions and is a deep-water perennial; there are several species on
the Pacific Coast. Chrysmenia occurs in Florida and California. Gen-
eral methods are applicable to both genera.

Fia. 61 .
—Laurencia apectahilia: median longitudinal section of a sperrnatangium with
spermatia. Fixed with 1 % chroiu-acetic in soa water for 20 minutes; stained with iron
hematoxylin and erythrosin,

Champiaceae. —Considerable work has been done upon Gastroclonium

coulteri (Lomentaria which occurs abundantly on the Pacific Coast
ovalis),
and is easily manipulated.It is perennial and can be collected at any
time, but the plants often show the effects of their inability to withstand
the buffeting of the waves. It is heterothallic, but the spermatangial
plants are very difficult to find. Fix the terminal 5 mm. of the short
lateral branches and microtome longitudinally at 10^. These will show
the group of approximately 15 apical cells and probably carpogonial
 «

RHODOPHYTA 311

filaments or gonimoblasts. Somewhat older portions of the branche^s

may be selected and sectioned transversely for the carpospores. Tetra-
sporangial plants are less abundant than the cystocarpic they may show ;

'".I
^ '

'V ^ ^ ^ ^ V
V ' '

f»
,

‘ *
'"I" •*

Fkj. 52 —
Lanrencia virgala: loniscitudinal section of apex of frond with very young
.

(!arpospo!ophyte. Fixed in chrom-ncotic in sea water; stained with iron hematoxylin and
fast green.

1^2
l--X» >r:

Fig. 5.3 .
—Laurencia virgaia: longitudinal section of apex of frond with developing carpo-
sporophyte. Fixation and staining as in Fig. 52.

either tetrasporangia with four tetraspores or parasporangia with 15 to 20

paraspores.
Two species of Lomentaria occur side and may be
on the Atlantic
treated like Oastroclonium. One Champia also occurs on the
species of
East coast and is so small that whole mounts may be prepared.
 CHAPTER XXV
MYXOTHALLOPHYTA
MYXOMYCETES
The Myxothallophyta are the simplest of the plants commonly known
as ^Vfungi^^(MacBride and Martin 1934). They are characterized by an
assimilative phase which consists of a naked, multinucleate, and mobile
mass of protoplasm known as the ^^plasmodium^’; and a reproductive'
phase which in most cases consists of a membranous spore case. The
latter frequently contains, in addition to the spores, a capillitium of
netted or free threads and sometimes bears, inside or on the outside,
calcareous deposits.
Occurrence. —Myxomycetes occur in all except the coldest regions,
thriving best in damp habitats. Most of them
are very small and the
free-living forms arc exceedingly fragile structures. Some of the latter
can be found in warm, moist locations at almost any time of year. The
amoeboid and plasmodial stages occur in the upper layers of soil and in
decaying surface vegetation. The beautifully colored saprophytic forms
are easily collected. Stemonitu, the most widespread and readily recog-
nized form, grows on rotting wood, and its sporophore is dark brown in
color. Other forms are found on beams in damp cellars and on other
fungi, such as agarics, boletes, and Polyporus.
Collection and Preservation. —
These delicate organisms must obvi-
ously be handled with great care.
If the organisms are not to be killed and fixed in the field, they may
be placed in boxes for transportation to the laboratory. An ordinary
pasteboard box with a piece of cork linoleum covering the bottom serves
satisfactorily. The pieces of wood or other substrate carrying the
organisms may be pinned to the cork to hold them in place. Damage to
the organisms causes either cessation of development or abnormal growth
of the fruiting bodies, and the greatest care, particularly, must be taken
not to let them become dried. It is usually impossible to bring immature
fruit to normal maturity in the laboratory; if the myxomycete is in the
immature fruiting condition, leave it where it is, watch carefully until it

matures, then collect, and place immediately in the killing fluid. For
herbarium purposes only mature fruiting material should be collected.
Most botanists simply let the organisms dry on the substrate and later
glue or sew them to the bottom of small boxes, one species to a container.
The boxes in which slides come are ideal for the purpose. ]Entire colonies
m
 M YXOTHALLOPH YTA 313

may be preserved in a mixture of 5 cc. formalin and 10 cc. glycerin in 100

water, but there is always the danger that colors other than
cc. distilled

brown or black will disappear in time.

Fixation. —A weak chrom-acetic fluid is good. About 1 g. chromic
acid and 3 cc. glacial acetic acid to 100 cc. water have also proved to be
satisfactory. Allen's modification of Bouin's fluid has been strongly
recommended (Howard 1931a, b). Formalin-aceto-alcohol has worked
very well, but it ^s probably not to be recommended for the finest details.
For dehydrating, avoid xylol and similar fluids which tend to harden the
tissues so excessively that they crack during microtoming. Infiltration
is not difficult, but avoid leaving overlong in the paraffin oven.

Free-living^ forms growing on rotten wood {e.g., Trichia) should not

be scraped off the wood, as damage inevitably results. Cut out suitable
portions of the wood bearing the plasmodia and sporangia, and work
up as if they were one. Rotten wood presents no difficulties whatever in
embedding and microtoming.
Sections need not necessarily be very thin, save for cytological details.
F"or general morphology, IOm is not too thick; half that thickness is thin
(jiiough formost purposes.
—
Simple Whole Mounts. Put fresh fruiting material on a slide, wet
with alcohol, and then, if necessary, with 2 to 3% potassium hydroxide
to restore plumpness. The hydroxide may be mixed with a little glycerin
if the material is required for prolonged examination. Permanent prep-
arations may be made by placing the material on a circular coverslip in a
drop of Amann's medium:
Phenol 20 g.
Lactic acid 20 g.
Glycerin 40 cc.
Distilled water 20 cc.

Cover with another covershp of slightly smaller diameter, set aside until
the medium has hardened sufficiently, and then the whole may be inverted
over a drop of balsam on a slide.
Whole mounts of the forms which are naturally dark colored are
readily made. Kill in 95% alcohol for a few hours, and wash out with a
change of 95% alcohol. Transfer to a deep watch glass and give two or
three changes of hygrobutol, then add balsam diluted with hygrobutol.
Evaporate to a mountable consistency. To show up the capillitium,
however, it is necessary to get rid of most of the spores. Place the
myxomycete in a warm place for hour, then, by tapping smartly
against the substrate, displace the spores, and when enough have been
removed, proceed to the killing.
Sections. —
All forms are very easily embedded and sectioned. Do
not leave the material in the oven too long since heat lends to harden it.
314 SPECIAL METHODS FOR THE VARIOUS PHYLA

Stainix^. —Most forms which are naturally dark colored can be

mounted been described above. Stemon-
entire without staining, as has
itis is an example of the types which make beautiful whole mounts without
staining.
For the plasmodium iron hematoxylin is the best stain. Resting and
germinating spores, killed and fixed, acquire a sharp and precise stain with
crystal violet (Cotner 1930). Acid fuchsin is said to be an excellent stain
for the plasmodial strands, and rose bengal is recommended for soil forms.
Spores may be readily germinated in aqueous dilutions of the vital
dyes (Howard 1931). Use dilutions of about 1:20,000. Place a large
drop of the dye solution on a slide, sow the spores in the drop, then place
the whole in a humid chamber. The myxomycete will go through its
normal course of development, unless this is checked by contaminating
bacteria or protozoa, and abundant plasmodia may develop in about 72
hours after sowing the spores. To study the vacuoles, their size, distribu-
tion, and movement, use neutral red. The cytoplasm takes an intense
stain with methylene blue. Both the cytoplasm and mitochondria take
up Janus green B. These methods have been tried only with the
Myxogastres.
Cultivation. —
Spores of most forms will germinate as soon as they ar(?
thoroughly ripeiKHl, Vitality of spores probably does not extend over
three years. Germination taktis pla(;e in from 20 minutes to three or four
days, depending upon the species and the age of the spores. For the
details of spore germination, reference should be made to published papers
(Howard 1931).
Spores may be germinated in autoclaved tap water. Young i)las-
modia can be obtained in this fashion, and portions may be transfei'red lo
solid media for further developmental stages. An acidified oatmeal agar
is probably the best medium. Put 30 g. rolled oats or oatmeal, 15 g.
agar, and 1 liter water in a doubleboiler, and cook for 15 minutes. Trans-
fer the gruel to a flask, and autoclave for 15 minutes at 15 pounds pressure.
When sufficiently (;ool, pour into Petri dishes, flasks, or other suitable'

(containers.The medium should be definitely acid in reaction in order to

prevent excessive growth of bacteria. The tempei*ature at which thcc
plasmodium grows best is slightly above room temperature (about 20 to
26®C.).
Mannite agar forms a favorite substratum for the cultivation of
plasmodia. The formula is as follows:
Mannite 16 g.
Dibasic potassium phosphate 0 2 g.
.

Magnesium sulphate 0 2 g.
.

Sodium chloride 0 2 g.
.

Calcium sulphate P 1 g-
•

Agar 15 g.
Distilled water 1 liter
 M YXOTHALLOPHYTA 315

Autoclave, and pour into Petri dishes. This medium is unfavorable for
the growth of bacteria and most other fungi.
A great many other methods have also been proposed (e.g., Camp
1936).
The above methods yield an abundance of plasmodia but not in a
them to be of direct use for slide-making purposes. It will
eondition for
he necessary to grow the plasmodium directly upon the slides; this,
fortunately can be re.adily accomplished. Obtain a shallow wooden box
about 2 inches deep and a pane of glass largo enough to (*over it. Place

Fig. 54:.—C€ratiomyxa porioides: vertical longitudinal section of pillar with mitoses. Fixed
with alcoholic Bouin’s; stained with iron hematoxylin and orange G.

two or three layers on the bottom and

of blotting paper or drying felt
moisten thoroughly with sterile water. On
another piece of stiff absorb-
ent paper, cut so that it can be easily placed in and removed from the
box, arrange chemically clean slides side by side in rows. With a steril-
ized wide, thin, and soft-bristled paintbrush, cover the slides with a thin
film of the melted oatmeal agar medium described in the preceding para-
graph. Try to make the film as even as possible over the slides and
uniformly thick; as the plasmodium will grow rapidly, the film should
not be over 0.1 mm. thick. Place the paper with the slides in the box, and
cover until the agar solidifies. The dry paper will absorb sufficient water
from the lower dampened layers. Next, taking all possible precautions
to avoid bacterial and mold contaminations, remove tiny portions of
vigorously growing plasmodium from a culture by means of a platinum
 316 SPECIAL METHODS FOR THE VARIOUS PHYLA

loop, and place one Replace the cover, and

in the center of each slide.
set the giant moist chambej' in a warm In only a few
place to incubate.
hours the slides may be covered with plasmodia. When growth has
reached the desired stage, remove the slides, and plunge into jars of fixa-
tive (Allen^s Bouin has been recommended) for'several hours. Remove
from the fixative, wash, scrape off excess or unwanted growth, then
proceed to the staining. If the film of agar was thin and perfectly flat,
no difficulty in mounting in balsam should be experienced.

Fig. 66. —Ceratiomyxa porioidea: section of pillar with early spore formation. Fixed with
alcoholic Bonin’s; stained with iron hematoxylin.

The agar may be omitted and the plasmodium grown directly on the
slips, but growth maynot be quite so regular and abundant.
By exposing the plasmodia gradually to the air, sclerotization will take
place. After de.siccation has been completed, the sclerotia may be stored
for at least a year. Reactivation of the sclerotium and development of
new plasmodia may be brought about by wetting the material (but not
submerging in water). An abundance of oxygen appears to be necessary
for regrowth.

Exosporeae
The division contains a single genus. Ceratiomyxa, which in turn is
probably monospecific. It is actually an extremely common myxomycett^
 M YXOTHALLOPHYTA 317

but isnot often collected. It occurs on decaying wood. The plas-

modium is colorless when very young, but as it grows older, it turns white
or occasionally has a pink or bluish tinge. The plasmodium grows within
the wood; when ready to fruit, it breaks through at the surface and pro-
duces a stalk-line fru(*tification composed of membranous, columnar,
simple or branching sporophores on whose surface stalked spores are
borne.
Ceratiomyxa is an exceedinglj^ fragile plant : the slightest shaking, high
temperatures, or sudden drying will ruin the culture. It is better to fix

the material in the field rather than attempt to bring specimens to the
laboratory for the purpose. The spores, however, may be germinated
in the laboratory, using small culture vessels for the purpose, and the
entire culture may be killed by cautiously adding the killing fluid. Th(‘
most satisfactory reagent is a variation of Bouin\s fluid, made by using a
saturated solution of picric acid in 70% alcohol in place of the usual satu-

rated aqueous solution (Gilbert 1935). Embed material as usual and

microtome at 5/x. Staining should be with iron hematoxylin for all
stages (Figs. 54, 55).

Myxogastres
The forms whose beauty is so captivating belong among the Myxo-
gastres, which includes upward of 400 species. The general methods for
the Myxothallophyta apply.

Acrasieae

The Acrasieae resemble the other myxomycetes only superficiality.

They pass the whole of their vegetative period as naked amoeboids;
neither swarm spores nor a true plasmodium occur. In their fruiting
phase they merely become loosely aggregated together.
The Acrasieae occur in the soil, in decaying wood, and on dung or
other organic matter. Polysphondylium and Dictyostelium have fr(‘-
quently figured in morphogenesis investigations (Harper 1926, 1929;
Raper and Thom Growth appears to be most luxuriant on man-
1932).
nite agar to which shredded rat dung has been added just before
sterile
the agar solidifies. The medium should be adjusted to a pH of 6.0.
Permanent preparations may be made according to the general methods
cited above.
 CHAPTER XXVI
MYCOPHYTA (EUMYCETAE)
The Mycophyta include the vast group of saprophytic or parasitic
plants known as ^‘fungi/^ They differ from practically all other plants
in that they are unable to carry on photosynthesis.
The carbohydrate reserve is glycogen, never starch. In the cell wall
of primitive species cellulose predominates, but in the more advanced
species so-called ‘^chitin’’ is commonly found. Pectose and callose
also occur in fungal cell walls.
Cultivation. —Methods of cultivating the fungi are so numerous that
it is impossible in the space available to cite even the general methods.
A compilation of culture methods for all the fungi has apparently never
been made. Most fungi grow well on all ordinary bacteriological media
(Levine and Schoenlein 1930), but flourish better if a sugar is added.
Decoctions of horse dung, potatoes, beans, prunes, etc., may be sr)lidified

with agar and used as culture media. Or 1% Difco peptone plus 5%

crude dextrose solidified with 1.5% agar is excellent for nearly all types.
Sabouraud^s medium is extensively employed for the culture of fungi:

Maltose 20 g.
Peptone 10 g.
Agar 16 g.
Glycerin 5 cc.
Distilled water '
1 liter

Dissolve the substances, adjust the pH to 5.5, filter, tube for slants or
tails, and sterilize at 15 pounds pressure for 15 minutes.
Still another medium is the Dox and Thom modification of Czapek's
solution agar, which is the standard medium on which to grow Aspergillus
(Thom and Church 1926) in controlled cultures; it is also suitable for fungi
in general, Actinomycetes, and Fenidllium^ but not for Mucor:

Sucrose 30 0 .
g.
Sodium nitrate 2 0 .
g.
Dibasic potassium phosphate 1 .
0 g.
Magnesium sulphate 0 5 . g.
Ferric chloride 0 6 . g.
Ferrous sulphate 0 01.
g.
Distilled water 1 liter
Agar 16 g.
318
 MYCOPHYTA {EUMYCETAE) 319

One difficulty in cultivating molds is to prevent or keep down the

growth of bacteria. Most of the proposed methods are unnecessarily
complicated, requiring special apparatus, difficult techniques, or time-
consuming procedures. A simple mechanical method (Raper 1937) is
one of the easiest. Fuse three small glass beads not over 0.5 mm. deep
to one end of a Van Tieghem ring (merely a piece of glass tubing about
1.5 cm. in diameter and 6 to 7 mm. in length), and place the ring in a
Petri dish with the beaded end on the bottom. Pour sufficient nutrient
agar into the Petri dish until about three-fourths of the depth of the ring
is covered. After the agar has solidified, place a bit of the inoculum on
the agar within the ring. The mycelium will grow down within the ring
and out into the surrounding agar between the beads, thence to the
surface outside the exposed top of the ring. The contaminating bacteria
will not be carried along by the mycelium, consequently small portions
of the latter may be removed and transferred to fresh media and bacteria-
free cultures obtained.
It has been recommended that citric acid be added: the addition of
about 5% of the acid should be sufficient. Tartaric acid also retards
the growth of bacteria. %
Make a solution containing 5 dextrose and 5 %
tartaric acid. After autoclaving, this mixture may be kept for some time.
When a culture medium is needed, melt a deep tube (about 10 cc.) of
ordinary nutrient agar (preferably containing meat extract and peptone),
and add to it 1 cc. of the dextrose-acid solution. This gives a final
concentration of about 5% dextrose and 0.5% tartaric acid, with a pH
of about 3.8.
Whenfungi are grown on an agar medium and both fungus and
substrate are to be fixed and sectioned, the addition of a little lampblack
to the agar just before sterilizing has been recommended, as making it
easier to locate the sections of the fungus. The lampblack has not been
found to have any effect upon the development of any fungus grown on
media to which it has been added.
Further cultural directions will be outlined for many of the species
described in detail below.
Fixation. — All the fleshy and parasitic fungi are excellently fixed with
formalin-propiono-alcohol. A weak or medium chrom-acetic or a weak
chrom-osmo-acetic also serve well. About 1% saponin or 0.2% ethyl or
methyl acetate is advantageously added to all aqueous fluids in order to
lessen the surface tension. Gelatinous forms are apparently better
fixed in aqueous than in alcoholic fluids, since the latter may sometimes
cause such a contraction of the gelatinous substance that the hyphae are
distorted. Care should be taken not to overdo the fixation.
For mitochondria in fungi, any fijqng fluid giving a basic fixation
image may employed.
320 SPECIAL METHODS FOR THE VARIOUS PHYLA

Many plant pathologists claim that it is impossible to obtain adequate

preservation of both fungus and host in the case of parasitic species, and
there appears to be considerable ground for their complaint. The trouble
apparently has been due principally to the older dehydration methods.
—
General Staining. Most fungi are easily stained with all the various
hematoxylin schedules. Iron hematoxylin is particularly favored as a
cytological stain. The coal-tar dyes as a rule stain well but are fre-
quently difficult to differentiate satisfactorily, particularly in the spores.
Differential Staining of Fungus and Host. — Quadruple staining com-
binations are the most satisfactory on parasitic species since the mycelium
iswell brought out in the host tissues. Many methods have, in fact, been
proposed for distinguishing mycelium in woody tissues, but all of them
have the disadvantage of requiring much experience to obtain a really
first-class differentiation (see Stoughton 1930, Vaughan 1914).
Advantage may be taken of the oxidizing or reducing powers of th(‘
mycelium silver nitrate in weak sohit ion stains mycelium brown or orang(‘
:

and the wood a light brown.

The following is a rapid method of detecting mycelium of wood-rotting
fungi :

1. Boil infected blocks (about 1-cm. cubes) for 30 minutes or longcu’,

and soak for several hours to a few days in equal parts of glycerin and
70% alcohol, then cut freehand sections in a sliding microtom(\
2. Flood sections with Bismarck brown (2% solution in 70% alcohol).
Time varies according to the material, but 1 or 2 minutes usually suffices.
Drain excess stain, and wash with distilled water.
3.

Flood sections with a saturated aqueous solution of methyl viohd-

4.
for 2 minutes (or use a diluted stain and increase the time).
5. Wash out excess stain with distilled water. Mount a specimen
section in water, and examine microscopically. If the brown is too faint,
begin the staining process again with step 2; if the violet is not strong
enough, return to the violet stain. Wash again thoroughly before
proceeding to the following step.
6. Flood sections with 50% dioxan, pour off immediately, and replace

with 70% dioxan. After 10 seconds replace the 70% dioxan with pure
dioxan. Give two more changes of pure dioxan, then mount the sections
in balsam. The sections should never be placed directly in pure dioxan
as the latter will precipitate dye all over the sections. Hyphae are
stained deep violet; cell walls of the wood are yellow to brown. The
bordered pits and medullary rays of conifers are usually stained violet.
The following is an excellent method for paraffin sections of infected
tissues (Margolena 19326) bring slides down to water, and stain for 1 hour
:

in a solution of 0.1 g. thionin and 100 cc. of a 3% aqueous solution of

phenol. Rinse in water, counterstain in 0,5 %
light green in 96% alcohol
 MYCOPHYTA {EUMYCETAE) 321

until the sections appear green; wash with water, which will remove all

the green color except from the xylem, pass through 95% alcohol and
absolute alcohol, and again counterstain in a mixture of orange and G
erythrosin (1 part saturated orange G in absolute alcohol to 2 parts
saturated erythrosin in (*love oil). Clear in xylol, and mount in balsam.
(Mtin and spore walls are yellow, cellulose yellowish-pink, cytoplasm
pink, lignified elements green, middle lamellae reddish, hyphae and spores
purple.
Another excellent method for sections (Ikata 1932) consists in staining
them 5 to 10 minutes in 1% methyl violet in 50% ethyl alcohol, washing
briefly in 50% alcohol, and then staining for 30 to 60 minutes in 1 eosin %
in 50% alcohol. Differentiate in a mixture of 2 parts turpentine, 1 part
cedar oil, and 2 parts phenol crystals. Next wash in xylol, then mount.
The cell walls of the host are stained violet, the chloroplasts and granules
red, and the hyphae an intense red.

PHYCOMYCETAE
Members of the Mycophyta in which the asexual spores are produced
in indefinite numbers within a s})orangium are irududed in the Phyco-
inycetae. No single distinctive type of spore is produced. In most
species th(‘ zygote resulting from gametic union produces a thick wall
and a resting period precedes germination. The mycelium is generally
nonseptate but may occasionally be transversely septate. Some genera
do not produce a mycelium. There is, in any event, no production of a
macroscopic plant body.
All the Phycomycetae are either saprophytic or parasitic and many
genera are aquatic in habitat.

Plasmodiophorales
The two more prominent genera are Plasmodiophora^ in which tlu^

spores at maturity lie free in the host cell, and Spongosporaj in which the
spores are united in a solid sponge-like ball at maturity.
The single species of Spongospora^ S. suhterranea, is the cause of
powdery scab of potatoes. Accounts of the life history are contradictory.
Portions of infected tubers may be fixed in a medium chrom-acetic fluid.
For the myxamoeba stages thin sections of very recently affected tubers
are needed. For nuclear details stain in iron hematoxylin and fast green;
for general morphology any triple combination may be used.
Plasmodiophora hrassicae occurs in the roots of Brassica oleracea and
related species, causing readily apparent malformations (Fig. 56). For
the myxamoeba, roots of seedlings should be used if there is reason to
suspect that they are infected. Roots that have begun to swell will show
the later stages; spores will be found in badly swollen roots. Formalin-
 —

322 SPECIAL METHODS FOR THE VARIOUS PHYLA

aceto-alcohol has given excellent fixation of all stages when followed by

tertiary butyl alcohol dehydration. N
avashin’s fluid or a medium chrom-
osmo-acetic fluid might also be tried. The stains recommended for

Spongospora may be applied.

Fig. 66. Plaamodiophora brassicae: cross section of young root of Brassica oleracea
with developing plasmodium. Fixed with formalin-aGeto-alcohol; stained by a triple
combination.

Chytridiales

Members of this order, commonly called ^^chytrids,^^ are regarded as

the lowest of the fungi by some authors (Fitzpatrick 1930).
The plant body is unicellular, or with only a weakly developed
mycelium. Most of the species are parasitic. In some species the entire
cell becomes transformed into a reproductive body; in other species, the

cell becomes differentiated into a globose fertile portion and a rhizoidal

vegetative portion. A simple and quick method of obtaining chytrids

is to place fresh or dry pine pollen in pond water. The pollen presumably
carries zoospores. The grains may be removed after 2 or 3 days and
examined.
 MYCOPHYTA (EUMYCETAE) 323

Rhizidiaceae. —The unicellular parasitic plant body is differentiated

into a walled fertile portion, which lies and a naked
external to the host,
branched rhizoidal portion. The species, of which there are both marine
and fresh-water types, grow upon other aquatic fungi, on algae, or on
pollen grains. Whole mounts are probably the only kind of permanent
preparations that can be made; most of the published observations appear
to have been made from living material.
—
Olpidiaceae.^ The entire unicellular plant body, which lies completely
within the host cell, is fertile. Treatment is predicated by the nature of

Fig. 57 .
—
Synchytrium amsinckiae: cross section of Amsinckia leaf with three uni-
nucleate summer spores. Fixed with forrnalin-aceto-alcohol; stained with iron hematoxylin
and fast green.

the host; whole mounts should be made when possible. Iron hematoxylin
is the preferred stain for nuclear details.
Synch3rtriaceae. — Most of the species are parasitic in the epidermal
cells of Angiosperms. The whole of the plant body lies within the epi-
dermal cell, and all of it goes into the formation of an aplanospore which
in turn is transformed into zoospores.
Synchytrium is the best known of the Ch 3'^tridiales. Its range is
cosmopolitan. Navashin^s fluid, preceded or not by a 10-minute immer-
sion in Carnoy’s fluid, appears to give very good fixation; formalin-aceto-
alcohol causes some plasmolysis. Cut out portions of infected leaves,
fix, embed, and section transversely at 10/x. Iron hematoxylin gives an
excellent nuclear stain but- care should be taken not to leave the cyto-
plasm, which is somewhat dense, overstained (Fig. 57).
 324 SPECIAL METHODS FOft THE VARIOUS PHYLA

Cladoch3rtriaceae. —
In Physoderma are placed the species which cause
merely a discoloration or slight thickening of the infected parts of the
host, while those which produce galls or cause pronounced deformations
are placed in Urophlyctis. In both genera, sections of portions of the
host bearing the parasite are required. In selec^ting material of f/ro-
phlyctisj choose the youngest possible infections, since spore formation
begins early in the development of the malformations.
—
Woroninaceae. The family is composed of unicellular parasites,
each of which infects a single cell of the host. The entire body becomes
fertile during reproduction. The species infest primarily Saprolegnia,
Achyla, Pythium^ and similar aquatic fungi, and a species of Woronina
inhabits filaments of Vaucheria. Whole mounts of the host, if carefully
stained with iron hematoxylin and critically examined under high-power
lenses, should reveal the parasite if it is present.

Blastocladiales

The plant body is a true mycelium. Anisogamous sexual reprodiu*-

tionis known in one of the two genera comprising the order.

Allomyces has received considerable attention and is an easily stiulied,

but not so easily collected, form (Hatch 1935). To induce productioi)
of sexual mycelia, dry sterile agar cultures of rnycelia at either 40 to
45°C. for 1 to 2 hours, or at room temperature for a few weeks to several
months. Add distilled water to the dried culture to soften the agar, tb(Mi
cut out portions of the agar 1 cm. square, and inoculate four halves of
hemp seed cotyledons in aqueous culture. The sexual mycelia will hav(‘
developed sufficiently in three or four days. Gametogenesis may b(‘
observed in hanging-drop cultures. Or the sexual mycelia may be fixed
with a medium chrom-acetic or other fluid which on trial gives good fixa-
tion. For staining, the Feulgen reaction is preferable.

Monoblepharidales
Included in the Monoblepharidales are the only fungi in which active
sperms are retained. All the species in the two genera are aquatic
saprophytes, commonly found on twigs. There is little mention of the
^
order in the literature, although material is easy to obtain (Sparrow 1933)
and it would appear that whole mounts are easily prepared by the general
methods. Growth of mycelium and production of zoospores are abundant
on pea broth (Cotner 1930).

Ancylistales
The parasitic mycelium is small, sparingly branched, and grows within
a single hostcell. Fresh-water members of the Chlorophyta principally
 MYCOPHYTA (EUMYCETAE) 325

are attacked. Whole mounts of the host may be made; staining should
be in iron hematoxylin, very carefully differentiated.

Saprolegniales
The mycelium is ‘well developed. The reproductive organs are
terminal on the hyphae: asexual reproduction is by biflagellate zoospores;

sexual reproduction is oogamous. Gemmae are also present in some

species.
Saprolegniaceae . —
Saprolegnia can generally be obtained by dropping
dead flies, meal worms, ant eggs, small dead fish, etc., into pond
bees,
water. Within a few days to a week the host should be well covered
with mycelium. Egg masses of Tritvrus and frogs are often attacked by
Saprolegnia. A portion of the mycelium can be transferred to peptone
agar (1 %
peptone and 1 %
agar) the outer ends of the resulting mycelium
;

can be transferred to a fresh culture dish and a pure bacteria-free growth

obtained. Pea decoction (1 pea to each 20 cc. of water) or soybean agar
are excellent culture media. Saprolegnia and related genera are very
sensitive to the minutest traces of copper, consciquently glass-distilled
water should be used in making up cultui-e media.

Fig. b^.- -Pythium gracilc endophytic in Vauchvria UrrmtHs. Fixed with fornialin-aceio-
alcohol; stained with iron hematoxylin and dehydrated with hygrobutol.

Two methods of inducing the formation of oogonia and antheridia

have been described. One is to transfer highly nourished mycelium to a
0.1% solution of leucin; the .sex organs should appear in about 24 hours.
The other method to cut ordinary corn into small pieces, boil for 20
is

minutes, and place the cooled corn in Petri dishes. Nearly cover the
corn with pond water. The sex organs may appear in about 4 days
after inoculation.
There are nine other genera resembling Saprolegnia in a general way
(Coker 1923). All may be treated alike microtechnically. Many species
are well fixed in 10% aqueous formalin, particularly if mitochondrial
investigations are being made. Formalin-aceto-alcohol has also proved
326 SPECIAL METHODS FOR THE VARIOUS PHYLA

satisfactory. For nuclear details, iron hematoxylin is the only satis-

factory stain, but it must be very carefully differentiated, as the nuclei

may lose their stain before the cytoplasm has become cleared of stain.
Dehydrate and mount the material very much as if it were Spirogyra
(page 110), using the hygrobutol method.
—
Leptomitaceae. The mycelium, unlike that in the preceding family,
is constricted at intervals. Treat material as if it were Saprolegnia.
—
Pythiaceae. The species are aquatic parasites, some causing “damp-
ing-off^^ of seedlings. Vaucheria is also frequently invaded by Pythium,
whose presence is readily revealed in whole mounts of the host (Fig. 58).
Cultivation may be on corn- or pea-decoction agar (Dissmann 1927).
Fixation may be with 1 % chrom-acetic or with formalin-aceto-alcohol,
staining with iron hematoxylin, not too far destaincd, with a counterstain
of fast green to aid in showing up the mycelium.

Peronosporales
All species assigned to the Peronosporales are parasitic on land plants.
Asexual reproduction is by the production of conidiosporangia that either
form biflagellate zoospores or germinate directly. Sexual reproduction
is oogamous.

—
Peronosporaceae. Species with a branched sporangiophore, each
bearing a single conidiosporangium, are assigned to the Peronosporaceae.
Since the mycelium and sex organs are intercellular, sections of the
host are necessary for these structures. For developing sporangiophores
sections are also needed, but for the mature fructifications whole mounts
are more satisfactory. If the affected part of the host is the leaves,
mounts of portions of a leaf with the parasite attached are easily made
according to the general methods described above.
Many of the genera can be cultivated artificially. The following
nutrient solution produces a good growth of all species of Phyiophthora
(Leonian 1930):
Proteose peptone 2 g.
Dibasic potassium phosphate 0 6 . g.
Magnesium sulphate 0 5 . g.
Succinic acid 0 2 • g.
Dextrose 6 g.
Distilled water 1 liter

Or the following, used in Petri dishes at the rate of 15 cc. in each, is

also excellent:

Dry malt extract 6 g.

Dibasic potassium phosphate 0 6 . g.
Magnesium sulphate 0 6 . g.
Agar 20 g.
Distilled water 1 liter
 ^

MtCOPHYTA (EUMYCETAE) 327

Autoclave both at 10 pounds pressure for 15 minutes. The optimum

temperature for growth is 25°C.
General staining methods may be used.
—
Albuginaceae. The sporangiophores are unbranched and form a
palisade-like layer beneath the epidermis of the host. Conidiosporangia
are formed successively. There is but one genus, Albugo (Cystopus),
Only sections are of any value. Portions of the infected host may be
fixed in a medium chrom-acetic fluid or in formalin-aceto-alcohol. Sec-
tions should not be over 8/x in thickness. Stain with iron hematoxylin,
and counterstain with orange G, or if preferred, a quadruple combination
might be tried.

Mucorales
The black bread mold is one of the commonest fungi that one
encounters. The generally unseptate mycelium is well developed and
branches freely. Asexual reproduction is generally by means of aplano-
spores produced in sporangia; sexual reproduction is essentiall}^ isogam-
ous. There are both affirmation and denial that nuclear fusion oceans
during the formation of the zygote.
Members of the Mucorales occur so commonly and are so easy to
obtain in culture that they have acquired a prominent position in botany
courses. Methods of cultivating will be given in some detail in the
following discussion.
Rhizopus, Mucor, and most of the other genera are difficult or impos-
sible to immerse in, or to dampen
with, water on account of the great
Alcohol should not be used for lowering the
difference in surface tension.
tension, but a somewhat innocuous medium should be tried. A soap
solution or a 0.5% aqueous solution of gelatin may be poured over
the mass of mycelium until most of the air has disappeared in the form of
bubbles, washed out with water, and finally placed in the killing solution.
—
Mucoraceae. Some 10 genera are included in the family, of which
those most likely to come to the technician's attention are RhizopuSy
Phycomyces, Zygorhynchus, and Mucor (Fitzpatrick 1930. Hennrici 1930).
The presence of a columella in the sporangium distinguishes the Muco-
raceae from the other families in the order. Rhizopus and Mucor which
are often confused, may be readily differentiated: in Mucor the sporangio-
phores arise directly from the mycelium; in Rhizopus the sporangiophores
arise in a fascicle from a node on the stolon, opposite a tuft of rhizoids.
In Petri dish cultures Rhizopus mycelium grows over the underside of the
lids, forming sporangiophores; Mucor does not attach itself to the lids.

Some of the mucors are pathogenic. In addition to the spores produced

in sporangia, round bead-like blackish chlamydospores ma^ sometimes
be found on the mycelium. The chlamydospores should not be mistaken
 :

328 SPECIAL METHODS FOR THE VARIOUS PHYLA

for zygospores. When grown in poorly aerated liquid media, spherical

yeast-like oidia may be produced. These oidia may reproduce by
budding.
All Mucoraceae are very easily grown on culture media. Sterilized
bread, horse dung, prune agar, or almost any of the sacchariferous agars
form convenient cultural media, or the following synthetic medium may
be employed

Distilled water 1 liter

Sucrose 30 0
. g.
Aminonhiiii chloride 0 0
.
g.
Magnesium sulphate 0 6
. g.
Monobasic potassium phosphate 0.6 g.

However, it is difficult to remove material from solid media, in an

undamaged condition, suitable for slide-making purposes. A simple
method of securing abundant and easily removed material is available.
Prepare a broth or decoction, whether synthetic or from such articles as
potatoes, prunes, or malt extract. Add agar to about a two-thirds
portion of the fluid, sterilize, and pour into Petri dishes as usual, but do
not fill the containers more than half full. Also sterilize the remaining
one-third portion. As soon as the agar has solidified, pour a layer of the
liquid portion over the agar to a depth of not over 2 mm. Inoculate.
The dishes must, of course, be kept level. When the desired degree of
growth has been attained, the entire mass of fruiting mycelium may be
lifted off, washed in water, and placed in the killing fluid.
Most of the Mucoraceae grow satisfactorily at room temperature.
Species of RhizopuSy however, can be roughly divided into three groups,
depending upon the temperature at which they grow most luxuriantly:
20 to 22°C., 30°C., 35°C. The intermediate group contains the most
vigorous species. The culture vessels may be kept in the dark, but
Rhizopus seems to grow better in moderate light.
In some species, or in entire genera, the mycelium is homothallic, in
others heterothallic. In the latter species the two distinctive types of
mycelium are classified as “plus^' (the more vigorously growing strain)
and minus.” In the homothallic species zygospores are produced when
neighboring filaments from the same plant fuse. In the heterothallic
species zygospores are developed only when filaments of opposite strains
come into contact (Fig. 59). The zygosporic stage will hardly be encoun-
tered in ordinary cultures since it occurs very rarely in nature. Cultures
ofknown plus and minus strain^ are available in practically all universities
which have a good mycologist on their staffs, or they may be secured
from the American Type Culture Collection. To produce zygospores,
a prune decoction agar (or use moistened bread), and inoculate
 MYCOPHYTA (EUMYCETAE) '
62 \)

one end with the plus strain and the opposite end with the minus strain.
The spores will develop where the two mycelia come into contact.
In genera, such as Sporodmia, which possess only one type of myce-
lium instead of two, the zygosporic stage is more easily secured. Sporo-
dinia may be cultivated on bread soaked in prune juice, on slices of beet
or carrot roots, or on prune decoction agar. Zygospore development
should commence in about four days.
Forrnalin-aceto-alcohol fixes the Muc.oraceae splendidly, but the
amount of formalin may need to be increased to twice the usual volume.
Allow the material to remain in the killing fluid for at least two days.

Fig. 59
.
—
Rhizopus nigricans: whole mount showing formation of gametes and zygotes.
Fixed with forrnalin-aceto-alcohol; stained with safraiiin and fast green. {From a prepara-
tion by Dr, Geo. H. Conant.)

Harris' hematoxylin with a counterstain of fast green brings out nuclear

details of sporangial development beautifully. Iron hematoxylin may be
used, but the results are not always satisfactory because the dense cyto-
plasm too often remains overstained. After they have begun to
become darker, the zygospores are impossible to stain intensely. Safra-
ni n and fast green has been successfully employed on material containing

mostly dark zygospores. For whole mounts the hygrobutol method han
been the most serviceable one.
—
Pilobolaceae. The best-known members of this family are tho
species of Pilobolus inhabiting horse dung. Freshly dropped dung m%y
be placed under a bell jar and abundant mycelium should appear within
330 SPECIAL METHODS FOR THE VARIOUS PHYLA

a few days. The sporangia are numerous and large. Treat as described
for the Mucoraceae.

Entomophthorales
Some of the Entomophthorales are saprophytic; others are parasitic
on various insects, particularly flies. The thallus ranges in size, accord-
ing to the species, from the vegetatively nonhyphal type to a profusely
branched mycelium. Asexual reproduction is by means of conidio-
sporangia that germinate directly; sexual propagation is by the fusion of

aplanogametes of equal or unequal size.

Basidibolaceae. —Complectoria is an intercellular parasite in the
prothallia of ferns. Whole mounts of the prothallia should reveal the
parasite if it is must be rather precise. Sections of
present, but staining
the prothallia to show the zygospores are easily made if the methods for
the treatment of the prothallia as described under the Pteridophyta
are followed.
Entomophthoraceae. —Entomophthora (Empusa) is the largest genus
in the order, consisting entirely of insect parasites. The host is invaded
by the mycelium and the fungus usually develops to maturity in from
five to eight days after infection. Special cultural methods are required
for artificial cultivation (Sawyer 1939). There are two methods of
making permanent preparations. One is to kill and fix the entire insect,
puncturing the abdomen and thorax and using a suction pump if the
first

material does not sink into the killing fluid. Then wash, stain (prefer-
ably in a hematoxylin), dehydrate, and infiltrate with balsam. With a
needle and fine-pointed scissors, dissect out small portions of the insect
bearing the fungus, and mount in balsam. The second method is to kill
and fix the infected insect as before, then, after washing out the killing
fluid, it can be
to soften the chitinous exoskeleton of the insect so that
sectioned after embedding. Potassium hypochlorite (eau de Javelle)
may be employed as the softening agent, but sodium hypochlorite appears
to be preferable as being less violent in reaction. Dilute the commercial
solution of either reagent with 4 to 6 volumes of water, and immerse the
insects for about 24 hours, or until the chitin becomes sufficiently soft-
ened. Wash with water, and treat cautiously with a weak solution of
acetic acid if precipitates appear to have formed. Then proceed to
paraffin infiltration following tertiary butyl alcohol dehydration.

ASCOMYCETAE
The Ascomycetae are so called because sexual reproduction is by
means of spores formed within an ascus immediately subsequent to
meiosis. Other types pf spqrei^ are also produced, but always asexually,
 V MYCOPHYTA (EUMYCETAE) 331

Morphology. —
The Ascomycetae include both saprophytic and para-
sitic specfes. Some groups lack a mycelium, some have a slight mycelial
growth, a few others possess a loosely interwoven mass of hyphae, but
most often the mycelium is more or less developed into a pseudoparenchy-
matous structure of definite and often
characteristic form. The hyphae
are incompletely septate; there a central perforation through which the
is

protoplasm of one portion may stream into that of the adjoining portion.
Each septated portion is generally uninucleate, but in a few genera, such
as Pyronema, it may be multinucleate. The mycelium may be homo- or
heterothallic.
—
Sources of Material. The commonest and most easily found of the
Ascomycetae are the so-called ^^laboratoiy weeds,” Penicillium and
Aspergillus^ the cup fungi and the powdery mildews. The latter are
abundant' in many localities; Taphrina can be found on almost any
unsprayed peach tree shortly after the leaves appear; in short, no matter
where one may be located, some ascomycete or other can be found without
much difl[iculty. As a last resort, material can be purchased from the
botanical supply concerns.
Most Ascomycetae, as ordinarily found, are too old for other than
display or herbarium purposes. It is a difficult matter to find sexually
reproductive phases.
Cultivation. —Cultures of most of the Ascomycetae may be grown with
a minimum of bother since their requirements are either simple, or, if

specific, are readily provided. Specific directions are cited below for a
number of the more interesting genera.
Little work, however, has been done on the nature and development
of very many species.
Fixation. —In general a weak or medium chrom-acetic fluid or for-
malin-aceto-alcohol serves satisfactorily. So much surface
is usually

presented for direct contact with the killing fluid that perfect fixation is
the rule rather than the exception. The only precaution to observe is
to dehydrate with a well-graduated series of fluids.
Staining. —For general morphology a triple or quadruple combination
serves well. For nuclear whether on material to be mounted
details,
whole or in sections, nothing surpasses iron hematoxylin; a counterstain
is generally undesirable, but if one is wanted, a weak counterstaining with

erythrosin or fast green will assist in revealing the ramifications of the

mycelium of parasitic species in the host tissues.
—
Sexual Reproduction. In most of the Ascomycetae an antheridium
is developed so that it lies opposite an ascogonium (the female sex organ).

The ascogonium in some species consists of more than one cell; if not
broadly rounded at the apex, the distal end may be prolonged into a
trichogyne. In other Ascomycetae the sex organs are not paired. In
 ,

332 SPECIAL METHODS FOR THE VARIOUS PHYLA

these species the antheridium is generally replaced by a spermogonium,

which greatly resembles a pycnidium and in which spermatia are pro-

duced. In some species spermatia are not produced in a definite spermo-
gonium. The ascogonium commonly has a multicellular trichogyne.
Material must be very carefully collected if the sex organs are to be
studied, and must be at a far earlier stage of development than seems to
be generally realized.
After fertilization the resulting zygote develops either directly into
an ascus or indirectly into one to numerous asci. The first method is
characteristic of the Protoascomycetae the second of the Euascomycetae.
;

There are in addition irregular methods of ascus formation in some species.

The entire group of asci and their enveloping hyphae form an asco-
carp. There are three general types of ascocarps: the cleistocarp, which
always remains closed; the apothecium, open and more or less bowl-
shaped; and the perithecium, with a pore-like opening at the apex.
Among the most suitable genera in which to find the sex organs, pro-
vided sufficiently young material can be secured, are Phyllactinia
Sphaerothecaj Pyronema, and Venturia.

Protoascomycetae

In this group the zygote develops directly into an ascus. The asci
are borne singly on the mycelium and are never in an ascocarp or sui-
rounded by sterile tissue (Dodge 1935).

Endomycktales
Saccharomycetaceae. —The family includes all the various types of
true yeasts which form endospores. So little isknown concerning the
yeasts that it is extremely difficult to identify unknown forms (Heinrici
1930). Only a few species have such striking characters that they are
instantly recognized. Some genera exhibit so many intergrading types
and certain species are so variable in certain of their characteristics that
even specialists on the yeasts have trouble distinguishing between them.
Consequently, unless one merely seeking to find yeasts in the different
is

types of situations where they occur,it would be better to obtain a culture

of known identity from a reliable source. The American Type Culture

Collection lists innumerable genera and species.
To show multiplication by true budding, use Saccharomyces ellip-
Boideus (found naturally on grapes) or S. cerevisiae (compressed yeast).
These forms may be induced to produce spores, as described below, but
it will be found much simpler to employ a species in which the spores are

regularly formed. For this purpose, Schizosaccharomyces octosporus

(Fig. 60) or some species of Zygosaccharomyces serve excellently.
 ;

MYCOPHYTA (EUMYCETAE) 338

Yeasts may be grown in liquid media or on nutrient agar; the spore-

forming species should be cultivated on nutrient agar only. Malt
extract broth (Difco), plain or solidified with agar, is preferable, but one
may use grape juice diluted with an equal volume of water or a 5 to 10%
aqueous solution of molasses.
Spores can often be found on th(‘ surface of cakes of commercial
yeast kept in the refrigerator for a week, but, on the whole, spore prodiu*-

Fig. 60 . —
SchizosaccharomycGs octospoms: whole mount showing ascospore fonnatioii.
The staining is darker in the photomicrograph than on the slide. Pui'O culture suspension
dried to slip, stained with iron hematoxylin and fast green.

tion in most yeasts

is a matter of great uncertainty. It sometimes occurs
if the culture kept overnight in the refrigerator (or at 9 to 13°C.),
is

then transferred the next morning to an incubator kept at 25 to 30°C.

germination of the spores will begin after 2 to 3 hours, or at most after
7 hours. Another method is to make a weak carrot infusion agar and
to add a little calcium sulphate just before tubing; tube, sterilize, slant,
and inoculate (Heinrici 1930). Or use Gowodkowa^s medium (Maneval
1924):

Dextrose 0 26.
g.
Sodium chloride 0 50. g.
Beef extract 0 30. g.
Agar 1 .
5 g.
Distilled water 100 cc.
334 SPECIAL METHODS FOR THE VARIOUS PHYLA

Most workers simply dry the yeast cells to chemically clean slides
preparatory to staining. Place 1 drop of water on the slide, mix in a
small amount of culture by means of a platinum loop, and, after the water
has evaporated, pass the slide through a flame eight to ten times to fix
the cells. Better adhesion of the cells to the slide may be secured by first
smearing a thin film of Mayer^s adhesive, diluted 1:10 with water, over
the slide. Still another method is to smear with full strength Mayer’s,

smear the yeasts on the adhesive (with the finger or a scalpel), and fix
the moist film with Bouin’s fluid. Basic and acid fuchsins are the dyes
most commonly used for staining such smears, and there are innumerable
modifications. The slide may simply be placed in a 1 %
aqueous solution
of the dye for a minute; wash off excess stain, and let the preparation
dry. A slightly better method is to stain in a stronger solution of the
dye for 1 minute, wash with 5% aqueous tannic acid for 20 seconds, then
wash with water slightly acidulated with hydrochloric acid, dry, and
mount in balsam. A 1 %
aqueous solution of methylene blue employed
for 20 seconds is also good, as is a mixture of equal parts of 1 aqueous %
solutions of acid fuchsin and methyl green.
The most precise method is to fix a large volume of culture with a
weak chrom-acetic fluid, wash thoroughly, and stain with iron hematox-
ylin and fast green or orange G run up as if for whole mounts.
;
It will
be necessary to use a centrifuge between clianges of fluids. Fixing fluids
containing alcohol or formalin should be avoided, as they appear to cause
flocculation of the chromatin. Mitochondrial fixatives give excellent
preservation. The mitochondria consist of long, wavy bodies. Vital
staining has been recommended (Heinrici 1930); suspend some of the
cells in water under a coverslip, and put a drop of 1 % aqueous solution
of neutral red at one edge. Dead cells will stain a uniform red, but in
living cells only the volutin vacuoles and granules will take up the dye.
The former stain light pink, the latter deep red.
Tonilaceae. —Placed in the Torulaceae are the so-called false yeasts
”

which do not form spores. They reproduce by budding and never form
a mycelium. They are difficult to identify. The red forms are very
common and are found particularly upon dairy products and on grapes.
Technically, they may be treated exactly like the true yeasts.

Euascomycetae
There are some fifty times as many species of Euascomycetae as of
Protoascomycetae. The sori are developed on ascogenous hyphae which
arisefrom a zygote. The asci are produced, generally in large numbers, in
an ascocarp.
In the following discussion only those families containing species of
fairly common distribution in the United States are included.
 MYCOPHYTA (EUMYCETAE) 335

Aspergillales

The asci are irregularly distributed in a closed ascocarp. A number

of saprophytic species of economic importance are included in the order,
—
Gymnoascaceae. The peridium is made up of loose floccose hyphae.
Species occur on earth, manure, feathers, cadavers, etc. Whol^ mounts
of small fructifications can be prepared, but sections should be made of
the ascocarps.
Aspergillaceae. —
The family is by far the most significant one in the
order. The ascocarps are small, sessile, and remain closed. Of the 13
genera, only the following are likely to come to the attention of the tech-
nician : Thielavia^ PenicilUum^ and Aspergillus.
Thielavia hasicola appears in wet weather to cause a root rot of numer-
ous Angiosperms, being especially serious on Nicotiana tahacum. In the
earlier stages the two types of conidia are abundant and the ascocarps
appear after the host is killed. Sections of roots at various stages of
growth may be prepared according to general methods; a quadruple
stain is good.
Aspergillus is more widespread in the tropics than elsewhere but is

nevertheless easy to secure. appears on bread on which

It frequently
Rhizopus has been grown, at about the time the latter has run its course.
The Aspergillus will appear more quickly if the bread is moistened with
grape juice or 10% aqueous sucrose rather than with water. Place the
slice ofbread on slightly dampened filter paper in a suitable moist cham-
ber, after (*xposing the bread to the air for about 2 hours, and keep at a
temperature between 25 and 37®C. Microscopical examination of the
growth which appears in a few days will be necessary to determine its
identity. Aspergillus is usually green in color, hut Mucor and Penicillium
also have a greenish coloration. If this procedure is unsuccessful, cul-
tures are obtainable from the supply concerns. Mildewed herbarium
specimens are a likely source for A. herhariorurn or a related species, which
are excellent ones in which to induce perithecial development. Once
Aspergillus has been secured, cultures may
be transferred to a simple
medium prepared by steaming 10 g. of either wheat or rice bran with
10 cc. water in a 400-cc. Erlenmeyer flask, stoppered with a cotton plug.
The proportions of bran and water may be varied to suit requirements.
Czapek’s medium is the standard for growth studies on Aspergillus
(Thom and Church 1926). Wliole mounts of the conidial stage are easily
made by standard methods; stain with either iron or Harris’ hematoxylin
and fast green. Acid fuchsin also stains well. The conditions under
which perithecia formation may be induced are imperfectly understood,
but the availability of an abundance of easily assimilable carbohydrates
appears to be the principal prerequisite. Agar media seem to be prefer-
336 SPECIAL METHODS FOR THE VARIOUS PHYLA

able: one might solidify either a 40% solution of sucrose in prunp juice
(obtainable in larger grocery stores), or a 20% solution of dextrose or
glucose in grape juice, in suitable containers and sow spores thereon.
The culture should be kept at a temperature of at least 25°C. The
perithecia are carried above the surface of the substrate and are loosely
borne in networks of hyphae. Cut out 15-mm. squares of the agar
containing either sex organs or perithecia, kill in a medium chrom-acetic
fluid, and embed the entire mass in paraffin (the agar gives no trouble).

Fig. 61 .
—PenicHHum glaucum: transverse section of orange rind with the parasite
growing in Only sections can show the penetrating mycelium and the nuclei in the
situ.
aerial mycelium and spores. Fixed with formalin-propiono-alcohol stained with safranin
;

and fast green.

Microtome perpendicular to the surface of the agar at lO/x; stain with

iron hematoxylin and fast green, or substitute safranin for the
hematoxylin.
PenicilUum is more easily obtained than is Aspergillus. It appears
more often than does the latter genus in aging Rhizopus cultures on bread.
Perhaps the best source, however, is decaying oranges which have been
kept slightly damp. Portions of the orange rind may be cut out, fixed
with formalin-propiono-alcohol, embedded, sectioned at lO/i, and stained
with safranin and fast green or a triple combination (Fig. 61). Details
are more clearly revealed in this fashion than by whole mounts: the
penetrating mycelium, for example, may be followed out. Most species
grow well on sacchariferous media of the type recommended for Asper-
 MYCOFHYTA {EUMYCETAE) 337

gillus. Perithecia are not often produced, and the species that form them
belong mostly to one section of the genus (Thom 1930). Some of these
species are known to be heterothallic and consequently produce perithecia
only when two different mycelia intermingle.

Erysiphales (Perisporales)

The species are almost all parasitic, usually superficially so. This
indicates that sections of portions of the host bearing tlu^ parasite in
various stages of development should invariably be made.

Fig. ^2. - Erysiphe cichoracearnm: cross section of loaf of Amsinckia with vegetative
hyphae of parasite. Fixed with fornialiii-propiono-alcohol; stained with iron hematoxylin
and fast green.

Erysiphaceae. — "i'his is a widespread and common family, (*onsiituting

the so-called powdery mildews.^^ The most prominent representatives
arc noted below. The life history of only Erysiphe has been completely
investigated.
Sphaerotheca pannosa Rnd S, humuli are veiy common on leaves of
Rosa, generally occurring in the conidial stage; the perithecial stage is
rare. Material of both host and parasite becomes rather hard during
dehydration, but soaking under water remedies the difficulty. Staining
is good by quadruple methods, or safranin and fast green may be used.

Erysiphe cichoracearum is cosmopolitan, infecting chiefly Asteraceac

and Cucurbitaceae, and is excellent for the vegetative hyphae, haustoria,
and conidial development (Fig. 62). Fix portions of the host leaf in
either formalin-aceto-alcohol or Navashin^s fluid, section transversely
at 10 m, aiid stain with iron hematoxylin. The cytoplasm of the hyphae
retains the stain more deeply than does that of the host. A counterstain
is inadvisable. E. aggregata, which is not so widely prevalent as the
338 SPECIAL METHODS FOR THE VARIOUS PHYLA

other species, is the most favorable one for the development of the
ascocarps. They begin to appear as the growing season of the host is
ending. Stems are better than leaves; fix short portions in a medium
chrom-osmo-acetic fluid, microtome the host transversely at 10m, and
stain with iron hematoxylin and orange G for critical cytological details,
or with a triple combination for general structure. A series of develop-
mental stages are usually obtained in a few sections. E. graminis is
common on various Poaceae but is not very satisfactory for sections.
Microsphaera alni occurs on numerous hosts in the northern states
but is usually collected on Syringa leaves. Leaves bearing perithecia and
their peculiar appendages may be fixed with 8% aqueous formalin for
24 hours, this washed out, then stained with 1% erythrosin in equal
parts of water and methyl cellosolve slightly acidulated with acetic acid,
dehydrated with hygrobutol or dioxan, and infiltrated with balsam; the
perithecia may be cut off with a small scalpel and mounted independently,
or portions of the leaves bearing the perithecia may be cut out and
mounted. Great care should be taken at all stages not to handle the
material roughly lest the appendages be broken off. However, sufficient
pressure may be exerted on the perithecia just before the coverslip is
applied in order partially to force out some of the asci. The leaves may
be fixed in formalin-propiono-alcohol, sectioned at 11m, and stained with
any desired combination for detailed studies.
Phyllactinia coryleae occurs on a multitude of hosts. It may be
treated like Microsphaera,

Phacidiales

Of the three families in this large, mainly saprophytic order, only the
following occurs in the United States.
—
Phacidiaceae. Rhytisma is the best known of the 18 or so genera.
It produces conspicuous black areas on the leaves of Acer, Conidia are
produced before the leaves turn yellow, while the ascocarps begin develop-
ment after abscission of the leaves. Remove portions of the leaves
bearing infected areas, fix with formalin-propionoralcohol, soak under
water for several days after embedding, microtome in the transverse
plane of the leaf at about 10m, and stain with a triple combination.

Pezizales

Members of the order are distinguished by their typically dish- or

saucer- to cup-shaped apothecia lined with a layer of sori. The apothecia
in size may
be barely visible, up to bodies 10 cm. in diameter; in con-
sistency they vary from fleshy or gelatinous to leathery, horny, or
oartilaginous.
 MYCOPHYTA (EUMYCETAE) 339

Helotiaceae. — Sclerotinia fructigena commonly occurs on mummified

drupes and pomes; the apothecia arise from sclerotia produced either
within or upon the latter. Cut off the sclerotia, and fix with formalin-
propiono-alcohol as the sclerotia are rather tough bodies, some soaking
;

under water will be necessary. Stain with a triple combination.

Among the other genera, cut the apothecia away from the substrate,
and treat as described below for Peziza.
Molhsieicesie. -Pseudopeziza is the most important genus, occurring
as leaf parasites. P. medicaginis is common on Medicago saliva and

Fig. 63 . —Pyronema confluens: cross section of portion of the fruiting body with spores.
Fixed with 1 % chrom-acetic; stained with iron hematoxylin and fast green.

related species. The very small apothecium develops siibepidermally,

breaking through only at maturity, consequently sections of the leaves
are indicated. Fabraea rnaculata resembles Pseudopeziza^ occurring on
the leaves of pear and quince which have wintered naturally.
P3rronemaceae. -—P^/^onema confluens has long been of interest to
botanists. It is not an easy plant to collect, save by experienced mycolo-
gists. It is a saprophyte and generally is to be found on soils which have
been burned over, sometimes on partially burned, damp wood, or on soils
which have been sterilized by heat or steam. The older fructifications
are immediately recognizable by their bright rose-red color; the apothecia
become confluent in groups. The sex organs constitute the most impor-
340 SPECIAL METHODS FOR THE VARIOUS PHYLA

tant phase, but the gi*eat difficulty is to collect the plants when they are
young enough for this stage. Growth is quite rapid: when grown in
artificial culture (Seaver 1909), the antheridia and oogonia begin to
appear in four to days after the spores have been sown, and the
five
ascocarps are mature within the next five days. The plants are very
sensitive to carbon dioxide, and strong light is required for the optimum
growth. Spores may be sown on sterile earth; put the culture in the light
at about 25°C. fructifications will appear within a week.
;
The following
is a still better method. Fill the lower half of a Petri dish with tin*
follo\\ing nutrient medium:
Agar 2g.
Inulih, pure 2 g.
Monobasic potaHHiinii phosphaie 0.05 g.
Ammonium nitrate 0.05 g.
Magnesium sulphate 0.02 g.
Ferric phosphate 0.001 g.
Distilled water 95 cc.

Place the dish inside another dish of somewhat greater diameter but of th(‘
same height, and fill the space surrounding the inner Petri dish with tlu^
same medium minus the inulin. Inoculate the inulin agar with spores
or a small portion of mycelium of the Pyronema, and cover the whole.
Fruiting will occur in a few days on the inulin-free portion. Fix material
with a medium chrom-acetic fluid, microtome in the vertical longitudinal
plane at 8 m, and stain with iron hematoxylin (Fig. 63).
Pezizaceae. —Members of the family are of two sorts: hypogeous and
epigeous. Very little has been described for the technique treatment of
the hypogeous types. The epigeous genera, including Peziza, Aleuria,
and related genera, form their brightly colored fructifications on the
surface of the ground and are found mainly in damp woods in the —
summer and autumn in the Eastern states and in the winter and spring
on the Pacific Coast. To secure the youngest stages of ascocarp devel-
opment, it is often necessary to dig beneath the surface near where
mature apothecia occur and to search carefully. Fix with a medium
chrom-acetic for the younger stages or with formalin-aceto-alcohol for
the older ones, and microtome vertically at IOm- Young stages should
be stained with iron hematoxylin, other stages with the same stain or
with safranin and anilin blue.

Tuberales
The Tuberales include hypogeous plants consisting of a colorless
mycelium and ascocarps that are more or less completely enclosed
apothecia. Nearly all species described from the United States are from
California: they are found under trees, mostly Quercus and Sequoia^ and in
 ;

MYCOPHYTA (JEUMYCETAE) 341

shaded locations under humus. They occur at depths of from 8 to 30 cm.

consequently they must be dug up by means of a digging rake with long
prongs. As the external surface in many species is mucilaginous in
nature, they are covered with debris and may easily be overlooked because
of their resemblance to clods or small rocks. The apothecia are more or
less globular, or flattened slightly in some species, smooth, warted, or
convoluted, and they range from 3 mm. to 10 cm. in diameter. Slabs
should be cut from opposite sides of small specimens, and larger ones
should be reduced to convenient smaller portions for fixation. Formalin-
aceto-alcohol is excellent, or a medium chrom-osmo-acetic fluid may

be used. The bodies always become considerably hardened during

dehydration and require soaking under water before they can be sec-
tioned easily. The sex organs have never been found, but it is not

difficult to secure sections showing young ascogenous hyphae. Micro-

tome this stage at 8/x, the later stages at 10 to 12/x. Stain all stages
with iron hematoxylin; apply a coxinterstain of fast green on those for
the ascogenous hyphae, but omit on later stages. Other stain combina-
tions have been unsatisfactory.

Helvellales
Members of this saprophytic order are characterized by a subter-
ranean mycelium and an aerial, usually stalked, ascocarp. The latter
assumes many characteristic and sometimes brightly colored shapes.
Helvella, Morchella^ and Gyrornitra are the more prominent genera and
are of wide distribution, preferring cooler and damp woods. The sex
organs have never been described, but there is a possibility that they
have been replaced by a fusion of paired nuclei in certain vegetative cells.
Portions of the hymenium of the ascocarp may be cut out, fixed with a
chrom-acetic fluid or with formalin-propiono-alcohol, and embedded so
that sections may be cut perpendicular to the external surface. Micro-
tome at lljjL] stain with iron hematoxylin or safranin and anilin blue.

ExOA SCALES
The asci are arranged parallel to one another in an open, loosely
palisade-like layer.
There are a large number of species of Taphrina {Exoascus), all of
them being aggressive parasites affecting leaves, twigs, and fruits to
cause all sorts of malformations. T, deformans, which causes peach leaf
curl, is the species with which the technician is most likely to deal (Fig.
64). It first appears on the leaves of Prunus persica shortly after they
unfold in spring and produces yellowish and red discolored malforma-
tions. Remove portions so that each is made up about equally of infected
342 SPECIAL METHODS FOR THE VARIOUS PHYLA

and uninfected tissues, fix with formalin-propiono-alcohol, microtome

transversely at lO/u, and and fast green, a
stain with either safranin
quadruple combination, or iron hematoxylin, although the latter does
not always react sufficiently well. If material is unavailable locally, it
may be purchased from the supply concerns.

Fig. 64 .
—
Taphrina deformans: cross section of portion of hypertrophied peach leaf
with the parasite growing on the surface. P^ixed with forinalin-aceto-alcohol; stained with
safranin and fast green.

Hypocreales
The by soft and brightly colored (white,
species are characterized
brown) perithecial w^alLs, which do not becomt'
red, yellow, violet, or
hardened during dehydration. There are hundreds of species, including
many formerly included in Fusarium of the Fungi Imperfecti, whose
technique treatment is essentially similar.
Each genus usually produces at least one form of free-borne conidia,
and, in some, several different kinds of conidia are present. The ascifer-
ous stage is often more or less suppressed. Many species which produce
asci do so only during the second growing season. Most of the species
are saprophytes, some are plant parasites, and others attack insects.
Neurospora, which has been the subject of important genetical
researches, belongs in the Hypocreales. Cultures may be obtained from
investigators on this plant. Related genera, such as Melampsora, are
also under controlled cultivation. Whole mounts of such genera are
readily prepared according to general methods.
Species of Nectria are both saprophytic and parasitic, even within the
same species, as in N. dnnabarina. It is usually found in wounds of
hardwood trees. The mycelium does not penetrate sound tissues; con-
sequently pieces of only the infected part of the stem need be removed
for sectioning.
 .

MYCOPHYTA {EUMYCETAE) 343

Epichloe typhina is abundant on various grasses; the somewhat fleshy

stroma completely ensheathes the stem and becomes a bright orange in
color. Microtome short sections of the stem transversely after first
soaking under water for several days. Stain by a triple or quadruple
method.
Claviceps abounds wherever rye and, to a lesser extent, wheat, oats,
and other grasses are grown. There are a number of biological races.
The ovary of the host is invaded at the time of flowering, and this organ
is soon covered with conidiophores on the surface. The mycelium
becomes metamorphosed into a compact sclerotium which matures at the
time the grains become ripe. The following spring those sclerotia that
have not become too far desiccated germinate to produce capitate out-
growths. The stroma (capitate portion) of each outgrowth contains
numerous perithecia in which the sex organs and later the asci are pro-
duced. The sclerotia and outgrowths may both be fixed with a strong
chrom-acetic fluid or in formalin-aceto-alcohol. The sclerotia may be
sectioned at lOg either transversely or longitudinally; the outgrowths
should be microtomed in the vertical longitudinal plane at the same
thickness. Fully mature sclerotia, however, become as hard as stone and
probably cannot be sectioned; they should be fixed just before they
commence hardening. A triple combination will stain both clearly, but
iron hematoxylin is better for the sex organs and ascogenous hyphae.
Hypomyces, parasitic on the underside of the fruiting bodies of the
Agaricaceae, is noteworthy for the extensive development of gemmae.
Remove portions of the host bearing the parasite for fixation microtome;

vertically.
Cordyceps is pai*asitic on various insects and their larvae and is most
abundant in the tropics. The fructifications are usually large enough to
be cut off and embedded separately.

Sphaeriales
This order is a very large one, embracing about 11,000 species, mostly
plant parasites. Little cytologi cal work has been done on the majority
of species; consequently thereis a dearth of technique information. The
peridium of the perithecia is darkly colored and leathery, hard, or car-
bonaceous and always becomes brittle after embedding has been
completed.
The family Mycosphaerellaceae has been most extensively investi-
gated, with special emphasis on the damagingly parasitic Venturm
inaequalis. Infestation of young leaves or blossoms is caused by either
conidia or ascospores developed the previous season. The hyphae are
confined to the region between the cuticle and epidermis of the apple and
other ppmaceous plants, except those of the pear. Conidia are then
 344 SPECIAL METHODS FOR THE VARIOUS PHYLA

formed, and the acervulus becomes exposed. Portions of the infected

host may be fixed with formalin-propiono-alcohol, sectioned transversely
at 10/4, and stained with a triple combination. The sex organs are
produced in October, and the ascospores mature in February in warm
locations or as late as May in regions where spring comes late; the peri-
thecia appear on the undersides of leaves or on decaying fruits and are
most abundant when protected by humus or sod. Perithecial stages are
difficult to section; methods applicable to woody stems should be used.
Xylaria is a cosmopolitan genus, generally inhabiting dead wood and
producing cylindrical, clavate, or branched fructifications. Conidia are
first formed in the autumn, followed in most species by perithecia the

following spring. Fix portions of the fructifications, and section trans-

versely at 10/4. Stain preferably with iron hematoxylin.

Dothidiales
Little technique information is available on the more than 1200
species, of which only a few are plant parasites. They are distinguished
by their firm black sclerotium-like stromata. The perithecia occur in
vast numbers in the external layer of the stroma, embedded in the undif-
ferentiated mycelium.
Plowrightia morbosay which occurs on the branches of both* wild and
cultivated species of plums and cherries, is the species ihost commonly
studied. The parasite requires two years for growth to be sufficiently
advanced for the production of conidia in the spring. Shortly before
the conidia disappear, perithecial development commences, and asci are
produced during the following winter. The stroma are rigid and brittle,
and only methods similar to those intended for hard woody stems can
be used successfully. Pieces of infected stem should be reduced to con-
venient small portions, and these should be microtomed in the transverse
plane of the stem. Either a triple or a quadruple stain combination
may be employed.
PhyUachora graminia may be encountered. The stromata are jet
black in color. It is readily amenable to treatment, but some soaking
of embedded material under water will be necessary.

Laboulbeniales
The Laboulbeniales are minute ectoparasites which produce their
fructifications only on the chitinous integuments of living insects. They
do not give rise to fatal epidemics, as does Cordyceps; their own existence
ends with that of the host. Most of the $pecies are limited to definite
insect genera, and even to definite areas of the insect^s body.
Botanists in general have given very little study to the Laboulbeniales,
^ipstly because they are hard to find in the United States, and partly
 MYCOPHYTA (EUMYCETAE) 345

because of the great difficulty of fixing the material. The plants are
surrounded by a thin, homogeneous, impermeable membrane. Studies
for the most part have been made on living material or on whole mounts
of material removed from the host. Powerful fixing fluids and strongly
penetrating stains should be employed, followed by any gradual balsam-
infiltrating method.

BASIDIOMYCETAE
The Basidiomycetae get their name from the fact that the spores, or
basidiospores, are borne on a special one- or four-nucleate structure known
as a basidium. Many species produce other types of spores in addition to
basidiospores.
The group includes the mushrooms, puffballs, rusts, and smuts.
Morphology. —The mycelium, which is always multicellular and
freely branched, may either consist of a single mass of hyphae or develop
into a characteristic macroscopic body in which definite tissues are
differentiated.
Sex organs are not produced in the Basidiomycetae. There are,
however, two distinct cytological phases: the haplophase (gametophyte),
in which the cells are uninucleate, and the diplophase (sporophyte), in
which they are binucleate. The change from diplophase to haplophase
occurs during the development of the basidium. The two divisions of
the life cycle may be combined in the same mycelium or segregated on
separate mycelia. In the former type of mycelium the earliest cells are
uninucleate, those later developed are binucleate. The diplophase arises
when a conjugation tube is formed between two uninucleate cells and the

contents of one migrate into the other. The nuclei, however, do not
cell
fuse. The conjugation takes place at various stages of development and
between different types of uninucleate cells.
Diplophase mycelia may produce spores, particularly in the rusts,
which upon germination always develop into mycelia with binucleate
cells. Only in the production of basidiospores does meiosis occur. Not
much work, however, has been done on the nature and development of
the mycelium of the fleshy fungi (Hein 1930) and of other types.
—
Sources of Material. The mushrooms and fleshy fungi, as a rule, are
easily collected in woods and fields in most regions during the rainy
season. At other times recourse must be had to preserved or fixed
material, or a visit may be made to a commercial mushroom growing
establishment for PsalUota campestris. For most general purposes this
cultivated form is quite satisfactory. The Uredinales and Ustilaginalea
must be collected at the appropriate time of year for each stage in the
life cycle. The botanical supply concerns are fairly well stocked with
material prepared for slide-making purposes.
 ^

346 SPECIAL METHODS FOR THE VARIOUS PHYLA

—Methods growing
CultiTation. of various phases in the life cycle
of the more significant species are given in the following discussion.
Preservation. —To preserve the fleshy fungi, soak in a mixture of
2 parts formalin to 1 part liquid phenol, and, after superficial drying,
suspend the specimens over strong ammonia until they set solid without
drying (Ewart 1933). The final appearance is somewhat like that of
candied fruit; the fungi keep indefinitely, are not attacked by insects,
and may be handled without damage. The impregnating material (tan
be removed by soaking the specimen in water or alcohol.
If preservation in a purely liquid medium is desired, use 10% aqueous
formalin to which is added 10% sucrose to assist in preserving the colors.
All fleshy forms may be dried over gentle heat, then stored in boxes
with naphthalene flakes to prevent insect depredations. The parasitic
forms may be preserved by methods suitable for the preservation of the
host.

Eubasidii

The basidia develop directly from terminal vegetative cells of the

diplophasic mycelium and are arranged in continuous or discontinuous
hymenia borne on a macroscopic fruiting body.
Methods of arranging the orders differ considerably (Gaumann-
Dodge 1928, Killermann 1928, Stevens 1921, etc.), but the one most
recently proposed is being followed here (G. M. Smith 1938, based on
Killermann 1928).

Agaricales
The basidia are exposed, nonseptate, and club-shaped. A few genera
are parasitic on woody plants, the rest are saprophytic. The fructifica-
tions usually develop as centrally stipitate pilei. The substance of the
fructifications is usually fleshy or fibrous, rarely tough, leathery, or horny,
but they generally become more or less hardened during technical
treatment.
Exobasidiaceae. — Exobasidiurriy a widespread genus, is parasitic,
mainly on the leaves of Rhododendron and related genera.
Vacciniurrij
It is peculiar among the Agaricales in that it does not form a definite
fructification. The affected host areas are red and gall-like: these may
be cut out, fixed with formalin-propiono-alcohol, embedded, soaked
under water for about a week, and sectioned at 10/4 in the transverse
plane of the host. Stain critically in iron hematoxylin, without a counter-
stain, for cytological details. The cytoplasm of the parasite is so dense
that it is difficult to differentiate by ordinary staining procedures.
 MYCOPHYTA (EUMYCETAE) 347

H]rpochnaceae. —HypochnuSy representative of a group of mainly

European genera, is parasitic on a variety of cultivated plants.
H.
ochroleucus forms a brown growth along the twigs and petioles of
felty
the host. It is occasionally abundant enough to be remoyed for treat-
ment as whole mounts, but sections are required to show the development
of the basidia.
Thelephoraceae. —The fructification of this very large family of
saprophytes is usually leathery or membranous, sometimes woody. A
species of l^helephora causes a root rot on oak trees resembling that of
Armillaria, The sterile mycelium
of Corticium vagurn solani (commonly
known as Rhizoctonia) is on an amazing variety of hosts. Tlu^
parasitic
sterile mycelium turns yellowish with age and forms brownish to black
sclerotial structures. The hymenophore, which frequently entirely
surrounds the green stems of the host near the ground, is a dark network
of hyphae which change to grayish-white during sporulation. Practi(;ally
no definite technical information is available, but general methods as
determined by the nature of the material should be applicable.
Clavariaceae. —All but oneof the genera are saprophytes; about half
possess small hymenophores, and the remainder have large
simple
branched hymenophores. The fructification is fleshy or waxy in most
of the genera, but a few are cartilaginous, horny, or leathery and haiiy.
Clavaria, a very widespread genus growing in the humus of damp woods,
has numerous colorful species commonly used for laboratory study.
Many species harbor a symbiontic alga. The fleshy hymenophore may
be divided into suitable portions, fixed with a chrom-acetic fluid or with
formalin-propiono-aleohol, and sectioned either transversely (the bettei*
plane) or longitudinally. The hymenial layer is so compact that sections
should not be over 8/x thick. Stain with either iron hematoxylin or
safranin and fast green.
—
Hydnaceae. The spore-bearing body is very variable in texture; it
may be fleshy, cuticular, leathery, corky, felty, or woody. In any event,
the body invariably becomes brittle after technical treatment. Most
of the species grow on rotting wood, forming shelf-like or resupinate
sporophores. The hymenium of Hydnurriy ('ommon in woods, is beset
with subulate spines, which carry the basidiospores. A group of these
spines may be removed, fixed, sectioned transversely at 8^, and stained
with iron hematoxylin.
Pol3rporaceae. —The so-called ‘‘pore fungi, including the genera
Boletus and Polyporus, make up this family.
Boletus possesses a fleshy, capitate, centrally or later^ly stipitate
sporophore, with a hymenium on the undersurface composed of tubes
that are adnate to each other. To show basidiospore formation, remove
348 SPECIAL METHODS FOR THE VARIOUS PHYLA

rectangular portions of the hymenium long before it is fully mature,

embed, section transversely at 10/i, and stain with safranin and fast
green. The other genera most closely related to Boletus are more or less
fleshy (Fistulina) or leathery and have the tubes separate.
Polyporus is a genus of over 500 species. The sporophore is usually
annual and may be simple or compound, stipitate or shelf-like, with the
pores developing from the base toward the outer margin. Several
species, such as P. sulphureusy are the cause of heart rot in native and
cultivated trees. Apparently the only preparations that are made of
these fungi are sections across the tubes. The sporophores are rather
thick, fleshy when young, becoming leathery or corky as they mature.
Material may be treated as indicated above for BoletuSy but it is occasion-
ally difficult to secure material that shows active spore formulation.
The other genera may also be treated like Boletus.
Agaricaceae. —This is the largest family, whose 9,000 species have
been described as being of ^^fatiguing regularity.^^ A single common
and easily collected species therefore will be described in detail, viz.^
PsaUiota {Agaricus) campestris.
The fruiting body —the so-called mushroom — is a stipitate fleshy
sporophore. This mature stage is not quite so interesting as the initial
developmental phases. It is not possible to collect these stages in the
field under ordinary conditions; it will be necessary either to visit a com-
mercial mushroom-growing establishment or raise them on artificial
media. Spores remain viable for at least a year if kept quite cold. In
tap water, germination occurs in about 24 hours. It appears to be
advisable to germinate the spores in sterile tap water first and then to
transfer to suitable receptacles containing various media, such as sterilized
dung, dung extract agar, malt extract, agar, soil mixed with shredded
horse dung, or corn-meal agar. The young ^‘buttons'' will appear in
about 2 to 3 months. In case the medium shows signs of drying out, add
sterilized water cautiously by means of a pipette. Remove the young
buttons carefully, and place in the fixing fluid: it is advisable to cut slabs
from opposite sides to facilitate penetration, but this is a very delicate
operation since the material does not cut easily. If the material was
grown on agar, cut out small blocks of agar bearing the buttons. The
killing fluid should be a weak or medium chrom-acetic, and the time of
fixation should be short (one investigator states that the finest cytological
fixation was obtained from material that remained in the fixing fluid
for 5 minutes). Closed stages require several hours, open button stages
about an hour. A suction pump, flowing gently, should always be used.
The material has always become excessively hardened by xylol and chloro-
form dehydration methods; consequently these fluids should be avoided.
14 would be better to employ an essential oil, such as that of cedar,
 MYCOPHYTA (EUMYCETAE) 349

bergamot, or origanum. All changes should be most gradual because of

the fragile nature of the individual hyphae. Embed in a hard paraffin,
and section buttons longitudinally until the prelamellar chamber is fairly
well developed, after which transverse sections through this region may
also be micro tomed. Sections should never be over Sn thick; 6 m is the
optimum thickness. If fully mature sporophores are wanted, the spores
should have been sown in large Erlenmeyer flasks on a substrate of
broth or horse-dung decoction plus beech or spruce sawdust. The fruit-
ing bodies begin to develop when the substrate becomes exhausted with-
out completely drying out. Iron hematoxylin, both with and without
a counterstaining of fast green, is the most commonly used stain, but the
following combinations have also been employed: aqueous safranin
followed by crystal violet in clove oil; aqueous pararosanilin plus a trace
of phenol, followed by fast green; for photographic purposes, mordant
with 5% aqueous tannic acid, then stain with pararosanilin. A triple
combination has been suggested, but care must be taken not to overdo
the orange G.

Lycoperdales
The group commonly known as .the Gasteromycetes composes this
entirely saprophytic order. Many well-known fungi are included: puff-
balls, stinkhorns, bird^s-nest fungus, earth stars, false truffles, etc.
Several are characterized by foul odors. The basidia are permanently
surrounded by sterile tissue, or become exposed only after development
has been completed.
There are 11 families and over a thousand species (Coker and Couch
1928), which differ considerably in external characteristics, but all may
be treated alike technically, at least in the earlier developmental stages.
Technical methods are the same as described above for Psalliota.

Dacromycetales
The Dacromycetales are minute, inconspicuous, gelatinous sapro-
phytes found on decaying wood and leaves. The order is characterized
by the freely exposed, nonseptate, Y-shaped basidia. Growth is quite;
slow.
The Dacromyces are scarcely over 3 mm. in diamete
fruiting bodies of *

and are yellow to orange in color. Cut them from the substrate with a
scalpel, microtome in the vertical plane at 6m, mounting all section-;
serially in order, and stain with iron hematoxylin.

Tremellales
The order consists of a very few parasitic species and a number oF
fleshy or gelatinous saprophytes found on fallen branches and decaying
360 SPECIAL METHODS FOR THE VARIOUS PHYLA

wood. The basidia are fully exposed and septate in a nearly vertical
plane.
The gelatinous types extremely difficult to fix adequately.
are
Strictly fresh material should be collected in the field, and placed imme-
diately in the killing fluid. Large portions should be reduced to smaller
pieces before fixation. Either of the following fluids may be used
(Whclden 1934):
(1) Chromic acid 7 g.
Glacial acetic acid 7.6 cc.
Distilled water 1 liter

(2) Saturated picric acid in 70% ethyl alcohol 100 cc.

Saturated aqueous mercuric chloride 5 cc.
Glacial acetic acid 7 cc.

Microtome in the vertical plane; for stages up to fusion of the two

nuclei in the youngest basidia, cut at 4ju, thereafter at 8 to 10/x. Stain
with iron hematoxylin and counterstain very lightly with either orange
G or fast green.

AuKICULAJII A LE8
The Auriculariales are gelatinous saprophytes with irregular and
expanded or capitate fruiting bodies. They become horny when dry.
The species grow mainly on stumps or fallen trees, and are most abundant
in the tropics. Reduce the fructification to convenient portions, fix
with a weak chrom-acetic fluid, microtome vertically at 8 m, and stain
with iron hematoxylin. A counterstain is not desirable because of the
dense cytoplasm.

Hemibasidii
The life cycle alternates between a haplophase and a diplophase.
The basidia are produced by the germination of a special resting spore.
The life cycle is polymorphic. All species are parasitic.

Uredinales
The special one- to several-celled spore, the teleutospore, which pro-
duces the basidiospores, is formed by the metamorphosis of the terniinal
cells of subepidermal hyphae.
The teleutospores are binucleate: if no other cells of a binucleate type
are produced, the species is described as microcyclic; if one or more
additional types of binucleate spores are developed, the species is described
as macrocyclic. Species that are macrocyclic include, in addition to
teleutospores and basidiospores; (1) binucleate aecidiospores originating
upon a haplophasic mycelium and producing a diplophasic mycelium
upon germination; (2) binucleate uredinospores borne upon a diplophasic
 :

MYCOPHYTA (EUMYCETAE) 351

mycelium and producing a diplophasic mycelium upon germination; and

(3) uninucleate spermatia borne upon a haplophasic mycelium, which
may possibly produce a haplophasic mycelium upon germination but
generally assist in the initiation of a diplophasic one. Microcyclic
species usually produce only teleutospores and basidiospores but occa-
sionally develop spermatia. The entire vegetative mycelium is haplo-
phasic in some species, but in others certain portions may be haplophasic
and others diplophasic.
—
Melampsoraceae. Little technical information is available concern-
ing the majority of the species.
Cronartium ribicola forms blisters on stems of species of Finns
these constitute the uninucleate aecial stage. Pines which show infection
with this usually fatal parasite should be examined closely, and the
youngest possible branches which appear to have the sites of early growth
stages may be removed in short lengths and treated as described for
Finns stems (page 426). The writer has never seen preparations of the
aecial stage which were adequately fixed or had a really satisfactory stain
differentiation. Triple combinations have usually been used, but such
are apparently the worst combinations that might be selected. The
uredosori and teliosori are produced on the leaves of \vild and a few culti-
vated species of Ribes, During recent years a systematic destruction of
plants of Ribes has been under way in an effort to check the spread of this
destructive fungus; consequently material of the telial and uredinial
stages is becoming increasingly difficult to procure. If it is available, fix

with formalin-propiono-alcohol, microtome at IOjjl in the transverse

plane of the Ribes leaf, and stain with safranin and fast green.
One species of Fncciniastrum commonly occurs on Hydrangea and
another on Agrirnonia. Only the uredinial and telial stages are present.
Treat as for Cronartinm.
Biological specialization is unusually complicated in Melampsora,
The aecial and spermatial stages occur on a wide variety of gymnosperms
and Angiosperms; the uredinial and tefial stages occur in abundance on
Populus and Salix. Material may be dealt with as described for Cronar-
tium stages on Ribes leaf.

The aecial stage of Coleosporium solidaginis^ a macrocyclic rust, ociairs

On FinnSy the uredinial and telial stages on Aster, SolMago, and the culti-
vated aster,' CalUstephuSi It is a very common rust. The usual methods
are applicable.
Pucciniaceae* —^Teleutospores are either disposed singly, mostly
stalked/ not -connected together, occasionally one-celled; or united to
fohn an umbrella-like head on a compound stalk.
It is needless to say that Puccinia graminis is the most extensively
investigated and most widely studied species in the family, although
362 SPECIAL METHODS FOR THE VARIOUS PHYLA

there are numerous other species in the genus (Arthur 1934, Holway
1906-1924). P. graminU
most abundant, in all its stages, in the plains
is

states and the wheat-growing provinces of Canada. Persons located in

other regions will experience difficulty in collecting material and identi-
fying it accurately; in such cases, the safer course is to obtain material
from the botanical supply concerns, which have gone to considerable
trouble to stock really good material.
The haplophase occurs on the leaves of Berberis and Mahonia, and
the aecial and spermatial sori are frequently present in great abundance.

Fig. 66 . —Puccinia graminis: section of a telial sorus on wheat leaf. Fixed with formalin-
aoeto-alcohol; stained with a quadruple combination.

Fix portions of. the leaves with formalin-propiono-alcohol, soak the

embedded material under water for two or three days, and microtome in
the transverse plane of the leaf at lOyu. Stain with iron hematoxylin,
and counterstain with orange G; this is the only combination which
two nuclei in the aecidiospores, but no
satisfactorily differentiates the
basic stain is known which always gives perfectly clear ffifferentiation
of the entire spermogonium (pycnium). The pycnia are evidently
developed before the aecidia; at least they are more abundant on leaves
which show the younger stages of aecidia formation. The pycnia are
usually formed on the upper eide of the leaves^ the aecidia on the abaxial
 MYCOPHYTA (EUMYCETAE) 353

The diplophase consists of a number of biological races and occurs

parasitically on the leaves and stems of many grasses and cultivated
cereals such as Triticum^ Secale, Hordeum^ and Avena. The reddish-
brown uredosori appear in late spring, followed by the development of
teleutospores in late summer. The latter usually first appear in the
uredosori, then in teleutosori. Both uredospores and teleutospores are
binucleate, but the former are unicellular with four to five germ pores, and
the latter are twp-celled with a single germ pore to each cell. Portions of
the host containing diplophasic stages may be fixed with formalin-aceto-
(or propiono-) alcohol. The host tissues are hard to section; the use of
10% hydrofluoric acid for a day or two has been recommended, but it

does more damage to the fungus than it softens the host tissues. Soaking
the embedded material under water for a week or longer is the better
procedure. Sections should be lO^t in thickness. A quadruple stain is

recommended; a variety but all structures are

of colors is produced,
clearly differentiated —
the invading hyphae should be stained green to
indicate that the combination has been correctly applied (Fig. 65).
Uredospores germinate promptly when they come into contact with a
suitable host plant. The teleutospore must overwinter or be subjected
to prolonged freezing temperatures before it will germinate. Most
authorities state that it germinates within a few hours, but others claim
that months are required for basidiospores to be produced. The teleuto-
spores of some species other than P. graniinis should probably give better
results if one desires to prepare whole mounts of the basidiospores.

Germinate the spores in sterile water; kill at the optimum time with a
medium chrom-acetic, then stain with Harris^ hematoxylin and fast
green or with a carmin stain, dehydrate with hygrobutol or dioxan, and
infiltrate with balsam. Handle the material with extreme care to avoid
breaking off the basidiospores.
Other species of Puccinia may be treated exactly as described above
for P. graminis, but the nature of the life cycle of each species should
first be ascertained.

Kunkelia (Caecoma) nitens has frequently figured in research investi-

gations. It is one of the two orange rusts occurring on Rubus, Sections
of the so-called ^‘caecome^^ stage are easily prepared. The uninucleate
spores may be germinated on sterile tap water in Petri dishes the temper-
;

ature should be kept below 25°C. To show the basidiospore develop-

ment, attach the germinated spores to clean slides by means of Mayer^s
adhesive, fix in a weak chrom-acetic fluid, harden in 70% ethyl alcohol,

then stain by a triple combination, dehydrate, and mount in balsam.

Phragmidium commonly occurs on wild species of Rosa and contains
a great many species. Prepare sections as described for the diplophasic
 354 SPECIAL METHODS FOR THE VARIOUS PHYLA

stages of Puccinia. The very large genus Uromyces may also be treated
as for Puccinia.

USTILAGINALES

The special spore producing the basidium is a chlamydospore formed

by direct metamorphosis of an intercalary cell of a diplophasic mycelium.
The order contains the so-called smuts.
—
Ustilaginaceae* The basidium formed by the germination of the
chlamydospore becomes transversely divided into uninucleate cells, each
of which may produce an indefinite number of basidiospores.
There are about a dozen genera, but only Ustilago is sufficiently well
known. There are over 200 species in this genus.
U. avenae infects the leaves of Avena, then the mycelium grows
through the leaf and stem tissues until it reaches the ovaries, where it
produces sori. Sections of the infected portions of the plant may bo
prepared without difficulty. The spores produce a well-developed
promycelium in about 24 hours after being sowed in sterile tap water,
and this may be transformed into whole mounts by the hygrobutol
method. If. levis is not easily distinguished from U. avenae and occurs

on plants of the same host as the latter; the sori are formed in the spike-
lets. U. hordei occurs on Hordeum vulgare and produces the sori in the
spikelets. Prepare longitudinal sections of the young spikelets to show
the development of the spores. The latter germinate readily in water
and produce abundant epibasidia. The equivalent species on Triticum,
is U. tritici and occurs wherever wheat is raised. The chlamydospores
should be germinated in plain water; they do not produce epibasidia in
nutrient solutions.
The corn smut, U. zeae^ is widely prevalent. The sori, no matter
where produced on the plant (they are usually found in the ovules, which
become enormously hypertrophied), are generally prominent. If a
stand of Zea mays should show indications of the presence of IJ. zeae,
young ears may be opened and ovules showing early stages of develop-
ment removed and fixed individually. It is difficult to make out the
details of chlamydospore development, which takes place somewhat
late in the growth of the sori. Kill with formalin-aceto-alcohol, section
at 10/x in any plane, and stain with safranin and fast green. The chlamy-
dospores germinate poorly in water, hence weak nutrient solutions
should be used. In such media the conidia are freely produced by the
basidiospores.
Tilletiaceae. —^The basidium does not become transversely divided
into uninucleate cells; a definite number of basidiospores are produced
at the distal end of each basidium.
 MYCOPHYTA (EUMYCETAE) 355

Urocystis cepulae produces its sori on the leaves and occasionally in

the bulb of Allium cepa. General methods for sections are satisfactory.
The chlamydospore is completely surrounded by sterile cells; the latter
are tinted, while the viable spore is reddish-brown. It remains viable
in the soil for many years. Upon germination in water, the basidio-
spores arise on a short promycelium and then give rise either to conidia
or to a new infective mycelium.
In Entyloma the chlamydospores enter through the stomata, germinate
in the stomatal chamber, and produce the basidiospores on tufts of
promycelia which emerge through the stomatal opening. Sections of
the host should therefore be prepared.
Tilletia includes the stinking bunts, common on Triticum in the Pacific
Northwest. The bunts include two species, T. foetans and T. tritici^
which are rather similar in appearance. The host can be infected only
within the first six to eight days after the seeds are sown, but it is not
possible to determine definitely whether infection has occurred until the
wheat flowers appear. The mycelium can be found near the growing
point of the host stem apex at almost any stage; longitudinal sections of
this portion therefore can be prepared and stained with a quadruple
combination. The chlamydospores may be germinated in water to show
file development of the basidia and the primary and secondary conidia.

If desired, whole mounts may be made of these structures by standard

methods.

FUNGI IMPERFECTI
The Fungi Imperfect! constitute a wholly group which serves
artificial
as a more or less temporary repository which a perfect t stage
for species in
such as ascus, basidium, or zygote has not been discovered. There are
some fungi which never form spores of any sort: these are also included
in the present group.

Mycelia Sterilia
Sclerotia, rhizomorphs, and various other forms of mycelium occur.
Technique treatment consequently depends upon the form assumed by
the mycelium. The general methods for parasitic species are readily
applicable since most of the species are plant pathogens.

Sphaeropsidales
Members of this order are mostly leaf-spotting fungi, though some
grow on fruit and stems, causing rot, cankers, and blight.
The conidia are produced in pycnidia or in various modifications of
such a structure. The pycnidia are usually tough structures, occasion-
ally becoming black, hard, and very brittle- Others are waxy. The
356 SPECIAL METHODS FOR THE VARIOUS PHYLA

embedded, but microtoming is frequently exceed-

fructifications are easily
ingly difficult. material should be soaked under water
The embedded
before sectioning is attempted. Staining may also be difficult on dark-
colored forms; differential acidification should be resorted to. Quad-
ruple stain combinations are excellent.

Moniliales
Many of the species or forms formerly included in this order have been
transferred to the Ascomycetae or Basidiomycetae but the perfect stages
have not yet been found for even closely related forms; consequently the
same genus is to be found in two very different fungal groups. The
Aspergilli and Penicillium are examples. The very diverse order con-
tains a multitude of forms of which most are saprophytes.
—
Moniliaceae. The hyphae occur in more or less fragile, loose cottony
masses; they and the conidia are clear or bright colored.
Oidium and Monilia are frequently confused. The present tendency
is to use the name Oidium for those forms that produce free cells by a dis-

articulation of the mycelium itself and to refer to Monilia those species

that produce free cells by budding from the mycelium.
Oidium lactis is an omnipresent mold, especially troublesome in tiie
dairy industry. It probably causes the ripening of Limburger cheese.
In milk and artificial liquid-culture media it produces a white, firm felt-
like mass. When grown on solid media, the mass is firmly adherent.
In older cultures the aerial hyphae bear conidia. The conidia- or o’idia-
bearing mycelium may be worked up like Rhizopus or Mucor (page 329).
Oidium dermaiitidis causes the so-called “blastomycosis’^ disease in
mankind. Tl^e organism, which can be cultivated on Sabouraud’s agar
if not too heavily contaminated with bacteria, assumes all forms from a

budding yeast-like oidium stage to a typical mycelium. Technical

treatment therefore depends on the form, but on the whole permanent
mounts are best made from a considerable quantity of the organism
worked up in bulk. Staining may be with iron hematoxylin or acid
fuchsin.

Melanconiales
The so-called “anthracnose” diseases are produced by species placed
in the Melanconiales. The mycelium is internal in the host; true pyc-
nidia are never developed. The conidia are variable, being borne on
conidiophores which form a stratum in immersed or erumpent black or
light-colored acervuli.Many important plant diseases are caused by
members of the order. General methods may be employed, conditioned
by the nature of the material*
 MYCOPHYTA {EUMYCETAE) 357

LICHENES

Lichens occur everywhere under very variable environmental condi-

tions,and material should therefore be readily accessible. The lichens,
however, have a well-deserved reputation for being formidable subjects
from the technique standpoint. This is due to their tendency to become
excessively hardened after dehydration, to buckle or wrinkle during the
microtoming, and to be difficult to stain sharply. They greatly resemble
the Rhodophyta in these respects.
The lichen plant consists of a fungus and an alga in more or less inti-
mate association, and these two components are the same in every plant
of a given lichen species. The algae are mostly referable to other free-
living species, but none of the fungi can be so referred. The fungi
in all save three genera belong in the Ascomycetae, the exceptions to
the Basidiomycetae.
—
and Preservation. ^Lichens ordinarily occur where there is
Collection
considerable moisture. They make their greatest growth during rainy
seasons, then become desiccated when dry weather arrives, and become
revived when the rains again come. Crustose species should be cut
away from the substratum for fixation, but for preservation a portion
of the substratum should also be removed. Foliose species usually must
be cut from the substratum by clipping through the rhizines, while
fruticose species are simply lifted off. Plants too large to be run up in
their entirety should be reduced to smaller portions.
Most dry the lichen thalli, either with or without
collectors simply
pressure, then store in envelopes or small boxes. The plants may be
preserved in formalin-aceto-alcohol to which are added about 0.2% cupric
sulphate and about 5% glycerin.
Fixation. —
Formalin-propiono-alcohol is the only fixing fluid which
the writer finds worthy to be recommended. Dehydration should be
by very gradual stages, but the length of time in each change should be
short rather than prolonged. Xylol as a dehydrating medium should
be avoided.
—
Microtoming. Practically all species should be sectioned, ordinarily
in a transverse plane, at 10/x. If the material has become hardened, soak
the embedded pieces under water for two or three days. When the
sections are placed on the flood of the adhesive, they will buckle con-
siderably and may even become loosened from the paraffin matrix, but
—
one should not be dismayed by such mishaps simply proceed as usual,
keeping the sections arranged in as natural a position as possible. After
the sections have dried to the slides, coat with a dilute celloidin solution,
as it is extremely difficult to retain many species on the slides during
the staining.
 :

358 SPECIAL METHODS FOR THE VARIOUS PHYLA

Staining. —Safranin and fast green have commonly been used by

technicians recently, as a substitute for the unsatisfactory cyanin and
erythrosin of the older botanists. Fairly good differentiation is usually
secured (Fig. 66). The best staining, however, is to be gotten from a
quadruple combination. When correctly applied, the various portions
of a foliose lichen such as Sticta pulmonaria should be colored as follows
upper cortex light purple; algal layer: cytoplasm pale reddish-orange,
nuclei bright red; medulla orange; lower cortex bright purple; rhizines
purplish-orange; pycnidia bright green, spores orange; asci purple, spores
darker, nuclei red; paraphyses orange-brown.

J'lG. bG.~-Phy8cia sp. cross section of thallus with longitudinal sections of two apothecia.
:

Fixed with formalin-propiono-alcohol; stained with safranin and fast green.

Classification. —
Many genera form such characteristic thalli that
they are easily recognized as such, but species determination is a difficult
task, especially in the large group Cladonieae. A manual covering all
species found in the United States is available (B. Fink 1935), as arc
various locality lists {e.g., Torrey 1934).
In this connection, mention should be made of two tests used to
distinguish species of Cladonia, which are apparently applicable to other
genera. In one, a 6 %
aqueous solution of potassium hydroxide is applied
to dry material: in some species there is no reaction; in others, various
color reactions are produced. If a reaction occurs, it indicates the
presence of a bitter-tasting substance, fumarprotocetraric acid. In the
other test, a small amount of paraphenylenediamine (paradiamino-
benzine) is dissolved in a watch glass in alcohol. The proportions have
not been determined exactly; the efficiency of the solution is judged by
—
testing it first on a very bitter-tasting species if the reaction occurs, the
 MYCOPHYTA (EVMYCETAE) 359

solution, which is stable for only a short time, is all right. If the bitter
acid is absent, there is no reaction or a pale permanent-yellow stain
results. If the acid is present, a yellow color quickly appears and deepens
to either orange, orange-red or brick-red, according to the species, as the
solvent evaporates.
Whole Mounts. —Thalli which the association between
of species in
the two components is made into whole mounts by standard
loose can be
procedures. The most satisfactory stain will have to be determined by
experiment on the species concerned. Erythrosin, acid fuchsin, and
other acid stains may be tried. The thallus should be dissected to a
slight extent just before the coverslip is applied.
 CHAPTER XXVII
BRYOPHYTA

The Bryophyta, although cosmopolitan, reach their greatest develop-

ment in the tropics. Species preferring the temperate zone are sometimes
to be found in widely scattered localities. Funaria is unquestionably the
most widespread of all, with Ricciocarpus natans a close second. Mosses
grow far north of the Arctic Circle, and species of Riccia have been
reported from desert regions: in short, Bryophyta of one sort or another
occur everywhere and, at the proper growth seasons, are readily accessible
to the technician. If unfamiliar with field collecting of Bryophyta, one

can, as a last resort, always find Lunularia or Marchantia and various

mosses in any commercial greenhouse. Professional botanists usually
despise materials from such a source, but they are very useful to the
technician.
Since general technical methods are essentially similar for the entire
phylum, these will be presented first. Structural peculiarities which
demand special treatment, particularly with regard to planes of section-
ing, will be mentioned under the families or genera concerned.
Collecting. —Some species are easily collected or preserved in the
field, but it is tedious work getting other species into a suitable condition
for embedding and sectioning. Aquatic forms like Ricciocarpus natans
are simply dropped into the killing fluid, but if the rhizoids are long and
abundant they should be trimmed with scissors. Practically all other
forms require reduction to smaller portions. In the case of those that
adhere firmly to the substrate, a large chunk of substrate should be dug
up, the whole brought to the laboratory, and the plants there cut off, by
means of a scalpel, as close to the substrate as possible. It is of little use
trying to wash the plants free from the substrate since so much grit will
adhere that the sections will may be
be torn and the microtome knife
badly nicked. Species which grow in large masses on the trunks of trees,
such as Porella, should be fixed immediately after removal since they dry
out very rapidly; the desired portions can be removed later.
It should always be borne in mind that most Bryophyta are essen-
tially semiaquatic organisms and must be handled accordingly. They
should never be allowed to become dry before being killed. Some species
can, of course, survive desiccation better than others, but material that
 BRYOPHYTA 361

has been allowed to dry out and then revived by soaking invariably gives
poor preparations. Genera such as Riccia^ Fossombroniaj SphaerocarpoSy
and various mosses can be removed together with generous portions of
the substrate, placed in trays, pans or shallow pots, kept watered and in
a cool and partly shaded place, and grown on until ripe spores are pro-
duced. This cannot be done with epiphytic species.
There are few Bryophyta that cannot be preserved entire in formalin-
aceto-alcohol and the desired portions removed at any future time for
further treatment.
Fixation. —
Formalin-aceto-alcohol and formalin-propiono-alcohol
have proved to be entirely satisfactory for all stages of both sporophytic
and gametophytic development over the entire range of the Bryophyta.
For special purposes, particularly for critical cytological studies, a chrom-
acetic or chrom-osmo-acctic fluid, with the proportions of each reagent
carefully adjusted by experiment, may sometimes be necessary. On the
other hand, a few investigators claim that only Carnoy^s fluid will give
satisfactory fixation of chromosomes in the Bryophyta (see e.g., Lorbeer
1934, whose methods might be followed by those particularly interested
in the cytological aspects).
A water suction pump should always be used on the Bryophyta since
the tissues contain considerable air. The proper procedure in getting
rid of air, and consequently obtaining perfect infiltration with paraffin,
is enough air at the time
to extract just of killing to cause the material to
sink, then after 24 hours or longer to pump out all remaining air possible.
If one attempted to exhaust all the air at the beginning of fixation, some

plasmolysis would result, and certain structures would actually be

collapsed. The sporophytes of Polytrichurriy for example, will be crushed
inwardly. If only part of the air is exhausted, the fixing fluid will ordi-
narily so harden the tissues that the remainder of the air can later be
extracted with no damage resulting. •

—
Embedding. Certain investigators contend that the delicate thalli
of some species must be embedded in paraffin of a low melting point,
preferably not over 43°C. Experiment has shown, however, that this
was necessitated because rather weak killing fluids were used, and these
did not bring about sufficient rigidity of the tissues to permit paraffins
of higher melting points and coarser structure to penetrate without caus-
ing some damage. With more modern methods of dehydration and
infiltration, no damage is likely to occur, and a medium Parlax permits
perfect infiltration. The older sporophytes of some mosses, notably those
of Funaria and Polytrichurriy have a tendency to become incredibly
hardened; consequently they should be embedded either in a paraffin of
around 62®C. or in hard Parlax, which can be further hardened by cooling
with ice water at the time of microtoming.
362 SPECIAL METHODS FOR THE VARIOUS PHYLA

The time in the paraffin oven should be short, particularly if the

killing fluid contained chromic acid, because the heat renders the tissues
of a few species somewhat brittle.

The great majority of the Bryophyta are sectioned at lO/x, both for
general morphology and for development of the sex organs and sporo-
phytes. Very young antheridia and archegonia should be cut at 6 )la;

this thicknessshould also be used for details of sperm formation in the

antheridia and for the early sporogenous cells of the sporophyte. Those
who claim that sections 1 and 2^ thick are the only useful ones are in
a class with microscopists who consider the diatoms to be the only objects
worth examining.
Orientation of the objects for microtoming always a difficultis

proposition. Suggestions regarding specific forms be given in the will

following discussion, but there are nevertheless some species {Riccia and
Anthoceros in particular) which one can only microtome with general
reference to the orientation of the thallus and trust to luck to get perfe(;t
meKiian longitudinal sections of the reproductive organs. This is not so
discouraging as it sounds, however, since the proportion of desirable
sections is actually quite high.
Staining. —The
most precise staining for antheridia and archegonia
on all Bryophytato be obtained with iron hematoxylin and a counter-
is

stain of fast green or orange G. The greatest care must be exercised

during the destaining because most species give up the stain with unusual
rapidity. Sometimes only a few seconds in a 1% ferric ammonium
sulphate solution are permissible. In those species whose gametophyte
is a thallus and whose sporophyte is wholly or mostly embedded therein,

the same stain combination is preferable. In other types this combina-

tion generally is to be selected for the antheridia, but not always for the
archegonia and sporophytes. It is excellent for the sporophytes: of
Funaria bu^ poor on those of Polytrichum. With the latter, safranin
and fast green are superior. The archegonia of Marchantia and some
other genera are notoriously difficult to stain sharply; Harris^ hema-
toxylin preceded by differential acidification,and followed by coun-
terstaining with orange G, is the only combination which gives clear
results.
Most commercial technicians use a triple combination, but the net
effect is agaudy splash of colors which generally fade completely in two
or three years. In the hands of experienced technicians, such combina-
tions can be manipulated to give beautiful and useful results, but the
novice should first get considerable experience by using the stains on more
suitable objects, such as anthers or root tips.
On sections of stems and leaves of the mosses safranin and fast green
or a triple combination will be satisfactory,
 BRYOPHYTA 363

Whole Mounts. —The prime requisite to making satisfactory whole

mounts of the thalli, flat portions, or leaves of any of the Bryophyta is
to secure adequate fixation of the material, since the thin and delicate
structures have a pronounced tendency to curl up tightly during the
later stages of dehydration or during the infiltration. A strong fluid is
better than a weak one, and an alcoholic better than an aqueous fluid.
Stain with Harris’ hematoxylin, counterstain with fast green, and
follow the gradual hygrobutol method. Many technicians stain in the

Fiu. fi7 .
— Riccia (jlauca: cross section of thalhis with longitudinal section of a t-wo-
celled and an older anthtiiidiurn. Fixed with fornialin-aceto-alcohol stained iron hema-
;

toxylin and fast green.

but the slight additional effort required for the hema-

fast green alone,
more than repaid by the clearer internal differentiation.
toxylin will be
Mount the material as soon as the balsam becomes thick enough. If
they are available, mount in depression slides.

HEPATICAE
M ARCHANTIALES
Ricciaceae. —In this family the sex organs are developed in a longi-
tudinal strip running the entire length of the thallus. There are five
genera, and but six of the species are included in the largest genus,
all

Riccia. This genus is nearly cosmopolitan. Ricciocarpiis natans and

Riccia fluitans are the only aquatic species. R. natans is more common
in the eastern part of the United States, with terrestrial species of Riccia
more abundant in the Far Western states.
Ricciocarpus natans commonly grows in pools and ponds* which are
dried up during part of the year. It commences growth along the edges
of the body of water as a terrestrial plant and becomes floating after the
364 SPECIAL METHODS FOR THE VARIOUS PHYLA

rising water level causes the plants to become detached. In some

regions, however, the plants always remain on muddy banks and are
said to become unusually large in such situations. Development of the
sex organs commences while the plants are still small and attached to the
substrate; for the best preparations for the origin of the antheridia it is
necessary to collect material at this time or shortly after the plants
become floating. After the plants have been floating for a week or longer,
it will be nearly impossible to get very young antheridia. Microtome the
thalli transversely at 10 to 12)u; examine portions of the ribbons under

Fig. 68 .
— Riccia glauca: cross section of thallus with longitudinal sections of two young
archegonia. Fixation and staining as in Fig. 67.

the microscope, and select those that show median sections of the sex
organs, embryos, and sporophytes. If the thalli are cut longitudinally,
median sections of the sex organs cannot be secured. For the apical
cells, cut off most of the sides to within 1 mm. of the central depression,
and section perpendicular to the flat surface at lOju.
Riccia fluitans always grows submerged but is very rarely found in a
fruiting condition. Whole mounts of the thalli are useful.
The terrestrial species are quite firmly attached to the substrate, and
it willbe necessary to cut through the rhizoids horizontally to get the
plants off without too much adhering grit. Most of the species of Riccia
are homothallic, but two are heterothallic, viz., R. bi8choffi,i and R. curtim
(McAllister 1916, 1928). Transverse sections of entire plants may be
cut, at an optimum thickness of lO/i, for all developmental stages (Figs.
 BRYOPHYTA 366

67, 68). Since the sex organs are commonly close together, a few sec-
tions will afford a number of stages. The transverse sections will fre-
quently show the apical cells, which occur in a transverse row of three or
more attached laterally to one another, but vertical longitudinal and
horizontal longitudinal sections may also be cut. The latter will afford
fine views of the sex organs and sporophytes in transverse section.
Meiosis takes place in the sporocytcs, and it is not at all difficult to
catch this phase in some species, notably R. glauca and R, sorocarpa.
One haploid chromosome is extremely small in nearly all species. Stain
meiotic stages critically with iron hematoxylin.
Targioniaceae. —
Targionia is abundant in the coastal region of
California and Oregon, and widely distributed in other states. The
principal character distinguishing Targionia from the other Marchan-
tiales of the United States is the enclosure of the archegonia and sporo-
phytes by an involucre.
The antheridia occur on short lateral branches. Cut these off, and
microtome in the vertical longitudinal plane at lOjW. The archegonia
originate very close to the apical (^ell. They are strictly dorsal in position
but appear to be on the ventral side because of the overgrowth of the
thallus. While the presence of sporophytes can always be determined
by looking for the characteristic purple-colored involucre, the presence
of archegonia can definitely be told only by sectioning. The tips of the
thalli should be cut off, about 3 mm. from the apex, and most of the lateral
wings of the thallus eliminated. Section in the vertical longitudinal
plane at 12jui. The archegonia are rather few in number. Ordinarily
only one of them develops into a sporophyte, but it is not uncommon
to encounter two equally well-developed sporophytes or a sporophyte
and an embryo. The sporophytes are rather difficult to stain sharply.
—
Marchantiaceae. ^All Marchantiales in which the archegonia ar(‘
borne on a stalked archegoniophore belong in this family. Some genera
have a stalked antheridiophore; in other genera the antheridia are
partially or completely embedded in the thallus, and may be localized or
produced continually or intermittently.
Two genera reproduce vegetatively by means of gemmae. In
Marchantia these are developed in cup-shaped depressions, and in Lunu-
laria in crescent-shaped cups. To demonstrate the origin and growth
of the gemmae, select thalli in which growth of the gemmae has not pro-
gressed to the point where they break loose easily. Cut out portions
bearing receptacles, and microtome in the vertical longitudinal plane of
the thallus at 10/x. Stain with iron hematoxylin and fast green or a
triple combination. Whole mounts of mature or germinating gemmae
are easily prepared: fix with formalin-aceto-alcohol, stain lightly with
366 SPECIAL METHODS FOR THE VARIOUS PHYLA

alcoholic fast green, dehydrate with hygrobutol, and infiltrate with

dilute balsam.
Marchantia is the most widely distributed genus and if not readily
available locally (Evans 1917), material can always be secured from the
supply concerns. The antheridia are borne on a stalked receptacle
which can be distinguished from the archegoniophores in that it is flat
on top and has no involucres. The earliest stages of antheridial develop-
ment are to be found when the receptacle first becomes definitely recog-
nizable at the apex of the thallus. Do not try to remove such young
receptacles from the thallus, but cut out narrow transverse portions of the
thallus bearing the receptacles. Fixation has always been excellent with
formalin-propiono-alcohol. Section the thalli at 8 to lOfx in the trans-
verse plane, which will give longitudinal sections of the receptacle and
antheridia. After the stalk becomes 3 mm. or more in height, the
receptacle may be removed from the thallus and sectioned longitudinally.
The youngest The
antheridia occur at the periphery of the receptacle.
archegonia likewise appearwhen the archegoniophore is barely recogniz-
able as a button’^ at the apex of the thallus. At this time they point
upward but rapidly become turned over, to point downward, by an
upward growth and swelling of the central region of the archegoniophore.
There are actually eight archegonial receptacles on each archegoniophore
in Marchantia, An involucre (a ^‘ray’O is developed between each
receptacle after the periphery of the archegoniophore has become
inverted. By the time the involucres are readily recognizable, most of
the archegonia are mature and may have already been fertilized. . How-
ever, in vigorously gromng plants, archegonia continue to be produced
until the first two or three produced in each receptacle contain young
embryos. The very youngest archegoniophores should be manipulated
and fixed as has been described for the young antheridiophores; even the
somewhat older stages are more easily oriented for microtoming if
left attached to narrow portions of the thallus. Section at 9 or lO/i for

the younger phases and 11 or 12^ for the mature archegonia. If sec-
tioned exactly parallel to the longitudinal axis of the stalk, most of the
archegonia will be medians. Stain with Harris^ hematoxylin, first
applying differential acidification, and counterstain with orange G. Up
to the time that the sporogenous cells and elaters are differentiated in
the young sporophyte, entire archegoniophores may be sectioned. These
stages should be stained with safranin and fast green. As the sporo-
phytes begin maturing, the archegoniophores should be cut into portions
which appear in triangular outline when observed from above, by cutting
through the involucres with a very snaall scalpel. If one attempted to
section an entire archegoniophore bearing mature sporophytes, very few
of the latter will be in median section. Microtome the sporophytes
 BRYOPHYTA 367

longitudinally at lO/i; stain in a triple combination, although safranin

and fast green will do just as well, the elaters having a particular affinity
for the green. Meiosis occurs in the sporophytes but is an extremely
difficult stage to obtain.
Conocephalum is a larger plant in all respects than Marchantia and is
frequently to be found where the latter does not grow (Fig. 69). Sterile
plants can be distinguished from those of Marchantia by the absence of
gemmae cupules. Treat exactly as described for Marchantia.

Fig. 69 .
— Conocephalum conicum: median longitudinal sortion through two mature
sporophytes. Fixed with formalin-aceto-alcohol; stained with iron hematoxylin and fast
green.

Lunularia, whose sole species, L. cruciataj is an advent from Europe,

is common in and around greenhouses, and will almost invariably bo
found only in the gemmiferous condition.
Asterella is very common in Oregon and California, Cryptomitrium
less so. The antheridia are borne in more or less rounded, cushion-liko
receptacles a short distance back from the apex of the thallus; these areas
are easily recognizable in both monoecious and dioecious species. Cut
out portions of the thallus bearing such regions, and microtome trans-
versely at 10/i. Treat the archegoniophores as in Marchantia.
 BRYOPHYTA 369

There are about a dozen other less well-known genera. The direc-
tions cited for the better known genera in the family will serve as guides
for the treatment of these less known genera.

Sphaerocarpales
Sphaerocarpaceae,—SpAacrocarpos occurs in the Gulf and Pacific
Coast regions; in central California it is, in very wet seasons, so abundant

Fig. 72 . — Sphaeroearpos criatatus: vertical longitudinal section of gametophyte with four-

celled embryo. Fixation and staining as in Fig. 70.

that it covers acres of bare ground or soil that had been plowed the

preceding summer. It is readily grown from spores and has been the
subject of significant genetical investigations. Preserved material can
be obtained from the supply concerns. When collected in the field, the
best procedure is to cut through the rhizoids, wash thoroughly in running

water to get all possible grit out of the involucres, then fix the plants
without attempting to separate the colonies to any great extent. The
species are all heterothallic, and the antheridial plants are often diflScult
to recognize in the earliest growth stages. They are not so abundant as
370 SPECIAL METHODS FOR THE VARIOUS PHYLA

the arcbegonial plants. In many species they can be distinguished by

their purplish color. Fixation should be with formajin-aceto-alcohol;
chromic acid solutions have generally given atrocious results. Microtom-
ing should be done with safety razor blades in a suitable holder, as plenty
of trouble will be encountered with grit which has lodged within the
involucres or among
the rhizoids and it will nick knives badly. Micro-
tome in the vertical longitudinal plane at lO/x for all stages. Series of
stages in the development of the antheridia, embryos, and sporophyles
are very easy to obtain, but good archegonia sections are rare (Figs. 70,
71, 72). Iron hematoxylin with fast green is the only combination that
can be recommended. If a triple combination is used, only a few seconds
in the safranin should be allowed.
Riellaceae. —
Riella grows entirely submerged in pools; one species is
known from Texas and South Dakota. In case material should be avail-
able, section parallel to the flat wings. Sporophytes are readily recog-
nized, but one will simply have to section considerable material to get the
sex organs. Staining has been found to be extremely difficult. Harris^
hematoxylin has given fairly good results.

JUNGERMANNIALES
By far the larger number of genera and species of Hepaticae are
included in the Jungermanniales, but little information of value from the
technique standpoint has been published.
The apical cell in the order is always solitary and is easy to find in
some species, but in others it is difficult to cut sections in the proper plane.
Both true gemmae and other vegetatively reproductive bodies some-
times called gemmae'^ are present. Gemmae cannot usually be col-
lected so abundantly as in Marchantia, consequently whole mounts are
impracticable. Sections of the gemmiferous branches or receptacles are
more useful. The gemmae are frequently to be found in sections pri-
marily intended for other structures.
If the gametophyte is thallose, the antheridia are borne either on
special branches or dorsally along the midrib of the main axis. If the
gametophyte is foliose, the antheridia occur in the axils of the leaves and
may be solitary or as many as four in each axil.
In both thalloid and foliose genera the archegonia occur either on the
main axis or on lateral branches and originate close to the apical cell.
The Jungermanniales are separated into two suborders, in one of
which the apical cell is transformed into an archegonium (Acrogynae)
and in the other it is not (Anacrogynae). ^

Acrogynae

The number of foliose Jungennanniales is very large, but remarkably

littte technique information has been reported for the group. There is
 —

BRYOPHYTA 371

scarcely anything in the literature concerning even so common a genus

SiS Porella,There are also very few descriptions of the development of
the antheridia and archegonia. Some species are homothallic; others
are heterothallic, with the sex organs produced upon either the main axis
or on short lateral branches; but many species, particularly those from
the tropics, apparently never fruit. The species in most of the genera

Fig. 73. Pordla navicularis: median longitudinal section of antheridial branch with
antheridia in several developmental stages. Fixed with formalin-aceto-al<?ohol; stained
with iron hematoxylin and fast green.

are extremely diflScult to identify, since splitting of both species and

genera has been carried to such an extreme that only a specialist of long
experience could understand the involved taxonomic literature.
Porella is the only genus with which the technician is likely to work.
Plants of this genus are common on the bark of trees, principally ylZnws,
and sometimes form extensive mats. Others grow on shady banks of
creeks. The sex organs occur on side branches which are easily recog-
nized even in the earliest developmental phases. Remove these branches,
372 SPECIAL METHODS FOB THE VARIOUS PHYLA

fivingeach sex and the sporophytes separately. A strong fixing fluid is

necessary; formalin-propiono-alcohol has given far better results than
chrom-acetic fluids. Microtome parallel to one flat surface at 8jtt for
antheridia and 10 m for archegonia and sporophytes. It will be almost

impossible to get perfectly median sections of the older and mature

antheridia because the organs are usually pushed around by the stalks,

Fig. 74 . —PoreUa navicularis: longitudinal section of archegonial branch with archogonia.

Fixation and staining as in Fig. 73.

which do not have sufficient room for straight growth and hence become
twisted (Figs. 73, 74).

Anacrogynae
Riccardiaceae. —Most of the genera are thallose. Riccardia and
Metzgeria are common in coastal Oregon and Washington, less so
in California; the first genus also occurs in the northeastern states. In
Riccardia the sex organs are produced on short lateral branches; in
Metzgeria the branches arise from the ventral side of the thallus. Remove
these branches, embed separately, and microtome in the vertical hori-
zontal plane.
Codoniaceae. —
few genera are foliose, but most of them are thal-
lose. The family is immediately recognized by the globose sporophyte
borne on a relatively long seta.
Pellia is a well known and widespread thallose genus. It has two
species in the United States. P: epiphylla is monoecious, and P. neesiana
is dioecious. The latter is especially abundant in coastal Oregon, form-
ing dense mats. Only material taken from moist habitats, or material
wl^h is damp at the time of collection, should be Gxed, For the anthe-
 8

BRYOPHYTA 373

ridia and archegonia cut the thalli into portions not over 5 mm. in length,
fibcwith formalin-aceto-alcohol, and section in the vertical longitudinal
plane. Since both sex organs are somewhat obliquely oriented, with the
apices directed toward the apex of the thallus, transverse sections are
valueless. As soon as the sporophytes become recognizable, but before
the seta commences rapid elongation as the sporophyte matures, cut out
narrow longitudinal portions of the thallus together with the involucre
enclosing the young sporophyte, embed these portions on their sides and
microtome parallel to one side, thus obtaining longitudinal sections
through the curved portion of the seta. After the sporophyte emerges
from the involucre, cut it olf through the seta at any convenient point.
Meiosis takes place comparatively late in the development of the sporo-
phyte, but it is difficult to catch this important phase. The spores
germinate while still enclosed in the capsule, and they are shed as six- to
nine-celled ovoid masses. The spores at the discharging stage are so
gorged with reserve foodstuffs that thin sections are necessary — m is
—
about right and staining must be critically controlled. Iron hematox-
ylin is excellent, but a triple stain will better reveal the beautiful mitotic
figures occurring during spore germination.
Fossombroniaj also widespread, generally occurs in patches of a few
to 35 to 50 plants intermingled with other bryophytes. The plants are
distinctly foliose and may be homothallic or heterothallic. The pres-
ence of antheridia, archegonia, or embryos generally cannot be told
from the outward appeai*ance of the plants. The latter are more readily
removed from the substrate than are most othei* terrestrial bryophytes.
Fix the plants entire, microtome in as nearly a vertical longitudinal
orientation as possible, then examine the ribbons under the microscope,
and pick out the satisfactory sections. Those species which are more
prostrate than erect may sometimes give good sections when microtomed
transversely, but longitudinal sections are preferable. As soon as the
sporophytes emerge, they may be handled separately.

Calobryalks
Members of the order are seldom to be encountered. They are so
much like the mosses (FAibiya) that they may beHreated similarly.

ANTHOCEROTAE
Of the Anthocerotae found in the United States, Anthoceros is wide-
spread in the northern states and west of the Mississippi River, being
especially common in coastal California, and Notothylas has been found
in Texas and California.
These and the other genera of the Anthocerotae are superficially much
alike and can be dealt with by technical methods common to all. The
374 SPECIAL METHODS FOB THE VARIOUS PHYLA

plants grow on steep banks along roads, especially where there is dripping
water, near waterfalls, on rocks and boulders in damp situations, and on
rotting logs in the tropics. Some species have such a peculiar dark green
color that they can readily be distinguished by this character from the
thalli of other Hepaticae, with which they frequently grow intermingled.
The gametophytes are dorsiventral thalli. They are homothallic in
all genera. Both antheridia and archegonia are completely embedded in
the thallus, as are the embryos and the youngest stages in sporophyte
development. It is difficult to determine from external examination
what stages might be present, up to the time that the sporophytes are
well along in development, and it is impossible to know whether sex
organs are present without making sections. The gametophytes are
easily separated from the substrate.
A single apical cell is present, but it is a matter of sectioning and
examining hundreds of sections to locate it.
literally
The thalli may
be fixed entire in formalin-aceto-alcohol, a medium
chrom-acetic, or in Navashin^s fluid. The writer^s experience leads to
a preference for the fluid first mentioned. Many investigators have
complained that excessive brittleness results following dehydration with
xylol and similar fluids; if the tertiary butyl alcohol schedule is followed,
there should be no brittleness.
Microtome in the vertical longitudinal plane; 8// is usually the opti-
mum thickness, but since the older archegonia are rarely oriented exactly
perpendicular to the dorsal surface of the thallus, it would be better to
cut at lOfx if these are particularly desired. The ribbons should be
stretched in water, examined under the microscope, and the satisfactory
sections picked out, since about three-fourths of the ribbon from a given
thallus will be found devoid of sex organs. One must search very care-
fully for the youngest archegonia, as they are easily passed over. Some-
times several archegonia and a series of stages in the development of the
antheridia can be found in a single section from a large thallus. If

Nostoc colonies occur in sections otherwise lacking desirable structures,

these may be mounted separately as many instructors find them valuable
in courses on the algae. For the early sex organs and the Nostoc colonies,
iron hematoxylin and fast green are superior; for later stages a triple
combination might be substituted if preferred. It is entirely a matter of
chance that one obtains stages in the older embryo showing the differ-
entiation of the archesporium (such as those shown by Campbell 1918,
Fig. 70E; G. M. Smith 1938, Fig. 46ff) and any technician who takes
pride in his art will feel considerable excitement, as did the writer, when
this stage is found for the first time. As soon as the sporophytes have
attained a length of about 2 mm. above the surface of the thalli, the latter
should be trimmed away from each sporophyte in such a manner as to*
 BRYOPHYTA 375

leave a narrow zone around the foot. These portions should be embedded
with the sporophyte flat, can be microtomed longitudinally. It
so that it

is actually difficult to get perfectly median longitudinal sections of the

entire sporophyte, since the foot and involucre are curved and the capsule
is sometimes bent as it emerges from the involucre. Spores are produced
early and develop rapidly; since the basal cells of the sporophyte remain
embryonic, there is a continuous production of spores and elaters so
long as the gametophyte remains capable of nourishing the sporophyte.
Maturing portions of long sporophytes should be bisected and embedded
in bunches of four or more for sectioning both transversely and longi-
tudinally. The younger stages should be sectioned at lOfx and stained
with iron hematoxylin and fast green; the older stages at different thick-
nesses, from 8 to \2ix^ and stained with safranin and fast green or a triple
combination.

MUSCI
The plants commonly knownas mosses belong in this homogeneous
group. In general the methods cited in the introductory paragraphs
to the Bryophyta are sufficient for the Musci, but additional details for
various structures are given below.
—
Protonema. The spores of most mosses are viable for several years.
Unruptured capsules are the best source for spores for germination pur-
poses. The culture medium found to be most consistently satisfactory
is made up as follows:

Distilled water . . 1 liter

Ammonium nitrate . . 1 g.
Potassium sulphate 0 .
5 g.
Magnesium sulphate 0 .
5 g.
Calcium sulphate 0 .
5 g.
Ammonium phosphate 0 .
6 g.
•Ferric sulphate 0 .
01 g.
10% aqueous potassium hydroxide A few drops

Sterilize in an autoclave for from 30 minutes to 1 hour at 5 pounds pres-

sure. Pour sufficient of the medium into sterile Petri dishes. Open
the capsules by means of sterile needles, and scatter the spores as evenly
as possible* over the surface of the medium. Keep the cultures in bright
or diffuse light, at room temperature (averaging 20®C.). The time
required for germination varies from a few hours to a week. The
protonema will usually grow rapidly and branch profusely but will
rarely produce gametophoric buds To induce growth
in liquid media.
of the gametophores, obtain some
from the habitat of the species,
soil
if possible, otherwise use any good loam to which some wood ashes hav()

been added. Put in 2- and 3-inch clay pots, soak with the nutrient
376 SPECIAL METHODS FOR THE VARIOUS PHYLA

solution, then sterilize inan autoclave at 15 pounds pressure for 3 hours.

After sterilizing, place the pots in earthenware plates in which water to
irrigate the plants may be kept. Place the whole in a Wardian case or a
similar container to avoid contamination by air-bome spores and to
keep the plants in a humid atmosphere. The protonemata may be
removed from the culture dishes by means of pipettes and transferred
to the surface of the pots. In controlled growth studies, care should be
taken to place only a single protonema in each pot.
Whole mounts of the protonemata at any stage of growth are easily
made. Kill and fix with formalin-propiono-alcohol or a weak chroni-

Fig. 76 .
—
Mnium affine: longitudinal Beetion through central portion of an antheridial
head with antheridia in various stages of development. Fixed with fornialin-acoto-
alcohol; stained with iron hematoxylin and fast green.

acetic fluid, stain with iron hematoxylin, counterstain lightly with fast
green, and follow a gradual hygrobutol method.
—
Gemmae. A considerable number of Eubrya produce multicellular
gemmae in groups at various points on the aerial portions. Solitary
subterranean gemmae, known as ^‘bulbils,’’ are produced on the rhizoids
of either the protonemata or the sporophores. When borne at the tips
or along the midribs of leaves, detach the latter, and mount entire.
The same procedure may be followed in the case of gemmae borne on
the branches or at the stem apices. If the plants are not too large, those
bearing subterranean gemmae may be mounted entire, otherwise the

r izoids may be cut off. Fix all types of material in a weak chrom-acetic
 BRYOPHYTA 377

fluid,wash thoroughly, stain critically with Harris^ hematoxylin, counter-

stain with fast green, and infiltrate with balsam by a gradual hygrobutol
method. The bulbils of some species have a distinctive bright color
that is not affected by fixation or staining; they may even be mounted
unstained if fixed with formalin-aceto-alcohol and taken directly through
hygrobutol into balsam.

Fig. 76. —
Mnium ajjlne: longitudinal section of archegonial head with ai chogonia. This
isa typical section, illustrating the difficulties in obtaining perfectly median sections of he
t

arohegonia. Fixation and staining as in Fig. 75.

—
Antheridia and Archegonia. Tips, which by dissection and examina-
tion under a high-power binocular microscope are known or strongly
suspected to contain the sex organs, are snipped off by means of fine-
pointed forceps and dropped into the killing fluid (Figs. 75, 76). For
ordinary preparations the more robust species are sufficiently well
fixed by formalin-aceto-alcohol, but for critical studies of spermato-
weak chrom-osmo-acetic
genesis a fluid is needed. The following formula
may be recommended:
 378 SPECIAL METHODS FOR THE VARIOUS PHYLA

1% aqueous chromic acid 180 cc.

2% aqueous osmic acid 26 cc.

Glacial acetic acid 12 cc.
Distilled water 210 cc.

For the archegonia, a 0.25% chrom-acetic heated to 30°C. is excellent.

The most satisfactory stain combinations are safranin and fast green,
and iron hematoxylin, without a counterstain or with fast green. The
archegonia! heads must be very carefully oriented for microtoming in
order to obtain perfectly median longitudinal sections.
Sporoph3rte. —
The sporophytes of some genera give no trouble, but
those of others are likely to be exasperatingly difficult. Formalin-
aceto-alcohol is usually much better than a chrom-acetic fixing fluid,
and the dehydration should not be rushed. For the earlier stages, up
to and including the stage where the spores are rounding up, iron hema-
toxylin is the preferable stain, but safranin is excellent for maturing
spores and for the structure of the sporophyte.
It is interesting to follow out the origin of the plastids during sporo-
genesis (Senjaninova 1927). First fix in Lewitzky’s fluid for two to
three days:

10% aqueous formalin 90 cc.

1% aqueous chromic acid 10 cc.
then transfer to
1% aqueous chromic abid 30 cc.
2% aqueous osmic acid 8 cc.

for nine days. Wash thoroughly, dehydrate, and embed. Microtomt'

the sections at from 2 to S/x. Bleach, then stain with iron hematoxylin.

Sphagnobrya
Sphagnurrij the peat moss, grows in moist places where the water is
quite acid and is restricted mainly to the North Temperate Zone. The
sex organs have been reported to be difficult to find, but this probably
came about because searches for material were made at the wrong time
of year. Some species are heterothallic and others homothallic. In
the Middle West and on the Atlantic Coast, the antheridia begin develop-
ment in August; the heads of the antheridial plants are decidedly globose
and. vary in color from yellow-brown to red-brown or almost black.
The archegonial heads appear early in September, are flatter on top
than the antheridial heads, and may be distinguished by the yellow-brown
to red-brown conspicuous bud in the center of the head. The archegonia
are fully mature early the next spring, and fertilization takes place at
this time. Plants with sex organs are said to be more readily found
are those with sporophytes, but this may be only a regional difference.
|S!i Remove the short lateral branches bearing the sex organs individually,
it will be much less trouble to obtain median sections by sectioning
 BRYOPHYTA 379

these smaller portions than by attempting to microtome the entire mass.

Little has been reported regarding the proper fixation of the sex organs
in Sphagnum, but it appears that a 1 %
aqueous chrom-acetic fluid diluted
with three times its volume of water and heated to 30°C. gives excellent
fixation (Bryan 1915). Formalin-aceto-alcohol has been found wholly
satisfactory for the sporophytes. The lateral branches are wider in one
direction than in the other; microtome parallel to one flat surface at
lO/x for the earlier stages and 12/x for later development. The long and
somewhat twisted stalk of the antheridia will make it difficult to securt;
median sections of the entire organ. Iron hematoxylin usually,
perfectly
but not always, stains brilliantly; a counterstain in fast green is desirable.
Safranin and fast green may also be used.

Andreaeobrya
In Andreaea the cespitose plants are strictly saxicolous and are usually
found in cold regions or on high mountains but descend to sea level in
New York state. Their color is dark brown to blac^k. The plants are
readily distinguished by the longitudinal dehiscence of the mature capsule
into four valves. The gametophytes are usually homothallic, but the
antheridia and archegonia are borne on separate branches in terminal
groups.
Material is generally quite brittle, consequently special methods,
involving the use of hydrofluoric acid, are required. The younger
portions, including those bearing the s(>x organs, should not give much
trouble if fixed with formalin-aceto-alcohol, embedded in a hard paraffin,
and then soaked under water for some time before being microtomed.

Eubrya
The true mosses are among the commonest and most widespread
l)lants, occurring almost everywhere. They are to be found in cold and
warm, fresh and brackish waters; on rocks, bare ground, tree
in caves;
trunks, on flower pots in greenhouses, and numerous
in other habitats too
to mention. Most mosses are gregarious in habit and are but little
mixed with other plants; consequently material as a rule can be collected
in great quantity with ease.
Despite the fact that there are many thousands of species of Eubrya,
the differences are of such minor importance that almost any available
moss will provide satisfactory material for the technician. It will be
found, however, that material of the same species from widely separate
localities will reactmore or less differently to the identical technical
treatment. Material of Polytrichum commune from Wisconsin is some-
what difficult to work with, the tissues having a tendency to collapse and
become hardened; that from central California is less troublesome, not
shrinking but becoming hardened; while material from the coniferous
380 SPECIAL METHODS FOR THE VARIOUS PHYLA

forests of Oregon is a delight to work with and gives no trouble whatever,

except for the older stems. Some species may prove to be objectionable
because the sex organs are too few in number; Funaria hygrometrica,
which is extremely common, has few antheridia and fewer archegonia
in comparison with Polytrichum or Mnium, and in Buxhaumia there is

generally only a single antheridium and but one archegonium. Pre-

sumably most species are heterothallic, but this condition may be more
apparent than real. In the homothallic species, the antheridia and
archegonia may either be freely intermingled in the same head, in separate
groups in the same head, or in separate branches on the same stem.
All factors considered, Mnium and Polyirichum (or Pogonatum) are
preferable for the sex organs, and these two genera together with Funaria
for the sporophytes. For bulbils Leptobryum pyreforme has proved to
be excellent, but on examination other species may be found to be
equally satisfactory.
Beautiful fixation has always been obtained with either formalin-
aceto-alcohol or formalin-propiono-alcohol. Chromic fluids may be
used, but they always cause brittleness and discolorations that are hard
to bleach.
The antheridial and archegonial heads are always microtomed in
the longitudinal axis. The first should not be cut at more than lOp
if details in spermatogenesis are desired; 12p is thick enough for general
purposes. Before the archegonial heads are placed in the killing fluid,
all leaves surrounding them should be carefully snipped off with fine-
pointed scissors. At best, it is difficult to orient these heads to get
most
of the archegonia in median longitudinal section. The archegonia an*
always long and are commonly borne on elongated stalks, and as they
get older, the jacket portion becomes bent and even convoluted. In
practice, one might try microtoming at 12/x; if the younger stages appear
to be satisfactory and at least a quarter of the nearly mature archegonia
have been sectioned through most or all of their length, then that thick-
ness may be considered as the optimum for that material. A series
of developmental stages of the sporophyte is more useful and instruc-
far
tive than merely sections of the mature capsule. Both transverse and
longitudinal sections should be prepared. In the latter the development
of the peristome may
be followed out. Some mosses lack a peristome
(e.g., Phuridium^ which is common on both coasts). In some species
the sporophyte is extremely difficult to section at all times (Funaria) j

and more or less shattering must be expected; in others the capsule

may become too hard to section, although earlier stages give no trouble.
To avoid these difficulties in large measure, all air must be exhausted
during or following fixation, infiltration must be very thorough, and
embedilpg should be in a hard paraffin.
 CHAPTER XXVIII
PTERIDOPHYTA
The ferns are a large, diverse, and extremely interesting group,
and in many respects they tax to the utmost the skill and ingenuity of
the technician. Indeed, they are exceeded only by the Rhodophyta
in the problems that they present. The beginning technician would do
well to acquire considerable experience on less difficult groups before
tackling the Pteridophyta. Ophioglossum^ IsoeteSj and Marattia are
the only genera all the* parts of which section easily and give no trouble
with the staining; consequently, if material of one of these genera should
be available, the beginning should be made on it. Thereafter progress
toward the more difficult genera can be started, taking up such as
Botrychiurriy Azolla, Lycopodium^ and Psilotum^ and progressing from
these to the Filicales and Equisetales.
Detailed directions for the various subclasses, orders, or species will
be given, since they differ considerably in the nature of the treatment
required, A number of general manuals are available, which contain
information of value to the technician (e.gf,, Ogura 1938, Verdoorn
1938).
Stelar Types. —The ferns have played a prominent part in the develop-
ment of the stelar theory. On the supposition that examples which
will illustrate each of the several types of steles in the stems and rhizomes
(G, M. Smith 1938) might be required, the following list has been
prepared.
Haplostele: Lycopodium cernuum (mixed type), Selaginella kraussiana,
Lygodium palmaium, Hymenophyllum.
Actinostele: Psilotum^ I socles, Lycopodium phlegmaria, L. inundatum.
Plectostele: Lycopodium adpressum, L. clavatum, L. vohibile, L. tri-
stachyum.
Polystele: Polypodium, Filix fragilis, PleopeUis simplex (probably
most species of the Polypodiaceae).
Amphiphloic solenostele: Dipteris (old plants), Marsilea vestiia,

Dicksonia punctiloba, Marattia (older stem), Pilularia,

Ectophloic solenostele: Ophioglossum (rare), Schizaea (older parts),
Botrychium virginianum (rhizome).
Dictyostele: Ophioglossum (uaual condition), Osmunda (older parts),
Ceratopteris (older stems), Botrychium virginianum (stem), Helrnin-
thostackys.
381
 —

382 SPECIAL METHODS FOR THE VARIOUS PHYLA

Polycyclic solenostele: not known in any North American pteridophyte.

Occurs in rhizome of Matonia pectinata.
Polycyclic dictyostele: Pteridium (older rhizome).
Mixed types (recapitulation) : Matonia pectinata, Cyathea, Gleichenia.

Fio. 77. Pailotum nudum: longitudinal section from near stem apex with two young
sporangia containing spore mother colls and enclosed by the bract. Fixed with formalin-
aceto-aloohol stained with safranin and fast green.
;

PSILOPHYTINAE

PSILOTALES

There isone family with two genera, PsUotum and Tmesipteris.

The latter occurs only in theSouth Pacific, but the former is widespread
in the tropics and subtropics and may even be found in fern collections
as it grows readily under cultivation. Anatomical material should be
obtainable from the supply concerns. The younger sporangia and stems
section easily, but the older sporangia and stems, as well as the rhizomes,
become greatly hardened, and embedded material must be soaked under
water for some time, whereupon it cuts without diflBculty. All stages
fix excellently with formalin-aceto-alcohol, but the dehydration and
 PTERIDOPHYTA 383

must be very gradual and prolonged; at least a day should be

infiltration
allowed for each stage of a closely graduated series of fluids. For some
unknown reason, most parts of the plants, especially the older stems and
rhizomes, have a marked tendency to shrink badly during the dehydration
process. Gametophytes are unobtainable. All parts give beautiful
staining effects with either safranin and fast green or a triple combination
(Fig. 77).

LYCOPODINAE

Lycopodia LE8

The order includes two living genera: the monospecific Phylloglossum

from Australasia, and the (‘.osmopolitan Lycopodium.
Species of Lycopodium o(;curring in the United States are usually
trailing or shrubby terrestrial plants, but a few are epiphytic with either
erect or pendulous sporophytes. The tropical species are mostly epi-
phytic and attain larger dimensions than do those from temperate
climates. Although Lycopodium should be present in most of the states,
it rarely occurs in abundance on the Pacific slopes and in the Southern

states, and colonies are rather sporadic and difficult to locate. If

definite locations arc not known, herbarium specimens in a local collection
should be examined and notes made of the exact loc^alities. In Canada
and the New England and Northern states it becomes as common as a
weed. Lycopodium is well represented in most fern collections, but the
species may be difficult to determine. Plants are easily obtained from
nurserymen. If all these sources fail, either preserved or embedded
material can always be procured from the botanical supply concerns.
The tropical species are less likely to give trouble during the microtoming
as they have softer stems and strobili.
—
Root. The root of Lycopodium has the typical differentiation char-
acteristic of vascular plants (Fig. 78). Fix with formalin- aceto-alcohol,
microtome at lO/x, and and fast green. The younger
stain with safranin
roots section readily, but if trouble is encountered in older roots from
the hardening of the xylem, they may be soaked under water for two or
three days.
Sections of older stems frequently show the adventitious roots, which
grow down through the cortex before emerging. Among the species
which have this feature are L. serratum and L. pithyoides.
—
Stem. Apical cells are not known to be present in the stem of
Lycopodium: instead, there is a mass of meristematic tissue. The
terminal portions of the stems may be detached about 5 to 8 mm. from
the apex, fixed in formalin-aceto-alcohol, embedded, and sectioned
longitudinally at lO/u, These will show the apical meristem, the develop-
384 SPECIAL METHODS FOR THE VARIOUS PHYLA

ment of the leaves, and the early differentiation into epidermis, cortex,
and stele. Serial transverse sections are also useful.
There great variation, even within the same species, in the organiza-
is

tion of the vascular tissues. A scries of preparations of stem sections

of as many species as could be obtained will form a collection of inesti-
mable value in any study of plant anatomy. Contrary to the common

Fig. 7S.- - Lycopodinm chivatum: cross section of a rhizopliore with exfoliated epidermis.
The heavily sclerized region of the inner cortex causes groat difficulty during microtoining.
Fixed with formalin-propiono-alcohol; stained with safranin and fast green.

impression, it is not at all difficult to microtome perfect sections of even

the oldest stems. The stems of all species become more or less hardened
during the dehydration; conselluently it is better to treat them all alike.
Cut the stems into portions not over 7 mm. in length, fix with formalin-
aceto-alcohol, dehydrate with tertiary butyl alcohol (being sure to use
the alcohol-paraffin mixture immediately preceding infiltration:
oil

this is the critical step),then after embedding, expose one end of each
piece of material under water for about a week for softer portions and
longer for the tougher pieces. Microtome at 12iu, and stain with safranin
 —

Fig. 79. Lycopodium davatum: cross section of vascular cylinder (ploctostele) of

mature stem. Fixed with formalin-propiono-alcohol; stained with safranin and fast
green.

Leaf. —The younger stages of leaf development are to be found in

sections of the stem apices. A single vein is present. If the leaves are
sufficiently closely appressed to the stem, the two may be worked up
386 SPECIAL METHODS FOR THE VARIOUS PHYLA

as a unit. If the leaves are over 1 cm. in length or are inserted at an

angle, they should be removed. Cut off short portions at both ends.
Embed bunches or layers. There should be no trouble with the
in
microtoming, even though the leaves of most species become rather
brittle.
Strobilus. — Strobili usually appear in June and mature in October.
The strobili in the various species, differ greatly in external form, but
there is little internal difference. Insofar as the technician is concerned,
the strobili of any one species are as useful as those of another. In
most of the species occurring in the United States, the fertile region is so
definitely delimited from the sterile it becomes a simple
portions that
matter to distinguish between the two. In some tropical species there
are alternating sterile and fertile zones, and in others sporangia are
found in the vegetative region subtended by foliage leaves.
In northern regions, L. davaturriy L. inundatum L. dendroideum,j

and L. ohscurum are the native species which have the most satisfactory
strobili. In the first two species the strobili become so long as they
approach maturity that they must be bivsected. The sporangia are
progressively developed from the base of the strobilus toward the apex,
and in most strobili not too far along in development a fine series of
stages are to be found.
For ordinary purposes formalin-aceto-alcohol fixes very well; but for
critical stages a fairly strong chrom-acetic or chrom-osmo-a(.*etic fluid
would be preferable. There may be a little plasmolysis at certain later
stages of spore development; to avoid this, individual sporangia will
have to be dissected out of the strobilus. If the sporophylls are large,
they may either be trimmed off with small scissors or slabs may be cut
from opposite sides of the strobilus. The strobili should not be bisected
longitudinally; if this is done, they will curl up tightly. The strobili
may be sectioned transversely if desired, but longitudinal sections are
preferable. In either case the sections may be cut at lOg for all stages.
When sections of strobili are mounted on the slides, it will generally
be observed that a milky substance runs out of them. This is due to
the fact that the spore mother cells and spores float in a viscous fluid;
the fluid is only partially coagulated during fixation, and the uncoagulated
portion becomes dissolved in the water on which the sections float.
The matter, fortunately, is of no moment since the spores are retained
in position and the dissolved matter does not become discolored by
stains.
Staining will occasionally prove to be troublesome. The earlier
stages and those involving maturation of the spores stain beautifully
with safranin and fast green. The intermediate stages are those that
give the most difficulty. Iron hematoxylin may be tried, but the
 PTERIDOPHYTA 387

differentiation must be carefully controlled, especially when the chro-

mosomes arc involved.
—
Gametophyte. Innumerable attempts to germinate the spores of
Lycopodium have been made, but most trials have been unsuccessful.
Two difficulties are attendant upon such attempts: (1) spores of certain
species do not germinate until after the lapse of from three to eight or
more years; (2) the young gametophytes, especially those which are
subterranean, cannot be carried beyond the 4- to 10-celled stage unless
they become infected with the characteristic symbiontic fungus. It
has been suggested that the first difficulty can be removed if some method,
possibly chemical, can be found to break the resting period. As for
the second difficulty, no one apparently has made a special study of the
fungus; at least, no information concerning its identity is available,
although it probably is an Ascomycete.
In some species the gametophyte is only partially embedded in the
substrate, the aerial portions (containing chlorophyll and bearing the
antheridia and archegonia. Some speci(*s have spores which germinate
immediately upon being shed, the sex organs are soon produced and the
young sporophyte is establish(cd the same season. No gametophytes
of this typ(‘ are apparently known which do not harbor a fungus.
The other type of gametophyte is subterranean, tuberous, and entirely
lacks chlorophyll. There are usually definite areas containing the fungal
symbiont. Spores producing this type of gametophyte require several
years for geermination, and the latter develop very slowly, sometimes
taking 16 years or longer for the dcwelopment of the sex organs. The
gametophytes are buried at depths of from 0.5 to 6 cm. Their probable
presence can be inferred by the finding of a few very young sporophytes.
They are generally found at some distance from the parent plants and
usually grow in the shade (Degener 1924; Spessard 1917, 1922; Stokey
and Starr 1924).
The majority of the localities in the United States where gametophytes
have been found are in Massachusetts, but others have been discovered
in Michigan and New Jersey. These are all regions where rains occur
during the summer. Whenever the gametophytes are found, they
usually occur in abundance.
Formaliii-aceto-alcohol has given perfect fixation of the aerial type
of gametophyte. Microtoming should be in the ver^cal longitudinal
plane at 11^. If the sex organs are at all present, they usually occur
in great abundance; the archegonia more so than the antheridia. Iron
hematoxylin is the only stain which has afforded satisfactory results
with the sex organs; fast green may be used as a counterstain, but orange
G is better on some species. For the embryo and young sporophyte a
triple combination might be tried.
388 SPECIAL METHODS FOR THE VARIOUS PHYLA

Selaginellales

The order includes one living genus, Selaginella, which differs from
Lycopodium in that it is heterosporous. The North American species
of Selaginella are found in habitats ranging from damp shade to xero-
phytic conditions. Some species are terrestrial and others epiphytic;
most of those occurring in the United States belong to the former group.
Selaginella is commonly planted in greenhouses as a ground cover
and in pots for table decoration. S. kraussiana is the species usually
employed as a ground cover. Tropical species are frequently to be
found in greenhouse fern collections, and these are the best source of
material, especially of the strobili, since they are less refractory than the
native species. The xerophytic species should be avoided, except for
special purposes.
—
Root. The roots are very small, are adventitious, and are usually
borne at the ends of the rhizophores. They present no technical diffi-
culties.
Rhizophore. —This structure is generally considered to be a part
of the stem, but its anatomical structure is similar to that of a root. It
is easy to manipulate, but occasionally becomes so hard that some soaking

of the embedded material under water is requin^d.

—
Stem. The stems of most species become too hard and brittle
to be sectioned without the expenditure of the utmost ingenuity. The
stem tips, whose apex may be occupied by either a mass of meristematic
tissue or a single apical cell, according to the species, gives no trouble, and
may be treated as described for the corresponding parts of Lycopodium.
As in Lycopodium, the tropical species of Selaginella have stems which
are easier to microtome. S. kraussiana is the easiest of all, but the
steles are of relatively small extent.
Stems of some species may be easier to section in celloidin, but con-
siderable success has been had after dehydration with tertiary butyl
alcohol and prolonged soaking in water. Fix with formalin-aceto-
alcohol or a strong chrom-acetic, microtome at ll/x, and stain with
safranin and anilin blue.
In the stems there is a space of considerable extent between the steles
and the inner face of the cortex; the space is bridged by trabeculae which
are easy to demon|trate in some species but not in others.
Leaf. —The leaves are so small and so appressed to, or parallel with,
the stem that sections of at least the younger stems show their structure
adequately.
Strobilus. —The is one of the most difficult
strobilus of Selaginella
microtome. Up to the time that the walls of the
of all plant organs to
megaspores become thickened in the younger strobili, the latter section
 PTERIDOPHYTA 389

without diflSculty, but after that stage the megaspores cannot be cut,
except in rare instances, without shattering. A few technicians claim
that embedding in celloidin removes the difficulty, but others have had
no success with this procedure. The best that can be done is to fix in
formalin-aceto-alcohol or a medium chrom-osmo-acetic fluid, taking
care to exhaust all air; to dehydrate over a considerable period (allowing
each change to react for at least a week) by the tertiary butyl alcohol
method; to leave in the paraffin oven for several weeks or even months;
and to embed in a hard Parlax or a paraffin melting at 58°C. After
embedding, the exposed material must be soaked under water for months
(see also Slagg 1932). The strobili of an unidentified species from
Jamaica sectioned easily after 45 days^ immersion; S. douglasii from
Oregon required 8 months’ immersion, but even then there was some
shattering. All methods designed to soften the spore coats by chemical
means are worthless.
In most species microsporangia and megasporangia are borne,
same strobilus, but in others only one
usually rather irregularly, in the
kind of sporangia are to be found in a strobilus. The distribution of the
sporangia follows four types of arrangement but occasional exceptions
may be encountered (Mitchell 1910): (1) a single large basal mega-
sporangium subtended by an especially large leaf; (2) several basal
megasporangia with microsporangia above; (3) strobili wholly either
megasporangiate or mic^rosporangiate; (4) megasporangia and micro-
sporangia disposed indiscriminately.
The strobili should be microtomed longitudinally. The claim has
been made that, in order to cut through the stalks of the vsporangia,
the plane of sectioning should be diagonal, from corner to corner; the
strobilus is square when viewed in transverse section. This may be
true of some species, but it does not appear to be so in others. The
strobili of some species are curved; consequently they can only be sec-
tioned parallel to either flat side. The thickness for general purposes
may be from 10 to 12iLC. It is difficult to keep the sections on the slides;
hence they should first be coated with a thin layer of celloidin and taken
through carbol-xylol for deparaffining, thence to 95% alcohol, and to
the staining. For the stages up to the rounding up of the spore mother
cells, iron hematoxylin is preferable; thereafter either a triple combination

or safranin and fast green will be preferable.

Gainetoph3rte. — Both microspores and megaspores commence develop-
ment into the respective gametophytes before being shed from the
sporangia. In most species development is not wholly completed at
the time of shedding. The stage of development at the time of shedding
of the megasporangia is far more variable than that of the microspores.
If the spores are not sufficiently advanced before shedding, it is a simple
390 SPECIAL METHODS FOR THE VARIOUS PHYLA

matter to sow the spores on filter paper or plaster of Paris blocks placed
in Petri dishes and moistened with distilled water or a weak nutrient
solution. After several days, spores may be removed daily and fixed.
It should also be possible to obtain sporelings in the same manner.
Sections to show the development of the androcytes should be cut at
4/i; those for the archegonia and embryos at 8ju. Safranin and fast green
constitute an excellent stain combination, but in the megagametophytes
the contents acquire a very intense color from the safranin. Triple
cell
combinations may be used, but tend toward gaudiness. Young spore-
lings are easily mounted entire, stained with Harris^ hematoxylin and
fast green, and dehydrated by a gradual dioxan or hygrobutol method.

ISOETALES
The single living genus,
IsoeteSy is essentially aquatic, and the locations
where might be expected can be found in any flora or in monographs
it

(Pfeiffer 1922). It is common over the United States but may be easily
overlooked because of its strong resemblance to grasses and sedges.
The plants are herbaceous, consisting of ligulate leaves borne upon a
corm-like stem, which is so short as to be externally indiscernible, and
roots borne upon a massive rhizophore below the stem.
—
Root. The roots may be snipped off, fixed with formalin-aceto-
alcohol, microtomed transversely at 12iu, and then stained with safranin
and fast green. Sections invariably look as if the roots were badly
disorganized, but this is the natural condition. The root tips are not
easy to collect; longitudinal sections will reveal the apical meristem.
—
Stem and Rhizophore. The two should be worked up together, since
it is useless to attempt to separate them. Take plants which have just
begun to produce sporangia, trim off the roots 1 or 2 mm. from the rhizo-
phore, and cut through the leaves just above the apex of the stem (the
apex has the shape of an inverted cone). Provided they are not too
large, even older plants may be used. Fix in formalin-propiono-alcohol.
In some species the stem is bilobate; in others it is trilobate (Fig. 80).
To show the ramifications of the interesting vascular system, serial
sections, both longitudinal and transverse, should be cut and all mounted
in order. For this purpose microtome at 15 or 16/x. Safranin and fast
green are the most adequate stain combination.
Leaf. —
Sections of young plants, prepared as noted in the preceding
paragraph, will show the structure of the leaves. However, there is no
reason why portions of the leaves cannot be removed from the plants and
run up independently.
—
The outer leaves on the stock are sterile, then come the
Sporangia.
megasporophylls, next the microsporophylls, and near the center are
the younger leaves with the youngest stages in sporangia development.
 PTERIDOPHYTA 391

However, plants will be found in which the older sporangia contain only
either megasporophylls or microsporophylls. The earlier ontogenetic
development is alike in both types.
Sections of young plants for the stem and rhizophore are very likely
to show the sporangia also. The material intended for demonstration

Fig. 80 . —laoetea nuttallii: cross section through middle portion of corm-like stem, show-
ing ramifications of the vascular system. Fixed with formaiin-propiono-alcohol; stained
with safranin and fast green.

of the sporangia should be prepared as described for the stem, but for
the earlier stages the sections should not be over lOju in thickness. While
safranin affords an excellent basic stain, iron hematoxylin may occa-
sionally prove to be more satisfactory. Unlike most aquatic plants,
Isoetes is easy to stain.
The megaspores, unlike those of Selaginella, give no trouble during
the microtoming; the real difficulty is to fix them properly. A fixative
of strong penetrating power apparently is required, and formalin-ace to-
392 SPECIAL METHODS FOR THE VARIOUS PHYLA

alcohol appears to be as good as an 3rthing.A triple combination or iron

hematoxylin plus orange G give good results.
—
Gametophyte. Germination of the spores begins immediately after
liberation from the sporangia, provided suitable conditions are present.
However, large numbers of the spores are incapable of germination; in
the case of the megaspores, one will have to undertake the tedious job
of separating the sound from the shriveled ones previous to fixation.
Bring portions of the with corms, megaspores, and young plants
soil

to the laboratory, place in suitable containers, and keep well watered.

It appears probable that the megaspores at least remain viable for about
a year. Transformation of the microspores into antherozoids occupies
about a week; the period elapsing from germination of the megaspore
to the well-developed embryo stage is about a month. To secure a
series of developmental stages, collections of germinating spores should
be made at frequent intervals during the respective periods. The
microgametophytes should be sectioned at fiju; the megagamctophytes
at 6 to 8/x. It is impossible to orient the megagametophytes and younger
embryos in the proper position for sectioning; hence one can only cut
blindly, but the number of usable se(^tions is surprisingly high.

EQUISETINAE
Equisetum, the only living genus of the Equisetinae, is well known to
every botanist, but there appears to be a general impression that it is
one of exasperating difiiculty to the tecdmician because the plants ani
encrusted with silicon. It seems not to be realized that the more interest-
ing and critical parts of the plants are not encrusted, or at most only
superficially so, and that microtoming of these parts is actually very
easy. The only serious difficulty is that all parts have an exceptionally
weak affinity for basic stains. Equisetum, on the whole, is a genus well
worth the technician's effort.

Eqitisetales

Equisetum a nearly cosmopolitan genus and affects a variety of

is

habitats. Most damp, shaded situations,

of the species prefer aquatic or
but others are almost xerophytic. Ponds, marshes, river and creek beds,
and shady banks are the best localities from which to collect material.
Some species are sporadic in occurrence, frequently forming very small
stands, but others form extensive mats and crowd out all other vegetation.
The aerial branches of a very few species are perennial, but those of
most species die down at the end of the growing season. The one
persistent portion of the sporophyte is the much branched subterranean
rhizome, which is usually a short distance below the surface but may
 PTERIDOPHYTA 393

descend to more than a meter. This rhizome in many species is the

only active means by which the plants spread over new territory.
—
Root. The adventitious roots are borne at the nodes of the rhizome
and in most species function for only one season. Therefore, roots
which plainly appear to have been formed recently should be collected;
the old roots are shriveled and not worth collecting. Root hairs are
abundant and conspicuous in the finished preparations. There is an
* apical cell at the tip of the root. The roots are not silicified and are
easily sectioned. Fix the tips in a chrom-aeetic or chrom-osmo-acetic
fluid and the older portions in formal! n-aceto-alcohol. Microtome the
former longitudinally at S/Lt, the latter transversely at 12 ^, Staining is
usually fine with a triple combination.
It is sometimes possibles to obtain longitudinal sections of the origin
of the roots by sectioning young rhizome nodes transversely.
—
Rhizome. There is a large apical cell, with three cutting faces, at
the tip of each rhizome. The apical cell of the rhizome is generally in
better condition, its divisions are easier to follow out, and it stains
better than is the case with the apical cell of the vegetative branch.
The; latter very apt to have the external wall indented, and it is difficult
is

to section in a satisfactory plane.

it The lateral apical cells of th(‘
vegetative stem are, in fac.t, usually in a better condition for study than
is the terminal apical cell. The tip of the rhizome is massive in many
species; slabs from opposite sides should therefore be cut off, and it would
be well to examine the sections under the microscope since only those
very close to, or containing, the api(‘al (*ell are worth mounting. Fix
in chrom-acetic or formalin-aceto-alcohol and microtome at 8/x.
The structure of the subterranean and aerial portions of the sporo-
phyte is nearly identical. The rhizomes arc only slightly silicified;
consequently if the aerial portion is too difficult to sec^tion, the rhizome
may be substituted. Otherwise, it would be preferable to utilize the
aerial branches. Fix portions of the rhizome internodes in formalin-
aceto-alcohol, section at 14g transversely, and stain with safranin and
fast green.
Aerial Branches. —These are of two types in most of the species.
The fertile when present,
branches, are flabby, not much silicified, and
each is terminated by a strobilus. The sterile, or vegetative, shoots
are formed and appear aboveground after the fertile branches have, as a
rule, completed growth.
In the other type the fertile and vegetative branches are combined
into one organ. The strobili are borne at the termini of the lateral
branches. In addition, there are one or two evergreen perennial species.
The vegetative shoots differ in the amount of silicification according
to the species. Some are slightly, others heavily encrusted. The
394 SPECIAL METHODS FOB THE VARIOUS PHYLA

former can generally be sectioned, after the blocks have been soaked
under water for several weeks, without evident tearing, but the heavily
encrusted types must be treated with hydrofluoric acid. Fix with for-
malin-aceto-alcohol, wash with two changes of 70% alcohol, then treat
with 50% hydrofluoric acid in 70% alcohol
for two days, finally washing thoroughly
with 70% alcohol, and proceeding to the
dehydration. The dehydration and infil-
tration should be very thorough. Safranin
and fast green are as good a stain combina-
tion as any; triple combinations have not
been satisfactory.
There is an apical cell at the terminus of
each branch. That at the end of the main
axis is the one which is meant when the
apical ceir’ is mentioned in most texts.
This cell invariably looks better in drawings
than it does in the actual preparation, as it

is rare that this cell is either perfectly fixed

or sectioned in exactly the right plane.
The external wall is generally indented.
The apical cell of the rhizome, as was men-
tioned above, is better organized and is

easier to fix. The apical cells of the pri-

mordial branches are just as useful as
the terminal apical cell, although few
botany instructors seem to be willing to
admit the fact. E, arvense is one of the
best species for apical cells. Stem tips
that have just emerged from the ground
Fig. 81 . —
Equisetum arveriae: may be cut off about 6 to 8 mm. below
median longitudinal section of a
young strobilus with spore mother the tip, fixed with a medium chrom-
cells. Fixed with formalin-aceto- acetic, and microtomed longitudinally at
alcohol; stained with iron hema-
toxylin and fast green. 8 to 10/x. Stain with safranin and fast
green.
Leaf. The leaves are small, chaffy or scale-like, and more or less
united to form a sheath around the base of the internode. In most
species they are not worth the trouble required to section them, since
they are heavily encrusted with silicon.
Strobilus. —The young strobilus is first recognizable at the apices
of the subterranean rhizomes in June or July; the sporogenous cells
are differentiated by the middle
August (Fig. 81); meiosis occurs late
of
in the same month, but the spores are not naturally shed until the follow-
 PTERIDOPHYTA 395

ing March or April. However, if strobili are brought into the laboratory
and kept in a warm place, they can be induced to shed their spores any
time after mid-September. In those species in which the strobili are
borne on the termini of vegetative shoots, they appear in early spring,
develop rapidly, and the spores are ready for shedding by mid-spring.
The strobili of all species are easily fixed and sectioned at all stages
of development. In those species in which the strobili originate on the
rhizomes, dig up the latter carefully, wash thoroughly under running
water, and cut off the rhizome apices. If the particular colony is known
to produce strobili, the latter arc fairly certain to be present at the
rhizome apices. Fix the earlier stages in Navashin’s fluid or in a medium
chrom-acetic ;
for the later stag(\s following ineiosis, formalin-propiono-
alcohol is good.
Section at 10 to 12ju. The younger stages should be
microtomed longitudinally, but transverse sections may also be made
of the later stages to show the relation of the sporangiophorc to th(i
central axis. For all stages up to meiosis, stain with iron hematoxylin
but omit counterstaining. The later stages are difficult to stain ade-
quately; a quadruple combination may be attempted.
Gametophyte. —The mature spores with their characteristic elaters
are easily mounted The simplest method
entire in their natural colors.
is mix a small quantity in a drop of melted glycerin jelly on a slip
to
and to add a coverslip carefully so as not to let any of the medium extend
beyond the periphery of the coverslip, then ring with Duco or other
ringing material. More substantial mounts may be made by placing
fresh spores in 95% ethyl alcohol for 15 minutes, then pour off the alcohol,
replace with Euparal or Diaphane diluted several times with 95%
alcohol, and place in a cool part of the paraffin oven. The alcohol
evaporates quickly; with a pipette draw^ up a small quantity of the
mixture, put 1 drop on a slide, azid add a circular coverslip 18 mm. in
diameter. Avoid jarring or shaking the spores, else the elaters becomes
detached. By either method the preparations will keep for at least
five years.
The spores of some species retain their viability for only 1 hour
after being shed, while those of others may remain viable for a few days.
Prothallia are easily raised artificially, provided fresh spores, secured
from about ready to discharge, are used. Boil shredded
strobili that are
sphagnum for about 1 hour, then pack tightly in sterilized moist chambers,
of a suitable type, to the depth of 1 or 2 inches. Press out the surplus
water. Cover the receptacles until cool, then sow^ the spores on the
surface of the sphagnum. Keep the moist chamber covered, and place
in a north window. No special precautions to prevent the appearance
need be taken, since
of fungi or algae it is possible to control these organ-
isms with potassium permanganate. If they should appear, add enough
 —

396 SPECIAL METHODS FOR THE VARIOUS PHYLA

crystals of the permanganate to distilled water to give the latter a

purplish color. Pour over the affected areas, then drain off all excess
solution. The solution may be allowed to remain for as long as 10
minutes in cases of bad infection, as the permanganate appears to benefit
rather than to harm the cultures. If only a few algae are present and
they do not seem to crowd the cultures, they may be allowed to remain.
It will be approximately a month after germination when the first sex

Fig. 82. Equisetum debile: longitudinal section of young embryo. Fixed with forrnalin-
aceto-alcohol; stained with safranin and fast green.

organs (usually the antheridia) are recognizable. The cultures can be

kept growing for years.
Equisetum arvense is the species whose spores are most easily ger-
minated in the above manner. The gametophytes are more like those
of the Filicinaeand whole mounts may be prepared in exactly the same
manner page 407). Other species produce cushion-like prothallia
(see
that are too thick for whole mounts. E. laevigatum is an example of
such species, but E. debile from India is better known. These massive
prothallia must be embedded and sectioned. They usually grow in
mud* It is impossible to get rid of all the enveloping mud; if the mud
 PTERIDOPHYTA 397

does not contain grit that might nick the microtome knife, do not bother
about it, but proceed to embed prothallus, mud and all. Fix with
formalin-aceto-alcohol or a medium chrom-acetic fluid, and microtome
in the vertical plane at lO/x. If the gametophytes are in good condition

(which is not always the case), a fine series of developmental stages of

antheridia, archegonia, and embryos is easily secured (Fig. 82). The
question whether the gametophytes of Equisetum arc homothallic or
heterothallic has not been settled; in any event, most of the gametophytes
in the majority of species will be found to be homothallic. In those
with the cushion type of gametophyte, the nature
species, like E. debt'Zc,
of each gametophyte can be determined only by sectioning. It will
sometimes be necessary to search very carefully for the archegonia.
Even if the gametophytes are sectioned blindly in the vertical plane, a
very large number of embryos and young sporophytes can nevertheless
be secured. All stages are difficult to stain. One might try placing
the slides with sections attached, after bringing them down to water,
in 1% aqueous chromic acid for 1 hour, rinsing thoroughly with water,
and then staining with safranin and fast green. It was nearly impossible
to differentiate the hematoxylins on all materials which were available
to the writer.
Gametophytes of Equisetum have been found in many localities,
most of those in this country being in Nebraska.

FILICINAE
The American ferns have been rather thoroughly studied taxonomi-
cally and can be readily identified from popular manuals, floras, and
special monographs. The ferns are widely distributed over the country,
but most of the species are included in the Polypodiaceae. There are
numerous dealers in native plants who advertise their local species; if
mature specimens are ordered, they are usually in a condition for immedi-
ate fixation, except occasionally for the sporangia. All the botanical
and most public gardens possess extensive fern collections; if the head
gardeners are approached, they are almost always willing to permit one
to collect stems, leaves, and sporangia. Material of many tropical
species is purchasable from the botanical supply concerns. Altogether,
it isnot a difficult matter for the technician to obtain, from one or another
of the sources just mentioned, suitable material of any desired type.
One is likely, however, to encounter trouble in securing stages of
sporangial development. It will be necessary to learn something of the
life history of the species concerned, particularly with regard to the

time of year when the sporangia are first formed. This period differs
considerably even within the same family. Those species which have
the sori at the periphery or on the abaxial side of the vegetative leaves
398 SPECIAL METHODS FOR THE VARIOUS PHYLA

commonly produce sporangia intermittently or continuously throughout

when in.cultivation indoors. Those which produce the
the year, especially
sporangia on special fertile spikes (as in Struthiopteris) usually have
the sporangia already formed as these spikes begin to appear from the
crown. Sporangia may develop slowly or rapidly, and sometimes may
even turn out to be sterile. Different species in the same genus may be
more suitable from both technical and teaching standpoints and more
useful than others (c.gf., Polypodium lineare^ a Hawaiian species, is the
best of all the numerous species in the genus for the origin and develop-
ment of the sporangia).
As has already been intimated, the ferns belonging to the Lepto-
sporangiatae are very difficult technically. The stems of most species
are rigid; the leaves are tough and leathery; the rhizomes in many species
are massive and surrounded by heavily sclerized leaf bases; but if the
proper precautions are observed, it is entirely possible to cut perfect
sections as thin as IQp of even the most formidable specimens. However,
material that has been kept in plain alcohol for years is worthless as the
various tissues have become almost completely disorganized.
Certain precautions need to be observed when collecting fern material
in the field. If roots and rhizomes of purely terrestrial forms are to b(‘
collected, the plants should first be loosened all around by means of a
spading fork. Any roots or rhizomes that appear to be too long to be
dug out in their entirety should be cut off as far from the base as possible.
Avoid all pulling or stretching, as it is very easy to cause the vascular
bundles to become separated from the surrounding tissues. Each clump
after removal from the soil should be thoroughly washed in a near-by
stream or under a water faucet, whereupon the desired root or rhizome
portions may be cut off with a large, sharp scalpel. If they are not to be

fixed immediately, they may be wrapped in damp newspapers for trans-

portation to the laboratory. Epiphytic species are easy to manipulate,
it merely being necessary to free them of adhering mosses, liverworts, etc.

Eusporangiatae

The group is so designated because the sporangia develop from more

than a single cell. The antheridia are embedded in the gametophyte.

Ophioglossales
The order includes three genera of perennial herbs. One genus,
Helminthostachysy ranges from India through Malayasia north to Japan.
Ophioglossum and Botrychium are world-wide in distribution, but are
more common in the Northeastern states than west of the Rockies in
the United States (R. T. Clausen 1938). Both occur usually as scattered
patches consisting of few plants, although in some especially favorable
 PTERIDOPHYTA 390

localities extensive stands are known. The plants are found in a variety
of habitats —moist meadows, shady fields, grassy thickets, rich swamp-
lands, and even on sandy beaches. If not already known, locations

c D
—
Fia. 83, Roots of the Ophioglossales, in transverse section, showing nature of vascular
cylinder and comparatively large cortex: A, Ophioglossum vtilgatum, B, O. pendulum; C, O.
reticulcUum, JO, Botrychium virgtnianum. Fixed with formal! n-aceto-alcohol stained with
;

safranin and fast green.

where material can be obtained are difficult to find; it would be well to

check herbarium specimens whose labels bear precise collection data.
If material cannot be collected in the field, the botanical supply concerns
are usually well supplied with excellent material.
The genera will be treated together in the following discussion, since
technical methods apply equally to all.
400 SPECIAL METHODS FOR THE VARIOUS PHYLA

Root. —^The adventitious roots are comparatively large, somewhat

fleshy in a few species, and usually gorged with starch (Fig. 83). Cut
them into portions 5 to 8 mm. in length, fix with formalin-aceto-alcohol,
microtome transversely at 12)u, and stain with safranin and fast green
or a quadruple combination. The material rarely becomes so hardened
as to require soaking; in any event, the blocks should not remain under
water for longer than a day.
There are no root hairs.
—
Stem. The stem is short, subterranean, and upright, although it is
dorsi ventral in some tropical epiphytic species. In some species of
Ophioglossum the stem is so short that one can only trim off the roots and
petioles and run up the whole mass. In old plants of Botrychium the
stem may become so long that it has been called a ^‘rhizome.”
The stem is easily manipulated, even when quite old. Section trans-
versely at 12/4, and stain with safranin and fast green. If necessary to
soak, do not leave the material under water after it becomes whitish-
opaque, or it cannot be cut at all; two days^ immersion is long enough.
The stem apex is particularly worth attention. There one will find
the buds of the leaves which will appear successively for the next three
(or four) years. The buds of the tropical species are naked; those of
the temperate zone are closely covered with hairs which may occasionally
give trouble. There is a single apical cell, but it is extremely difficult
to locate it and to be certain of its identity. In removing the apices
from the plants, cut them off carefully so as not to cut through th(‘ petiole
of the oldest one, lest it be lost. Wash the buds very thoroughly,
preferably under a small jet from a thick rubber tube attached to a
faucet, as the buds are full of fine grit. Fix with formalin-aceto-alcohol,
embed with one flat side down, and section across the flat face at lO/i.
Examine the sections before mounting, and discard those that do not go
through all three buds. Stain preferably with a quadruple combination.
In at least the oldest bud it will be possible to find the origin of the
fertile spike.
—
Leaf and Petiole. Both are very easy from the technical standpoint
and general methods are applicable.
Fertile Spike. —To obtain the very youngest stages, the plants
must be carefully watched. In early spring the plants may be dug up
and the leaves searched for the earliest stages, and, after this, collections
may be made at regular, frequent intervals in order to secure a complete
series of developmental stages. The spikes are readily fixed, sectioned,
and stained at all stages. Both transverse and longitudinal sections
should be prepared; the optimum thickness is 10//. Safranin and fast
green give a brilliant stain, but perhaps iron hematoxylin may be preferred
for the stages up to meiosis. The clusters of sporangia in Botrychium
 PTERIDOPHYTA 401

should be reduced to smaller proportions as soon as they become large

enough.
—
Gametophsrte. In both Ophioglossum and Botrychium gametophytc s
have frequently been found (R. T. Clausen 1938). They are usually
subterranean in habit but may be located so near the surface that small
lobes appear aboveground and develop chlorophyll. No one has suc-
ceeded in artificially gametophytes from spores beyond the
raising
13-celled stage. The gametophytes,
if one should be so fortunate as

to find them, may be fixed in a mtidium chrom-a(;etic or in formalin-

aceto-alcohol. If dorsiventrality can be distinguished, microtome in
the vertical longitudinal plane at lOju; otherwise try transverse sf'ctioning.
Staining is better with iron hematoxylin than with coal-tar dyes for th(‘,

sex organs, but for general purposes a quadruple stain may be attempted.

Marattiales
All species are tropical; material consequently is hard to obtain.
Leaves with synangia attached should be available from the supply
(‘.oncerns, but other organs probably are not. In any event, Marattio,
the principal genus, is one of the easiest of all Pteridophyta with which
the tec^hnician might work. Despite their large size, the stems s(H’tion
with surprising ease in paraffin, but the mature synangia may require
some softening under water. If material of Marattia is unavailable,
(iither Danaea or Angiopteris will serve equally well. The latter genus
might be found cultivated in conservatories or large fern collections.
The leaves and sporangia of thc^se two genera are more rigid than those
of Marattia and will require softening under water for a week or longer.
Some care must also be taken to avoid overstaining.

Leptosporangiatae
The plants which are more commonly understood when the term
^4*erns^’ ismentioned are included in the Leptosporangiatae.
The Leptosporangiatae are very widely distributed, but the majority
of the species are tropical. They range in size from the great tree ferns
of the tropics and subtropics to the tiny floating Azolla; from the fragile
filmy ferns'^ (Trichomanes and Hymenophyllum) of humid rain forests
to the tough and leathery xerophytic species of the deserts. In the
United States, however, most of the species are found in places where thc^
soil does not dry out completely during the rainless seasons, as in moist

woods, the shady sides of canyons and ravines, in fresh-water swamps

and marshes, and in deforested lands. Interesting species are to be
found growing under ledges and between boulders on high mountains.
Aquatic species are not so widely distributed: there is one species of
Azolla on the East coast and another in the West; Marsilea is commoner
402 SPECIAL METHODS FOR THE VARIOUS PHYLA

but less readily found, and Ceratopteris occurs in the regions immediately
adjacent to the Gulf of Mexico. Such a wealth of material is available
to the technician that the question becomes one of making a satisfactory
choice of the species with which to work. Each species, as a rule, is
more favorable in some respects than in others, but all have their indivi-
dual disadvantages. That is, each has an inherent technical problem
more or less peculiar to itself. These problems will be specifically
mentioned in the appropriate place in the following discussion.
The only characters which are constant in distinguishing one family
from another are those of sori and sporangia (G. M. Smith 1938, Ver-
doorn 1938). Almost every general and strictly local flora has a section
devoted to the Leptosporangiatae, and in addition there are several
popular manuals, all of which will be useful in identifying local species.
Warning should be given, however, that hybridization is rampant in
certain genera, and many species moreover are decidedly polymorphic.
Methods common to all Leptosporangiatae will be described first,
and these will be followed by fuller discussions under each family.
—
Root. The roots in general offer no special technical difficulties,
but those of some species are wiry (Filix) and consequently require
soaking under water. For the root tip and its prominent apical cell,
fix with a medium chrom-acetic, section some tips longitudinally and

others transversely at lO/x, and stain preferably with safranin and fast
green. For older roots, use formalin-aceto-alcohol, and microtomes
transversely at 12^.
Rhizome and Stem. —^The rhizomes and stems of certain speci(^s
offer very difficult technical problems, but the extreme beauty and
great utility of these structures are more than worth all the trouble
involved. Remove subterranean rhizomes carefully and wash free of
adhering soil. The stems of some species are covered with old leaf
bases, which should be trimmed away as much as possible since they
interfere with smooth sectioning. Any rhizomes or stems that can be
cut across with a sharp scalpel can be microtomed after embedding,
even if the portions should become hardened during the dehydration
and infiltration. The portions should not be over 1 cm. in length.
The rhizomes of certain species (e.gr., Pteridium) are filled with a mucilag-
inous substance whose dissolution during dehydration will cause more
or less plasmolysis. Fix with formalin-aceto-alcohol, embed in a hard
paraffin, and microtome transversely (and also longitudinally if desired)
at 12/x. All hard pieces qf material should be well softened under water,
but any that are congested with starch should not be left in the water
too long, otherwise hydrolysis of the starch occurs, and the material
cannot be sectioned. Safranin and fast green afford superb differentia-
tion; Harris' hematoxylin or crystal violet may be substituted for the
 ,

PTERIDOPHYTA 403

fast green. Triple combinations are usually too gaudy, but quadruple
methods are excellent.
The stems and rhizomesof the Leptosporangiatae are exceptionally
valuable in studies on the various types of steles to be found among
vascular plants. The stelar type in the young stem is frequently very
different from what is to be found in much older stems; even in the same
stem it may differ at various levels. In some species of Osmunda, for
(example, the portion of the stem first developed may be protostelic, but
the portions later developed may be dictyostelic. It is therefore neces-
sary to take into account the age of the plant when th(‘ material was
collected and also the portion of the stem that was selected.
Leaf. —In those species in which the leaves are all one type, it is the
common practice to make sections of the leaves and sporangia simul-
taneously. Microtoming is usually in such a direction (transverse or
longitudinal) as to go through the largest possible numb(‘r of sori. Where
the leaves are dimorphi(;, the structure of the fertile leaf is sufficiently
well shown in sections primarily intended for the sporangia, but the
sterile leaves must be worked up independently. The vegetative leaves
differ considerably in structure, but particularly in the extent to which
the cell contents are revealed. Practically nothing can be found in
mature leaves of Pteridium and Adiantum, as it is almost impossible
to stain them differentially; those of Polypodium or Cyaihea are far less
troublesome. The leaves of the filmy ferns an' only one (*ell in thickness.
To
obtain the earliest stages in leaf development, when the activity
of the apical cells is especially prominent, select young, tightly coiled
fronds which have just emerged from the crown; those which are about
1.5 to 1.8 cm in diameter are excellent. Piihyrogramma is one of the
best genera for the purpose. Microtome across one flat side of the
(‘ircinate frond at I2u- Safranin and fast green are excellent, but care
should be taken not to overstain with the basic stain sin(*e it is sometimes
not easy to differentiate.
—
Sporangia. The sporangia are as a rule borne in sori of different
types arranged in various positions on the leaf blade. In some sori
the sporangia develop simultaneously, in others they are formed in
basipetal succession, and in still others they are produced in an irregular
sequence (Fig. 84). When the sori are borne on the abaxial sides of the
leaves, at the periphery of the leaf, or at its apex (as in Hymenophyllum)
it is a simple matter to watch the plants for the first appearance of the

sori, and a series of developmental stages is easily secured. In some

of the species in which the sporangia develop simultaneously, leaves
bearing the earliest sori up to the mature spores can be found on the
same plant. Species with special fertile spikes are more difficult to find
with the earliest stages: the sporangia are already formed when the
 —

404 SPECIAL METHODS FOR THE VARIOUS PHYLA

coiled fronds oniergo from the crown of the plant and develop rapidly
as th(i coils unroll.

For sections of the leaves and sori, fix portions with formalin-aceto-
alcohol, microtome* in the plane which permits passing through as many
sori as possible (scatt(*red as in Cyrtomium)^ or transvc'rsely (arranged
in a linear series as in Y/oodwardia), or longitudinally (when arranged in
widely spatted rovrs, as in Polypodium) at 11 m- If the leaves have become
brittle or if the sj)orangia contain mature spores, soaking under water
becomes necessary. Safranin and fast green or a quadruple combination
stain adequately.

Fig. 84. Polypodium lineare: crosB section through a very young sorus showing indusia
and origin of the sporangia. Fixed with fonnalin-aoeto-alcohol; stained with bafranin
and fast green.

In some of the Filicales it is not so easy to produce slides showing

meiosis as it is in certain other species. Most of the Filicales have rather
high chromosome* numbers. Osmunda is the most suitable genus as
the spore mother cells are large, the chromosomes comparatively few
(n-22), large, and widely spaced. Stages in meiosis extend over con-
siderable periods: in the Northern states the spore mother cells appear
in the autumn in 0. claytoniana and 0, cinnamomeay and meiosis takes
place during the last half of April; in 0. regalis the stages appear con-
siderably later, meiosis occurring in early May. Fix material with a
strong chrom-acetic, microtome at 10m, and stain with iron hematoxylin
(which is not always so satisfactory as it should be) or with safranin and
fast green.
Sori scraped off the leaves may be mounted entire and give good
views of the sporangia. Species should be selected to shqw the various
 : —

PTERIDOPHYTA 405

types of annulus: rudimentary, equatorial, vertical, apical, and oblique.

Simply place the sori in hygrobutol for several hours, give a change, then
infiltrate with weak balsam, and evaporate down to a mounting con-
sistency. A more refined method would be to fix first in formalin-aceto-
alcohol, replacing this gradually with hygrobutol, and infiltrating with
balsam. not required. Small portions of leaves bearing son
Staining is

may also be mounted

entire, with the sori and sporangia remaining in
their natural positions. Fix with f ormalin-aceto-alcohol staining is ;

unnecessary, but if one is desired, wash out the fixative with 85% alcohol
and apply a strong solution of fast green in equal parts of methyl cello-
solve and 95% alcohol for several hours or overnight, then wash out with
two or three changes of equal parts of the two fluids just mentioned, and
infiltrate with highly diluted balsam, which should be brought to a
(;oucentrated condition quickly. Mount the leaf portions, sori side
up, in culture slides. The leaves will become very brittle in a short
time —no method that circumvents annoying propensity
this is known
therefore they should be mounted as soon as possible.
Gametophytes. —Spores are easily grown
of all Filicales in artificial
culture. The gametophytes are rarely found in first-class condition in
nature, and then only where the habitat does not become dried out.
In cultures, moreover, one may inspect the plants from time to time and
thus keep an accurate check on the growth stages.
Three general methods of cultivating prothallia are in use
1. A is half filled with broken pottery chips
shallow pot or fern dish
or fine gravel. Over a layer of rich loam to within 2 cm. of
this place
the top, then follow with a layer of clean plasterers’ sand about 5 mm.
deep. Water completely, and sterilize. When cool, scatter the spores
over the surface of the sand, and cover the container with a paru^ of glass.
Set in diffuse sunlight.
2. Procure a new porous clay flowerpot, wash thoroughly, and

pack tightly with damp sphagnum. Invert and place in a convenient

pan or saucer and sterilize. When cool, put sterile water to a depth of
about 2 cm. in the saucer. Scatter the spores over the sides of the pot,
cover the whole with a battery jar, and place in diffuse sunlight. Turn
the whole around occasionally so that all sides will become evenly
illuminated, otherwise the prothallia will acquire abnormal shapes.
3. The third method is better adapted for research purposes since
it affords complete control over the cultures. Sow the spores on a film
of nutrient agar (use Knop’s or Pfeiffer’s solution) about 5 mm. deep in
small Petri dishes under strictly aseptic conditions. For purposes of
cross-fertilizing or producing hybrids, each individual immature pro-
thallium may be transferred to a separate Petri dish, which transplanta-
tion should be performed before the archegonia appear. Fertilization
406 SPECIAL METHODS FOE THE VARIOUS PHYLA

may be effected when the archegoiiia are open, by flooding the culture
with nutrient solution and placing a prothallium with mature antheridia
in the solution. The elution is permitted to remain for about 12 hours
and is then poured off, and the antheridial prothallus removed. The
sporophyte will be visible in a week or two; after the root and cotyledon
have become w’'ell developed, the young sporophyte may be transferred
to soil in a small pot if it is desired to grow the plant to maturity.
The last method is probably the best one to follow if one wishes to
obtain an abundance of material of all stages in the origin and develop-
ment of the sex organs, embryos, and sporophytes. It may be necessary

Fig. 86. —
Filamentous fern prothallia. Fixed with formalin-propiono-alcohol; stained
with iron hematoxylin and fast green; dehydrated with hygrobutol and infiltrated with
balsam.

to modify the conditions in the case of a few ferns. As a rule, however,

one may sow spores on an agar substrate, and when the gametophytes
appear to have attained their optimum development, they may be
allowed to become slightly dry for a few days and then flooded with
nutrient solution for a period of about 12 hours. Beginning with the
pouring on of the solution, a number of gametophytes may be removed
at stated intervals and placed in a 1 %
chrom-acetic fluid. In this fashion,
given an abundance of material, a complete series of developmental
stages is readily obtained.
The discharge of the antherozoids and their entrance into the arche-
gonia is easily observed under the microscope. Place several fragments
of gametophytes with mature sex organs in 1 drop of water on a slide
and add a In a few minutes the antheridia will be
coverslip carefully.
observed to burst open, and the antherozoids will be seen congregating
around^ the archegonia in large numbers. The substance attracting the
 PTERIDOPHYTA 407

antherozoids is malic acid, which fact may be demonstrated by placing

some antheridial fragments in water under a coverslip and placing a drop
of a weak aqueous solution of the acid at one side of the cover. TIk;
antherozoids will promptly swim toward the acid.
The gametophyte of many ferns is at first either asexual or antheridial.
The antheridia may appear even during the filamentous stage, and are
usually very abundant. Considerable growth takes place before the
archegonia originate, customarily just behind the growing apex.
For sections, fix the gametophytes with 1% chrom-acetic. Micro-
tome them in the longitudinal vertical plane; it is better to try to obtain
the archegonia in median longitudinal section and to get the antheridia
by chance. For the antheridia alone, 5 to 8/i is thick enough; stain witli
iron hematoxylin. For the archegonia, lOju is satisfactory; staining
may be difficult, but a triple combination usually gives passable results.
For embryos and young sporophytes, section at lOju, and use safranin
and fast green. Cut all sections perpendicular to the anteroposterior
axis of the gametophyte.
For whole mounts fix in either 1% chrom-acetic or formalin-a(*eto-
alcohol. Stain with Harris^ hematoxylin, which will give brilliant
differentiation of the sex organs and embryos (Fig. 85). After diff(ir-
entiating the hematoxylin, upgrade to 85% ethyl alcohol, then leaver
overnight or even for as long as 24 hours in a strong solution of fast
green (dissolved in equal parts of methyl cellosolvc and 95% alcohol).
Overstaining rarely results; wash with two changes of 95% alcohol,
then dehydrate with hygrobutol, and place in diluted balsam. This
method is excellent with all developmental stages, but the counterstain
may be omitted on young embryos for the sake of greater clarity. In
mounting on slides, be sure to orient with ventral side up. The prothallia
should be mounted as soon as convenient, since they begin to curl up
and become brittle after remaining in thick balsam for more than thre(;
days.
Special directions for the gametophytes of the Marsiiea(*eae and
Salviniaceae will be given in the appropriate places under these families.

Filicales

Osmundaceae. —The cosmopolitan genus Osmunda does not occiu-

west of the Rocky Mountains in the United States. The stem is very
difficult to microtome because of the large masses of sclerenchymatous
leaf bases; prolonged soaking under water sometimes renders them suffi-
ciently soft to cut. The stelar portion of the stem is comparatively
small; the part formed is protostelic and later portions are dictyo-
first

stelic. The leaves are tough and leathery. Production of sporangia

 408 SPECIAL METHODS FOR THE VARIOUS PHYLA

differs according to the species: the sporangia are borne on slender brown
leaf segments—4n cinnamomea on two or three special leaves, in
0.
0. regalis in the terminal part,and in 0. claytoniana in the middle portion.
Transitional types are also present. All sporangia of a region mature
simultaneously. The mature spores contain chloroplasts, germinate
immediately, and do not retain their vitality long. The gametophytes
are long-lived, rather large, thick, and fleshy; the antheridia are produced
terminally or on the margins, the archegonia ventrally either over the
entire midrib or only along its edges. Only the younger and thinner
specimens should be selected for whole mounts.
—
Schizaeaceae. The genera are mostly tropical or subtropical,
but Schizaea occurs in the Pine Barrens of New Jersey and Lygodium
in rare instances from Massachusetts to Kentucky and southward. The
vascular organization differs according to the genus: in Lygodium^ which
is a climbing fern with a subterranean stem, it is a simple protostele.

Vernation is circinate, and the apical cells of the leaves are long-per-
sistent. The sporangia are of a simple type. The gametophyte is of
the typical cordate thallus type in all genera save Schizaea^ in which it is a
protonema-like branched filament containing a mycorrhizal fungus.
All the genera are difficult to manipulate in one way or another.
Schizaea is a tiny plant, except in the tropics, and considerable sporangial
material should be preserved. Older portions of all genera become dark
brown, and tannin deposits are sometimes so extensive as to interfere
seriously with staining. Older parts of Lygodium are more or less dis-
organized. Alcoholic killing fluids should be used. Safranin and fast
green should be employed for staining since other combinations have
not been satisfactory.
—
Gleicheniaceae. There are two genera inhabiting the tropics or
subtropics of the Southern Hemisphere. It is doubtful whether Glei-
chenia, the commoner of the two genera, is in cultivation in this country.
Stems and leaves with sporangia should be procurable from the supply
concerns. Most species possess long rhizomes. There is much variation
in the vascular organization. The son are naked and borne on vegeta-
tive leaves; the sporangia develop simultaneously. Fixation of all parts
is excellent with formalin-ace to-alcohol, but there is always some harden-
ing; consequently soaking under water is invariably necessary. Smooth
sections of even the toughest stems are then not at all difficult to micro-
tome. Staining, which is preferable with safranin and fast green, is
uncomplicated, except for younger stems and the rachis, which are hard to
differentiate.The prothallia resemble those of the Osmundaceae.
—
Hymenophyilaceae. This family includes the so-called filmy
ferns, with two genera, Hymenophyllum and TrichomaneB. The former
is found in tropical rain forests, but fortunately material can be obtained
 PTERIDOPHYTA 409

from the supply concerns. Trichomanes is abundant in Florida and

one species extends as far north as Kentucky, occurring on dripping
sandstone cliffs. The rhizome has a protostele. The leaves of many
species are only one cell layer in thickness; young stems and leaves are
very easily sectioned. The sori are marginal, and the sporangia slto
borne in basipetal succession on a receptacle the whole is surrounded by
;

an indusium. Pinnae with sori should be removed separately; embed

with one flat side down, and section parallel to this flat side at lOju.
Fixation of all organs is adequate with formalin-aceto-alc.ohol, and sharp
staining is given by practically all combinations. The spores are easy to
germinate, but cultures will require three or more years before they
commence producing sex organs. The monoecious gametophyte is
either a profusely branched filament {Trichomanes) or an irregularly
branched ribbon (Hymenophyllum); the sex organs are borne on short
adventitious branches.
—
Cyatheaceae. Th(‘ tree ferns are mainly tropical or subtropical
plants, but one n'pn'sentative {Dennestaedtia adiantoides) occ’urs in
Florida. Specimens of Cyaihea are common in greenhouse fern collec-
tions in the East and are widely planted outdoors in central and southern
California. Material is thus relatively easy to collect or to purchase,
but st(*m material is of rather doubtful attainability. Fixations should
be in formalin-aceto-alcohol; staining is usually easy but stains giv-
ing sharp contrasts should be selected. The sori are already
present wlum the circinately coiled fronds begin to unroll, but it is
not difficult to S(‘cure the earliest stages of sporangial development.
As the leaves develop, they become tougher and are rigid after being
embedded; consequently soaking is required. Prothallia develop very
slowly.
—
Marsileaceae. In the United States Pilularia occurs chiefly on
the Pacific Coast; Marsilea in the same region and along the Gulf Coast.
One species of the latter genus has become established in a few ponds
in New England. Material of Pilularia is difficult to procure, but Mar-
silea is easily raised from sporangia, which can be purchased if not
otherwise obtainable.
Marsilea sporophytes have a branched, creeping rhizome, whose
ramifications may cover a considerable area. The leaves and roots
appear at the nodes. Young nodes may be fixed entire and sectioned
in the vertical transverse plane (z.e., as if the internode was being cut
transversely) these will provide a great variety of interesting
;
and instruc-
tive tissues: origin of the adventitious roots, young leaves, petioles in
longitudinal section, and even the and the youngest
origin of the sori
developmental rhizome
stages, as well as sections of the proper. Fix
with formalin-propiono-alcohol, use the water suction pump, soak the
410 SPECIAL METHODS FOR THE VARIOUS PHYLA

embedded material under water for about three weeks, microtome

at 10m, and stain with either safranin and fast green or a quadruple
combination.
The vascular cylinder in the rhizome is an amphiphloic solenostele.
The broad flat leaves of Marsilea should be cut into convenient widths
for fixation with formalin-aceto-alcohol, and the awl-shaped leaves of
Pilularia into short sections.
The youngest sporocarps must be looked for in sections of the nodes.
As soon as they can be observed emerging, they should be cut out. They
are borne on peduncles inserted just above the bases of the petioles.
In Pilularia and in many species of Marsilea the sporocarps are solitary;
in other species of the latter genus they may number as many as 20.
As long as they can be cut with a razor blade or sharp scalpel, the sporo-
carps can be embedded and sectioned. Cut off a small segment at one
end to facilitate penetration of fluids, and fix with formalin-aceto-alcohol.
Infiltration with paraffin must be quite thorough. Soaking under water
appears to be only briefly needed by some species, a much longer time by
others. Serial sections are better than a few isolated sections. The
sporocarps should be microtomed perpendicular to their flat side trans-—
versely for the younger stages and longitudinally as soon as the sporangia
are definitely recognizable. (When nodes are sectioned for the origin
of the sporocarps, the latter obviously cannot be oriented in a definite
plane, but practically all sections are useful.) The optimum thickness
is IOm- Iron hematoxylin is the only satisfactory stain, and the solution
should not have been used before on other slides.
The sporangial initials at the apex of each receptacle become mega-
sporangia, and those lower down develop into microsporangia. It is a
fortunate matter for the technician that after the sporocarps become too
hard to be sectioned, the stages V^etween this period and the emergence
of the sporangia on germination are of little importance.
The sporocarps require a resting period of three months or longer
before they can be induced to germinate. The spores remain viable for
30 years or longer. The sporocarps should be filed at one end until the
whitish cavity is exposed and may then be placed in large Petri dishes
containing sterile tap water (distilled water should hot be used). The
gelatinous sporophore will be extruded within hour; the perfect ring
shape delineated in most textbooks does not always appear. The time
of emergence should be noted, since collections should be made at stated
intervals beginning as soon as all the sori have emerged. Development
proceeds rapidly; that of the microgametophyte requires from 12 to 26
hours, and that of the megagametophyte 14 to 22 hours, both at ordinary
room temperature. [At 28 to 30°C. antherozoids appear in about 6^
hours (Lang 1936).]
 PTERIDOPHYTA 411

For the development of the microgametophyte, collections should

be made at intervals of 30 minutes for the first 10 hours: at the first
hour, the nucleus is in the center; at 1 hour, it begins to move toward
one side of the spore; at \}^'2 hours, it has reached the side and prepara-
tions for the first division are evident; at 2 hours the sori begin to dis-
integrate, but enough spores are still caught in the gelatinous envelope
for the sori to l;»e fixed entire —
after this time the spores must be taken

—
Fig. 86. Marsilea vestita: sections of microspores 2)4 flours after germination, immedi-
ately before the reduction divisions begin. Killed with Sharp’s special chrom-osnjo-
acetic fluid; stained with iron hematoxylin and fast green.

up with a pipette; at hours the first mitosis occurs (Fig. 86); and
at the end of the seventh hour, the 16 androcytes are fully developed.
So close a series of stages has not been worked out for archegonial and
embryonal development. Matters here are complicated by the prev-
alence of parthenogenesis. Fertilization apparently occurs in about
24 to 36 hours. In four or five days the cotyledon has attained a length
of 6 to 8 mm. it has a bright green color.
;

For the microgametophytes fix with Navashin^s fluid made up in

the proportion of 13 cc. of Part A, 13 cc. of 4% formalin and 1 cc. of
1% aqueous osmic acid, or with the following (Sharp 1914):
1% aqueous chromic acid 25 cc.

Glacial acetic acid 1 cc.

2% aqueous osmic acid 14 drops

Distilled water 75 cc.
412 SPECIAL METHODS FOR THE VARIOUS PHYLA

Chromic fluids are in general worthlesson the developing megagameto-

phytes, but a fluid which completely satisfactory on these structures
is

is as yet unknown. Young embryos fix well enough with formalin-aceto-

alcohol. It will be tedious changing the dehydrating fluids after the
spores have become freed from the sori, but the cautious use of a cen-
trifuge will be of great assistance. For embedding, use small paper
trays or porcelain dishes about 5 mm. square, in order to, concentrate the
material as much as possible. Microtome at 8^, mount the ribbon seri-
ally, and stain with iron hematoxylin and fast green. The starch grains
stain so intensely that it is not easy to judge the destaining accurately.
Whole mounts of the megaspores with young sporophytes attached are
readily prepared: fix with formalin-propiono-alcohol, stain with Harris'
hematoxylin and fast green, and follow the hygrobutol method.
—
Polypodiaceae. This family contains the largest number of species and
is the dominant one in the United States, especially in the Pacific states.

Technical methods are essentially similar for all species and the
general methods outlined at the beginning of the Pteridophyta apply.
'Fhe one point to bear in mind is that any one species is not so equally
good in all respects as another might be. For instance, the rhizome of
Pteridium aquilinium is easier technically and has better organized
tissues than that of P. latiusculurn. The latter occuirs in the Eastern
states and the former on the Pacific Coast. The very finest material of
P. aquilinium comes from the humid forests of the Northwest. All
species of Polypodium are fine for the stem, but only a few have sporangia
which make good slides.Aspidium is excellent for the development of
the sporangia; Pteridium and Adiantum are among the worst.
—
Paxkeriaceae. The sole genus, Ceratopteris, which occurs from
Florida to along the Gulf Coast, is aquatic. It may be found in a few
botanical gardens or living specimens may be purchased from fern
specialists or dealers in aquarium supplies. General methods are appli-
cable, but the tissues are so fragile that great care with the dehydration
and infiltration is imperative.
—
Salviniaceae. Like the Marsileaceae, this family is aquatic and
heterospbrous, differing in that each sporocarp contains either micro-
spores or megaspores, but not both, and in that the plants float on the
surface of the water. There are two genera, Salvinia and AzoUa^ both
with only a few species each.
Salvinia is mainly an African genus, but S, natans is cultivated as an
aquarium plant. The sporophyte looks somewhat like a gigantic
Lemna. The leaves are arranged in threes; of these, two are floating
and one is submerged. The former are covered with papillae on the
upper surface and are densely matted with brown hairs on the underside.
The submerged leaves are finely dissected and give the appearance of
being roots, but true roots are absent. In general the methods described
 PTEIUDOPBYTA 413

below for Azolla apply to Salvinia, but it is easier to distinguish the

young sporangia in the latter.
Azolla is to be found in small ponds and the quieter parts of slowly
moving streams. In the shade the color is a pale green, bec^oming a
distinctive reddish shade in full sun. As a rule, only those plants growing-
in full sun produce sporangia, which generally appear in late summer or
early autumn. If unavailable locally, live plants may be purchased
from dealers in aquarium supplies or embedded material (which is
preferable to preserved material since the latter is likely to be sterile or
minus sporocarps) can be secured from the botanical supply concerns.
The plants may be fixed entire; if there are so many lateral branches
that the whole mass is too large, cut them off. The material should be
handled carefully since the maturing sporocarps are easily detached.
It is very difficult to get the sporophytes to sink into killing fluids. Use
a medium chrom-acetic, or formalin-aceto-alcohol, and add to the fluid
from 2 to 5% ethyl acetate, which should assist in lessening the surface ten-
sion on the plants. Use the water suction i)ump to remove air contained
in the leaves. Embed the plants singly, dorsal side down. Micro-
tome at lOg in the vertical longitudinal plane (t.c., lengthwise perpendi-
(mlar to the dorsal surface), as the greatest number and vari(‘ty of useful
sections will be obtained in this fashion. If vertical transverse^ or hori-

zontal sections arc desired, use small portions of the sporophyte; largt‘

plants cannot be accurately sectioned in these two planes because^ tlu'

tips are curved ventrally. Examine the ribbons under the mien'oscope,
discarding those that apparently have no sporocarps or which do not
come sufficiently close to the stem. In A. filiculoideSy the most
extensively studied species, the sporocarp will gtmerally be micio-
sporangial. Experience has shown that an older megasporangium will
be found in about 1 out of every 60 plants. The younger megasporangia
are relatively easier to find. Microsporangia in all stages of growth an*
usually abundant. Azolla is not easy to stain sharply or brilliantly for
all structures simultaneously. It would be better to concentrate on th ^

desired structures and to ignore the others. Safranin and fast green
constitute the best general stain combination; for mitoses in the micro-
sporangia, which are easy to obtain, use iron hematoxylin.
To obtain the later stages in the development of the sporangia,
especially for the archegonia and embryos, be necessary to place
it will

plants bearing nearly mature sporangia in a large glass receptacle, which

is then placed in full sunlight. After the megaspores have fallen to th('
bottom of the container, they may be removed and fixed at frequent,
intervals over a week or longer. It is, however, a gamble whether one-
will secure anything worth while.
The endophytic alga, Anabaena azollae, appears in almost every
section.
 CHAPTER XXIX
CYCADOPHYTA
Cycadales

The order includes one family, the Cycadaceae, with nine genera
and about 65 species. Zamia floridana is the only cycad that occurs
naturally within the confines of the United States; it occurs abundantly
in certain portions of Florida. Plants of other cycads are commonly
grown in large conservatories, particularly those in the parks of the
larger cities.
The cycads are strictly dioecious. The male strobilus is always a
compact structure in all the genera. The female strobilus may be either
a crown of loose sporophylls or a compact cone; all transition types
between the two extremes are also to be found.
It is a rather difficult matter to obtain material of the cycads, espe-
cially of the reproductive phases. To obtain a series of developmental
stages, one would have to make collections over almost an entire year.
vSome stages in the life history progress so rapidly that daily collections
are required, but for other stages one collection a week suffices. A
great number of plants are obviously needed to afford sufficient material
when a large number of collections are to be made. From cultivated
specimens one can usually obtain material at only one developmental
stage, except in the case of such as Cycas revoluta or C. circinaliSj when
it is possible to secure a series of stages at one time — provided on^ comes
at the right time. Preserved material, or even embedded material,
may be secured to a limited extent from the supply concerns. Leaves
and perhaps petioles can be readily obtained from a public park or a
commercial florist upon request, but in such cases one should check
up on the identification of the plants. If one can establish contacts
with a collector in regions where the various cycads abound, it might be
remain in good condition
possible to procure living cones as they usually
for two or even three weeks after removal from the plants.
—
Root. As compared to other seed plants, the primary root of cycads
is rather large in comparison with the stem. Root growth is frequently
aounaant, and the roots may attain considerable length. The structure
is tetrarch.
Cycad roots are exceptionally easy to embed and to section in paraffin
and to stain sharply. For morphological preparations, formalin-aceto-
4i^
 CYCADOPHYTA 415

alcohol fixes very nicely, but Navashin^s fluid should be used on root
tips. After safranin, anilin blue is a more satisfactory counterstain tlian
fast green.
On all seedlings and on specimens in greenhouses are to be found
peculiar apogeotropic (aerial) roots; these produce profusely branched
coralloid masses. Such roots contain bacteria in the form of bacteroids,
and midway between the vascular cylinder and the epidermis there is an
enlarged layer of cells, usually only one-cell wide, in which Anabaena
grows. The general methods reveal the alga clearly, but for the bac-
teroids, mitochondrial methods are indicated. A 10% solution of
neutralized formalin fixes well. If the usual mitochondrial staining
techniques fail to reveal the bacteroids clearly, resort may b(^ had to
with subsequent staining in safranin and fast
differential acidification,
green, since this procedure has worked well in the case of the ba(;teroids
in legume nodules.
—
Stem. The stems of most of the cycads are fleshy, with very little
wood present; consequently they are easily sectioned. It has been
claimed that stems can b(i most readily sectioned freehand while
th<'

fresh, but no trouble was occasioned in microtoming material that had

been in formalin-aceto-alcohol for 25 years. After killing and fixing
and with or without treatment with hydrofluoric acid, the stems or
stem pieces can be embedded in either paraffin or celloidin. Cutting
presumably should be easiest with a sliding microtome, even for paraffin
material, but the writer had no difficulty microtoming paraffin material
that had been in water for a fortnight.
Bowenia Knd Stangeria are tuberous, with all or most of th(^ st(*.m
located underground. Some species in Zamia, Macrozamia^ and Enceph-
alartos also have subterranean tuberous stems.
Petiole (Rachis).— If stems are unobtainable, petioles of most
cycads provide interesting material. In some species the vasculai’
bundles are arranged in characteristic positions. In Cycas revoluta, for
example, they are disposed in a flaring horseshoe manner when viewed
in transverse sections.
The petiole, when still young, is rather rigid and tough.
except
However, with formalin-aceto-alcohol, dehydrated with tertiary
if fixed
butyl alcohol, and embedded in hard Parlax or a hard paraffin and the
cut ends of the embedded pieces exposed under water until really soft
(for months if necessary), microtoming will be found to be easy. C\it
at 16 to ISjLt.

Leaf. —The vary considerably in size among the different

leaflets
species. The length may range from 7 to 40 cm., and the width from
1 mm. to 2.5 cm. In each genus the leaf type is characteristic (Lamb
1923), The leaflets of the adult plant are often quite different from those
416 SPECIAL METHODS FOR THE VARIOUS PHYLA

of juvenile plants. The vstructure of the mature leaflet is strongly

xerophytic. The epidermal cells are thick walled and heavily cutinized,
which adds to difficulties in sectioning leaves. All genera save Cycas and
Stangeria lack a midrib in the leaflets (Fig. 87). In Stangeria side veins
emanate from the midrib, but such secondary veins are absent in Cycas.
Bowenia is easily distinguished by its bipinnate leaves. There is a gland
at the base of each leaflet in species of Macrozamia.
Young leaves arc readily embedded and sectioned. The heavy
phlobaphene deposits and other substances in many species do not permit
a sharp stain differentiation, but results are generally satisfactory after

Fig. 87.— Zamia floridana: cross section of a portion of the leaf. Fixed with fornmlin-
aceto-alcohol ;
stained with a triple combination. The dark areas represent tannin-con-
taining colls.

either safranin and fast green or a triple combination. Thick, leathery,

mature leaves are quite difficult to section, but after prolonged immersion
in the softening effect of water, one can usually manage to obtain good
sections. The method of tying several mature leaves together and
sectioning them fresh in a sliding microtome, as recommended by some
technicians, is too crude to merit further discussion.

—
Staminate Strobilus. In some genera, such as Dioon, there is a single
terminal cone; in other genera, such as Macrozamia^ the cones are axillary.
The strobili vary greatly in size, ranging from 2 cm. in length in a species
of Zamia to the huge cone of Macrozamia denisonii which at maturity
becomes 80 cm. long and 20 cm. in diameter. Measurements, however,
are not of great import because the strobilus elongates greatly and
rapidly just before the pollen is shed. The microsporophylls also vary
greatly in dimensions.
The microsporangia are always borne on the abaxial (lower) surface
of the microsporophylls, in clusters of two, three, or as many as five in a
 CYCADOPHYTA 417

sorus. The sorus arrangement is like that in typical ferns; in fact, the
structure of the microsporangia remarkably like those of Angiopteris,
is

Development of the microsporangium is of the eusporangiate type.

The number of microsporangia on a sporophyll varies, being greatest in
Cycas and fewest in Zamia] Z. jloridana possessing only 25.

Fia. 88 .
— Zamia floridana: apiral x^ortion of a young staminate strobilus in longitudinal
section, at the time of origin of the sporogenous cells. Fixed with fonnaliii-aceto-alcohol;
stained with safranin and fast green.

The entire staminate cone, as can be judged from the dimensions

of the structure as cited above, is too large to be sectioned entire extjept
during the younger stages. Of course, the larger of the young cones
can be cut up into convenient portions, but for the later stages the
sporophylls should be removed and fixed individually. The sporogenous
cells appear first at the base of the strobilus, but the pollen grains mature
first at the apex and lastly at the base of the strobilus. Fonnalin-aceto-
alcohol has proved to fix adequately at all stages. For the finer cytologi-
 —

418 SPECIAL METHODS. FOR THE VARIOUS PHYLA

cal details, however, Navashin^s fluid, or a modification thereof, should

be substituted and only very small pieces of tissue should be fixed.
For staining, almost any combination will give satisfactory results,
although iron hematoxylin may be superior for cytological details and
safranin with fast green for general morphology (Fig. 88).
After the microsporocytes have rounded up and are more or less
free from one another, smears are readily made according to the general

Fig. 89. Zamia floridana: longitudinal section of an ovule through the apex of the
nucellus with young miorogarnetophytes. Fixed with formal! n-aceto-alcohol, stained with
safranin and anilin blue.

methods outlined in Chap. XIII. If the pollen, especially when at the

shedding stage, is obtainable in sufficient abundance, better results can
be secured if the material is worktsd up by a whole-mount method. A
carmin stain or the Feulgen reaction will give the sharpest staining.
Dehydrate with hygrobutol and infiltrate with balsam.
The microspore germinates before shedding. When shed, the pollen
grain consists of a genera;tive ceJl, a tube cell, and a prothallial cell.
The grains are likely to be somewhat shrunken at this time. Before
putting them into the fixing fluid, they should be left in water for about
20 nunutes to restore turgidity.
 CYCADOPHYTA 419

The pollen may be germinated in a 5 to 10% solution of sucrose or

in various sirups. Only the earlier stages of pollen tube development
may be obtained in this manner. The later phases must be studied in
sections of the nucellus.
The microgametophyte is a haustorium that penetrates the nucellus
and can be recognized as brownish lines radiating from the beak of the
nucellus. Cut across the young ovule in such a manner that the cut
is just below the lower portion of the nucellus, then remove those portions

from the upper half of the ovule in which the pollen tubes presumably
lie. Kill and fix in a chrom-acetic mixture in the proportion of about
2 cc. glacial acetic acid to 100 cc. of 1% aqueous chromic acid. The
material can be run into paraffin, or
it can be stained in bulk, dehydrated,

run into balsam, and the tubes with their sperms dissected out of the
nucellus just previous to mounting. For the latter purpose, staining
may be in aqueous safranin (about 5 hours), Harris^ hematoxylin, or
carmalum. Transfers in the dehydrating pro(*ess must be very gradual.
Sectioned material is most satisfactorily stained with iron hematoxylin
or safranin and anilin blue (Fig. 89).
Records indicate that in the region of Miami, Fla., pollination occurs
in late December or early January, the blepharoplasts appear in March
and swimming sperms may be found during the first week of June
(Chamberlain 1932). The interval between pollination and fertilization
is about five months. The body or spermatogenous cell divides immedi-
ately before fertilization.
Pistillate Strobilus. — The mature pistillate strobili are such large
structures in most of the species that it is impossible to cut sections of
the entire strobilus. More or less reduction in dimensions is required.
Even the individual megasporangia are so large in some species that
they cannot be sectioned entire when mature or nearly so.
Under ordinary circumstances it is impossible to obtain material
of the very youngest stages in the development of the megasporangia.
In the first place, the external appearance of the stem apices gives no

indication whether what is developing beneath the scale leaves is a

strobilus or merely a crown of leaves. In the second place, the plants
may be too scarce to run the risk of killing them by digging out the stem
apex. For the early stages portions of the ovule should be trimmed off
on opposite sides, but great care must be exercised not to cut within
2 mm. of the endosperm, which is so turgid that it ruptures easily. A
me(fium chrom-acetic fluid fixes splendidly. Staining may be in a
triple combination or with safranin and anilin blue. The same procedure
may be followed for all the later stages, including embryogenesis.
 CHAPTER XXX
CONIFEROPHYTA
Ginkgoales
The order was once cosmopolitan but is now reduced to a single
living representative, Ginkgo hiloha^ which probably no longer exists
in the wild condition but widely cultivated. Trees of G. biloba are
is

to be found almost everywhere in the United States, and anatomical

material is easily secured at any time. Material for cytological or
morphological purposes is more difficult to obtain at just the correct
times. Preserved or embedded material is readily purchasable from the
supply concerns, except for the critical stages between late free -nucleate
female gametophyte and young embryos.
—
Root. In most plants the root has a diarch vascular bundle, but
in the seedlings with three cotyledons it is triarch. The structure of the
mature root greatly resembles that of the mature stem. The root is
easier to section in paraffin than arc young stems but nevertheless requires
long soaking under water after being embedded. Safranin and ariiliu
blue or fast green is a good stain combination.
—
Stem. Two types of branches occur in Ginkgo, as in many other
conifers: long branches and short spur branches. Long branches always
appear first and grow for at least a year before spur branches are developed
on them. The anatomy of the two types of branches differs. In the^
long branch there are a comparatively small pith and cortex, there are
fewer mucilage cavities, and the wood is harder. Sections of spur
branches are easily cut in paraffin, but the embedded long branches
must be soaked under water for about two months before they can be
microtomed easily enough. If they are embedded in celloidin, they must
be treated with hydrofluoric acid immediately after fixation. Fix with
formalin-aceto-alcohol; stainingmay be with any desired combination.
The wood is rather difficult to microtome; resort should be had to

celloidin embedding preceded by treatment with hydrofluoric acid.

Freehand sections may also be cut on a sliding microtome. The vascular
cylinderis an endarch siphonostele. Annual rings are present and
usually prominent. In the tracheids of the secondary wood there are
one or two rows of scattered bordered pits on the radial walls. Both
bars and trabeculae of Sanio are to be found in the secondary wood.
The medullary rays are almost invariably only one cell wide and rarely
over four or five cells in height.
 CONIFEROPHYTA 421

Leaf. —
Portions of the leaf are readily embedded and microtome^.
In the leaves of the spur branches a palisade layer is absent, but one is
to be found in the larger leaves of the long branches. Formalin-propiono-
alcohol fixes well; staining is sharpest with safranin and anilin blue, or a
quadruple combination may be used.

Fig. 90 .
—
Ginkgo biloba: longitudinal section of two young ovules; the one at the loft is
sectioned rnedianly through the micropyle, pollen chamber and megaspore mother coll.
Fixed with formaliii-propiono-alcohol; stained with a triple combination.

Two endarch vascular strands ocanir in the petiole, which may be

treated like the leaf, but may require soaking under water before being
microtomed.
Staminate Strobilus. —The beginnings of the staminate strobilus
are first recognizable early in July by the swelling of the buds on the spur
branches of staminate trees. Development proceeds slowly until cold
weather sets in, at which time the microsporocytes have become differ-
entiated. The time at which growth is resumed depends upon the
locality: in the Palo Alto, Calif., region, this has occurred in mid-January
during mild winters, but may be delayed until early March in other
 422 SPECIAL METHODS FOR THE VARIOUS PHYLA

localities.^Once development has been resumed, it is very rapid.

Meiosis takes place at once; if meiotic figures are desired, one usually
has to anticipate the recurrence of growth and make collections before
it appears to have definitely started. Fixation should be in a strong
chrom-acetic fluid, and only small portions of a strobilus should be fixed.
For general morphology of the strobilus even entire strobili may be
preserved, and formalin-accto-alcohol is a better fixing fluid. Iron
hematoxylin is excellent for the reduction divisions, but safranin and
anilin blue are more useful for general topography.
Each sporophyll of the strobilus generally bears two microsporangia,
but as many as four and even seven have been reported. Development
of the sporangium follows the eusporangiate scheme. Throughout an
entire strobilus there is not so wide a series of developmental stages
present at any one time as might be expected, because of the rapidity
with which the spores are formed.
Pistillate Strobilus. —
The pistillate strobili are borne on the spur
branches at the apex. The ovules occur in pairs on long peduncles,
but one ovule frec^uently aborts early in development. The ovules are
first recognizable at about the time that the terminal bud on the spur

branch begins to swell. To get the earliest stages the tip of the spur may
be cut off, the outer scales removed, and the whole embedded and
sectioned longitudinally in a plane as nearly perpendicular as possible
to the two ovules. When the ovules begin to emerge from the bud, the
single integument has partially covered the nucellus. Between the
middle and end of April the megasporocyte has appeared, and it is also
at this time that pollination occurs (Fig. 90). The megasporocyte
undergoes meiosis, a linear quartet of megaspores being the inamediate
result. Irregularities, however, are not infrequent. In any event,
only one megaspore becomes functional.
In the peduncle which carries the ovules there are four vascular
bundles, in contrast to the two bundles in the leaf petiole. This fact
has led to the interpretation that the peduncle is a stem bearing two
sporophylls (the prominent collars at the base of each ovule), each
supporting a single ovule. If more than two ovules are present, the
number of vascular bundles in the peduncle is twice the number of
ovules.
—
Microgametoph3rte. The microspore germinates before being shed
from the microsporangium, as in the cycads. When shed, the mici^ospore
—
contains four cells two prothallial cells, one of which is aborted, a
generative cell, and a tube. cell. The lower two-thirds of the spore is
covered by the exine, the upper third by the intine alone. In the pollen
chamber of the ovule the generative cell divides into a stalk cell and a
body cell. Two blepharoplasts are developed in the body cell, which
 CONIFEROPHYTA 423

divides to form tvvo sperms. The blcpharoplasts eventually undergo

metamorphosis into cilia.
—
Megagametoph3rte. The megasporocyte undergoes meiosis, at about
the time of pollination, or in late April, to form a linear row of quartets.
The lower one becomes functional and enlarges rapidly, and free nuclear
divisions take place (Fig. 91). The nuclei are forced against the periph-
ery of the cell by a large central vacuole. At first the mitoses are
simultaneous, but later on some nuclei lag behind the others, and finally

Fig. 91 . —Qinkgo hiloha: early free nucleate female gametophyte. Fixed with formalin-
aceto-alcohol ;
stained with safranin and anilin blue.

a few fail entirely to divide again. These divisions occupy about two
months, or until the end of June or very early in July. At this time a
delicate membrane develops over the outer surface of the coenocytie
protoplasm, and cell walls perpendicular to this membrane are formed
between the nuclei. Wall formation progresses centripetally, but,
before the gametophyte becomes completely cellular, two or rarely
three archegonial initials have already made their appearance at the
apex. The two neck cells and the central cell are organized quickly,
and the commences growth and enlargement. Early in September
latter
the ventral canal cell and the egg become differentiated, a definite wall
424 SPECIAL METHODS FOR THE VARIOUS PHYLA
%

being formed between the two. Such a wall is not produced after the
same mitosis in the cycads.
Fertilization. —
Accurate and detailed descriptions of the fertilization
process have not been published.
—
Embryogenesis. Simultaneous free nuclear divisions follow upon
fertilization. Unlike conditions in the megagametophyte, the nuclei
are not pushed against the periphery by a central vacuole but are evenly
distributed throughout the egg cell. About eight mitoses occur, where-

Fig. 92 .
—Ginkgo biloba:median longitudinal section of nucellus with beak and pro-
embryos at beginning of cell formation. The typical shrunken appearance of the pro-
embryos is apparent. Fixed with medium chrom-acetic; stained with safranin and anilin
blue.

upon walls are simultaneously developed all through the egg (Fig. 92).
At first the cells are of the same size, but presently those at the basal
end grow rapidly and become small and numerous, those in the central
portion enlarge greatly (Chamberlain 1935). A readily distinguishable
suspensor is not developed. The basal portion soon grows rapidly and
becomes differentiated into cotyledons, stem, and root. The embryo
attains maturity in October.
All the stages of embryo development are easily fixed and sectioned.
As soon as cotyledon development has partially progressed, it is advisable
to dissect out the embryo and to work it up independently of other
ovular structures^ which will have become too hardened by this time to
 CONIFEROPHYTA 425

section easily. A strong chrom-acetic or even formalin-aceto-alcohol

fixes well; safranin and a suitable counterstain may be used. The cells
of the older embryo are so small and compact that somewhat thin sections
are required; about lOju is thin enough.
The seeds mature in November and germinate if planted at once.

As with the cycads, there is apparently no normal resting period.

CONIFERALES
I

Although the greatest natural concentration of the Coniferales is

to be found on the Pacific Coast and in the Northern states, conifers
nevertheless are to be found in practically every locality. If native
species are lacking, introduced forms are freely planted everywhere.
Material is thus readily acc^essible; even reproductive phases are not
difficult to collect, but they are unfortunately not always of a suitable
nature or desirable type. The most widely distributed genus is Pinus,
with Lanx, Pfcca, Tsuga, Abies, Thuja, Cupressus, and Tarrus of secondary
degree of distribution, particularly by landscape plantings.
There are 10 families, divided into two groups: Pinares and Taxares
(Buchholz 1934, Chamberlain 1935). In the Pinares the seed cone is
conspicuous and well developed, woody in most genera and fleshy
only in species of Juniperus, and the plants are mostly monoecious.
In the Taxares the seed cones are mostly small, poorly developed or
reduced to only a few scales, and fleshy; the plants are mostly dioecious.
Despite a very extensive bibliography on the Coniferales, it has
been claimed that “a complete life>history has not been worked out in
any of the genera in this large order (Chamberlain 1935). Finns has
been more extensively investigated than any other genus, and thus
more is known about it. In the other genera there has bc^en too much
emphasis on either wood anatomy or on reproductive structures, so
that in reality only some one isolated aspect of the complete life history
has been investigated.
The various organs and reproductive structures will be discussed
separately in the following paragraphs.
—
Root. Preparations of the mature embryo, in both longitudinal
and transverse section, will show many details of the primary root
structure. The seeds may be removed from mature pistilfkte cones
and the endosperm with the embryo removed from the seed. It is
difficult for fixatives and dehydrating fluids to penetrate the dense
endosperm, hence slabs should be cut from opposite sides to facilitate
penetration. Seeds may also be germinated and specimens fixed at
various stages of development. A strong chrom-acetic or formalin-
propiono-alcohol may be employed.
426 SPECIAL METHODS FOR THE VARIOUS PHYLA

To illustrate the origin of the stele in the primary root and the
development secondary roots, the seeds must be germinated in
of the
fine soil or in sphagnum. Begin fixing portions of roots when the hypo-
cotyl has attained a length of 2 cm. and continue until it is about 6 cm.
high. Root hairs are present but occupy a space rarely over 1 mm. in
length at the tip and are exceptionally fragile.
Longitudinal sections of the root tip of many species reveal, even by
ordinary fixation methods, characteristic features, but the results are
far more pronounced if mitochondrial fixatives are employed (Zirkle
1932). The tannin-containing vacuoles are well organized in the root
tips of the gymnosperms and form definite patterns. The tips may be
fixed in a mixture of 100 cc. of 10% aqueous formalin and 5 g. ferrous
sulphate for 48 hours to several days. Wash with water briefly, embed
as usual, section at not over lO/x, affix to slides, remove the paraffin with
xylol, and mount in balsam without further staining.
Portions of the fully mature root should be dealt with as if they were
stem sections, but pieces of younger roots can be cut without difficulty
until they are about 5 mm. in diameter. Avoid overstaining with the
basic stain.
Stem. —As long branches and spur branches occur in
in Ginkgo^
many such as AhieSj Cedrus, LariXj and PimiSy but
of the Coniferales,
the leaves on the spur branches are in fascicles. Structurally, the
vascular cylinder is endarch siphonostelic. Annual rings are con-
spicuous features of the mature wood of all but a very few species. The
transition between ‘^spring^^ and winter wood is abrupt in many
'

species, but in others there is a greater amount of “ spring w^ood than

of the other type. Spring'^ wood consists of larger, thinner walled,
and less hard cells; ‘^winter” wood is compact, with thick-walled cells.
In Phyllocladus the spur shoot is borne in the axil of a leaf. It
becomes flattened (i.e.j a cladode) and has the functions of a leaf. The
vascular bundles in these phylloclads are mesarch.
The meristematic stem and young stems up to a year old are easily
embedded and sectioned without special treatment. Cut into portions
about 1 cm. in length; it is unnecessary to trim off the young leaves along
the sides. Fix with formalin-aceto-alcohol, not in an aqueous fluid.
Soak the exposed ends of the embedded pieces of material under water
for about* 10 days, then sections as thin as IO/a are readily cut. Stain
with safranin and fast green or substitute Harris^ hematoxylin for the
green. Quadruple methods are also excellent.
Older stems, up to four years old of all species, that can be cut across
with a sharp scalpel can also be embedded in paraflin and sectioned if
taken at all stages, especially with the microtoming.
suflScient pains are
However, most conifer stems become too hard^ particularly from the
 CONIFEROPHYTA 427

heat of the paraffin oven, to section easily. If sufficiently rigid, young

stems may be cut freehand in a sliding microtome. It will generally be
impossible to cut sections of even thickness across the entire piece of
material, but small pieces in any event are just as serviceable and are
apt to be thinner in some portions than in others. Too often the cambial
region becomes impossibly crushed. The steam method for hardwood
sections may be useful on most conifer woods, but when all is said and
done, the celloidin method remains the most satisfactory of all.
Sections of live material cut freehand should be placed in formalin-
aceto-alcohol for 24 hours before being treated further. Well seasoned
wood of course does not need fixation. Useful stain combinations for
both types include safranin and anilin blue or crystal violet or Harris^
hematoxylin. The modern safranins stain so intensely in a short time
that the long periods rec^ommended by the older authors should be
greatly abbreviated, else it will be difficult to differentiate the stain
sharply. Special attention should be paid to the clear differentiation
of the bars of Sanio and the bordered pits.
Live materials to be embedded in either paraffin or celloidin require
fixation. The pieces should be trimmed down carefully in order that the
radial longitudinal sections may be microtomed exactly parallel with
the rays, and the tangential longitudinal sections precisely tangential to the
rays. Formalin-aceto-alcohol is the most useful fixing fluid. Several
days in the fluid is bettc^r than the usual 24 hours, and the material can
be left in the fluid indefinitely if a little glycerin is added to prevent
excessive evaporation. The stains mentioned in the preceding para-
graph are equally applicable to sections of embedded material, but most
workers apparently prefer safranin with Delafield’s hematoxylin.
Whether freehand or embedded methods were employed, each slide
should bear cross, radial longitudinal, and tangential longitudinal sections.
The longitudinal sections should first be examined under the microscope
in order to make certain that they arc either radial or tangential, as the
case may be, and not both radial and tangential.
The woods of all gymnosperms are easily macerated. An examination
of such slides will provide an idea of the nature of wood very different
from that given by sections. The acids of the macerating fluid sometimes
render staining rather difficult if they are not very thoroughly washed
out. A basic dye or a hematoxylin may be used, but it is of little avail
to attempt double staining.
—
Leaf. In the Coniferales the leaves are all simple; no compound
leaves occur. Wide variations exist in the size and character of the
leaves. Some species have very small scale-like appressed leaves, others
have fairly large needle leaves (as in Pinus coulteri)^ and still others
possess broad, thick leaves. The leaf of Sciadopitys consists of two
428 SPECIAL METHODS FOE ^HE VARIOUS PHYLA
needles fused along the posterior margins. Most leaves are rigid and
sharp-pointed. A few species have deciduous leaves, but in others the
needles persist for two years, or in some species for as long as 14
years.
Leaves of all ages are easily sectioned in paraffin. The youngest
leaves are to be found in cross sections of the stem apex, and these are
very desirable for comparison with mature leaves. The stem apices
are rather soft; after being embedded they need soaking under water for
only a day or two. Microtome at ISjit, and stain with safranin and
anilin blue or by a quadruple combination. In Pinus there are both
primary and secondary leaves. The latter are the needle-like leaves
and are borne in the axils of the scale-like primary leaves. At the base
the leaves are surrounded by a membranous sheath, which is deciduous
in those species whose needles have one vascular bundle and is persistent
in those with two bundles. The contents of the mesophyll cells in the
leaves of Pinus and in a few other genera are generally shrunken and
tend to overstain.
Species with rigid, appressed, scale-like leaves are difficult to section.
The best that can be done is to cut off portions of the young branches,
together with what leaves they bear, and, after embedding, to soak under
water for several weeks or even months until they cut satisfactorily.
Aqueous killing fluids are quite unsatisfactory. Excellent fixation of
all types has been obtained with formalin-propiono-alcohol.
—
Staminate Strobilus. Throughout the order the staminate strobilus
is a cone or is cone-like. The strobili are dominantly monosporangiate,
but bisporangiate strobili have frequently been described. They should
be regarded as teratological. Monoecism prevails, but some species
and even genera are dioecious. For example, the Araucariaceae are
dioecious, with the exception of two monoecious species, Agathis australis
and Araucaria bidwilli. So are the Taxaceae, with a few exceptions,
and in the Podocarpaceae the principal genus, PodocarpuSj is dioecious.
If one is not familiar with any species of which material is being col-
lected, it becomes necessary to determine whether the specimen is
monoecious or dioecious. The staminate strobili generally appear before
.

the pistillate strobili, but the remains of both can usually be found;
hence determination of sexuality is easy.
The strobili vary considerably in size, the smallest being found in
species of Juniperus and the largest in Araucaria. In the Cupressaceae
the arrangement of the sporophylls is cyclic; in all other families it is
spiral. The sporophylls also vary greatly in size, and the extremes
are to be found in the same genera just mentioned. The sporangia,
which generally number 2 but may be as many as 15, are borne on the
abaxial side of the sporophyll.
 CONIFEROPHYTA 429

The very youngest strobili are to be found at the apices of the stems,
partially or completely covered by the enveloping scales. Stem apices
intended for growth studies and for the origin of the leaves will occa-
sionally show the origin of the staminate strobili if they happen to
have been collected at a favorable time. The time at which the strobili
originate naturally depends upon the species. In some species, such as
old cultivated specimens of Sequoia sempervirens, strobili in various
stages of development can be found the year round. In most species
of PinuSy in the central California region at least, the strobili have pushed
out of the scales in early September and growth begins in earnest in
December. Meiosis usually occurs in early March but is governed
to a considerable extent by the severity of the winter months. The
strobili in C^ipressuSy in both the native species and in those commonly
planted in the region just mentioned, appear much later, and develop
more rapidly; the mature microspores are shed from late spring to mid-
summer. Many forms transplanted from the Southern to the Northern
Hemisphere appear to have difficulty becoming readjusted to the reversal
in the seasons and produce strobili at the wrong time. Altogether,
one must keep a close watch on the specimens, provided they are known
to be sufficiently mature to produce strobili, and to make collections at
appropriate periods in order to obtain a series of developmental stages.
For fixation of the early stages, formalin-accto- (or propiono-) alcohol
has been found quite satisfactory; a triple combination should give good
differentiation. Sections should be about 10 jj thick. 1 longitudinal
sections are more useful than transverse ones.
—
Microsporogenesis. To determine the stage of development in
the young strobili, dissect part of a microsporangium from the base of
the strobilus in a drop of iron-acetocarmin, and examine under the
microscope. If it shows division figures, the whole strobilus may be
fixed, whether cut into portions or not. If pollen tetrads are observed,

then examine a sporangium from near the apex; if microsporocytes are

found, the divisions are somewhere in between. In most spe(ues meiosis
commences at the bottom of the strobilusand progresses toward the
top, but in some species of Pinus with small strobili the divisions arc
nearly simultaneous throughout. appears that the longer the strobilus,
It
the wider is the variety of stages, ranging from microsporocytes to
pollen tetrads. The first division in the microspores, however, seems to
occur very unequally, even in the same microsporangium.
Strobili up to mature stages are easily embedded and sectioned,
fully
but in larg^ specimens equally good fixation cannot be obtained through-
out. Penetration of the fluid will be facilitated if slabs are removed from
opposite sides. In order to secure the optimum fixation for cytological
purposes, the strobilus should be bisected and the individual sporangia
430 SPECIAL METHODS FOR THE VARIOUS PHYLA

removed by cutting through the stalks with a tiny scalpel. For entire
strobili, fix in formalin-propiono-alcohol ;
for cytological purposes first
dip into Carnoy’s fluid for a few minutes then transfer to Navashin^s
fluid. Longitudinal sections of the strobilus are the most useful, but
transverse sections should be made for comparison. For the earliest
stages 10/4 is thin enough; for the later stages 12/i is satisfactory, but for
species with prothallial cells lO/x is better; for meiosis and divisions in
the microspores, the sections may need to be somewhat thicker. Safranin
and fast green are good for the earlier stages, but great care must be taken
with the differentiation of the safranin. For the middle developmental
stages a triple combination will probably be most useful. The chromo-
somes in the mitoses in the microspores stain beautifully with iron
hematoxylin.
The microspores of many species can be smeared exactly like similar
cells of the Angiosperms. After the microspores begin to round up,
they become too dry to adhere to the slides and must therefore be treated
as if for whole mounts. It is not at all difficult to secure pollen at the
shedding stage, if the trees are frequently examined. A wide paper
funnel may be fitted over the mouth of a bottle, and the pollen shaken
into the paper cone. Several times the amount of pollen that might
actually be needed should be collected, to make allowance for losses
during staining and dehydration. The pollen will be more or less
shriveled, but turgidity is quickly restored if the pollen is placed in water
for a few minutes. Fixation may be in either formalin-propiono-alcohol
or Navashin^s fluid. The material may be either embedded and sec-
tioned or treated for whole mounts. In the latter case, the contents
are usually too dense for the nuclei and chromosomes to be stained by
the usual methods. Feulgen^s reaction, when properly carried out, gives
superb staining. Dehydrate by going directly into hygrobutol gradually
after the staining is completed, thence into highly diluted balsam.
To embed the pollen, resort to a slow-speed centrifuge will probably
be necessary during the dehydration. Both dehydration and infiltration
may be carried out rapidly. Embed by pouring into a small paper tray
or embedding dish, which should not be over 5 mm. square, in order to
concentrate the material. Two less satisfactory methods are either to
pour the mass on a sheet of cold glass, or to solidify in a small vial, break-
ing the latter and removing the block after the paraflin has hardened.
Microgametophyte. —When the pollen grain of the Coniferales is

shed, the stage of development varies according to either the genus or

the family. In a few species, included in Taxus^ Cupressusj and in
JunipertiSf the microspore is uninucleate; when this is the case, pro-

thallial cells are not developed later. In these species, the first mitosis
occurs after the microspore has reached the nucellus and produces the
 —

CONIFEROPHYTA 43l

generative cell and the tube cell. In the other species the prothallial
cells and generative cell are developed before the microspores are shed.
The prothallial cells are recognizable with difficulty in some genera
(Pinus) but are quite conspicuous in others (Podocarpus). In most of
the Taxodiaceae and Cupressaceae and in all the Taxaceae prothallial
cells are absent. These cells are presumed to be vestiges of the tissues
which originally bore the antheridia.
In all the Coniferales the generative cell divides to form two cells;
in the Araucariaceae one of these cells aborts, and the other develops
gametes. In the other groups one cell assumes a stalk position, with the
other and larger cell attached at the other end. The nature or function
of the stalk cell is unknown, but in Microcachrys (Downie 1928) it has
the function of a spermatogcnous cell, dividing repeatedly, each time
producing a body cell which in turn forms two gametes.
Internal organization in the microspore follows a definite plan.
Differentiation of base and apex is the exact opposite of that occurring
in Ginkgo and the cycads. In the latter the prothallial end of the pollen
tube grows down into the nuccllus, carrying the whole pollen grain with it.
In the Coniferales the entire pollen grain and prothallial end of the pollen
tube remain where the former lodged on the nuccllus; the tube grows
down into the nucellus, carrying at first only the body cell, then the two
gametes produced by this cell.
The microspore, in most grows straight down from the tip
species,
of the nucellus to the archegonia. In Sequoiay Psevdotsugaj and Larix
the microspore lodges laterally and thus traverses the nucellus obliquely.
In the Araucariaceae, the microspore comes to rest at various locations
on the ovuliferous scale, or in its axil, or on the ligule.
Longitudinal sections of the entire young ovulate strobili almost
always show various stages of pollination and the germination of the
microspore. If a series of developmental stages of the ovulate strobili
are examined, all the different steps may be reconstructed. If the species

is a dioecious one, pollination and fertilization obviously will not be

found in ovulate strobili if staminate trees are not present in the imme-
diate neighborhood.
Growth of the microgame tophyte, generally called ^Hhe interval
between pollination and fertilization,^’ occurs over a highly variable
period, and may even be sporadic or with a resting interval, but commonly
requires months to a year or longer. The time of pollination is a highly
variable factor, but the duration of microgametophytic growth is rather
constant for the species. In other words, if the time of pollination is on
record from previously observed instances, the period required for
be effected can be computed with a high degree of accuracy
fertilization to
from the following table, citing instances recorded in the literature.
 jj

432 SPECIAL METHODS FOR THE VARIOUS PHYLA

Pinaceae: Abies halsamineaj 4~5 weeks; Cedrus deodara, 8 months;

PiniLSy 13 months in most species; Pseudotsuga, 2 months; Tsuga cana-
densis 6 weeks; Picea excelsa, 1 month.
j

Araucariaceae : Araucaria hraziliana, 6 months; Agathis australis

1 year,
Sciadopitaceae: Sciadoyitys, 14 months.
Taxodiaceae: Sequoia sempervirensj 6 months; Taxodium distichum^
3 months; Cryptomeria japonica, 3 months; Cunninghamiuj 3 months.
Cupressaceae Lihocedrus decurrenSy 2 months; Juniperus communis
:

12 months; J. virginiana, 7 weeks; Actinostrohus pyramidalis, 3 months.

Podocarpaceae Dacrydium intermediumy about 3 months.
:

Taxaceae: Taxus baccatUy 1 month in some localities to twice as long

in others; Torreya taxifoUoy months.
Since the species most likely to be available in the United States
are included in the above list, it serves as a fairly reliable guide in making
collections. It should be borne in mind that in those species in which a
long period elapses between pollination and fertilization there is generally
a rapid initial development of the microgametophyte. The latter grows
down as far as the free part of the nucellus and there remains until
growth recommences.
As has already been noted above, stages in pollination and growth
of the microgametophyte are to be found in sections of the ovulate strobili.
The same technical methods required by the latter are generally equally
satisfactory for stages in the development of the sperms, but somewhat
more care is required with the staining. It would be better if the paraffin
sections were examined under the microscope imjnediately after mounting
and before the water has entirely evaporated under the sections (after
drying it is too difficult to observe structures clearly), and those that
appear to contain the desired stages may be picked out and stained
particularly for microgam'ete formation. Safranin is usually good
enough, but if a critical stain is desired, iron hematoxylin may be applied
provided the sections are not over 12/i thick. A triple combination may
sometimes come out sharply enough.
—
Microgametes. Microgametes of the Coniferales are of two types:
naked nuclei or highly organized cells. The latter type is presumed to
be the most primitive form since it resembles the sperms of the cycads
and Ginkgoy but differs in that blepharoplasts which produce cilia are
lacking. These well-developed sperms occur in the Cupressaceae,
Podocarpaceae, Taxaceae, and Taxodiaceae. They are produced in
pairsand are equal in size in most of the genera but are greatly unequal
in CephalotaxuSy Taxus, and Torreya. In Cupressus arizonica (Doak
1032) and C. goveniana several small sperms are present. The sperms
are naked nuclei in the Pinaceae.
 ^

CONIFEROPHYTA 433

Obtaining slides showing the microgametophyte with the micro-

gametes is almost wholly a matter of chance. Material collected at
about the time that fertilization is presumably to be effected is most
likely to show these structures. A large number of ovulate strobili
will have to be sectioned, and the apex of every ovule should be carefully
scrutinized. Any satisfactory slides that might be found are of great
intrinsic value.
Ovulate Strobilus. —The point and time
of origin of ovulate strobili
are quite unpredictable in most Getting the very earliest stages
species.
is a complete matter of chance since it is impossible to distinguish between

purely vegetative buds and ovulate buds. The best that can be done is
to preserve a large quantity of suspected buds, to embed and section them,
and to examine the sections under the microscope before staining them.
If present, the ovulate strobili are easily detected, and the sections
containing them can then be stained. The ovulate strobili, in the
bisporangiate forms, appear to originate slightly later than do
the staminate strobili. After the ovulate strobilus has broken through the
enveloping bud, it is, of course, easily recognized.
The ovulate strobilus in the Coniferales is a compound structure,
in the sense that the ovules are not carried directly upon the axis of the
strobilus, as are the microsporangia. Save for the Cupressaceae and
in a few isolated genera, the bracts with their associated structures are
spirally arranged on the axis. The strobili vary enormously in size,
ranging from the small berry-like cones of Juniperus communis 6 to
8 mm. in diameter, to the huge woody cones of Pinus coulteri, which
average 40 cm. in length. They are also very variable structurally,
but at maturity most of them are hard, woody, and difficult to cut open.
The ovulate strobilus of all genera is readily sectioned in paraffin
or celloidin until the ovules are at the late free-nucleate stage. Remove
slabs from opposite with formalin-aceto-alcohol, dehydrate
sides, fix

slowly with tertiary butyl alcohol, embed in a hard paraffin (or in cel-
loidin), and soak exposed portions under water for about two weeks,
whereupon longitudinal sections as thin as lO/z should cut smoothly.
The tips of the scales will give the most trouble. If one will go to the
trouble of trimming off all projecting scale tips, especially from most
species of Pinus, a lot of exasperation will later be avoided. Transverse
sections can be cut just as easily but are of little service other than for
general structure of the strobilus. Stain carefully with safranin and
anilin blue or a triple or quadruple combination.
Megagametogenesis. —The initial cell of the megagametophyte
is the megasporocyte. It arises soon after the origin of the ovule by
the periclinal division of a hypodermal archesporial cell into a tapetal
cell and the megasporocyte.
434 SPECIAL METHODS FOR THE VARIOUS PHYLA

In the majority of species the megasporocyte is a single cell, but in

some species, such as Pinus muricaia and P. contorta, groups of mega-
sporocytes are regularly formed. Such groups are also present in Taxus,
where several megaspores may germinate and reach advanced develop-
mental stages, but a similar condition is not known in the species of
Pinus just mentioned.
It is not at all easy, on the whole, to obtain slides showing the mega-
sporocyte, and it is extremely difficult to get the meiotic divisions whereby
a linear quartet of four megaspOres is formed. The writer has sectioned
and examined several hundred ovulate strobili in many species of Pinus
but in all that material he has never encountered either a reduction
division or quartet, although the stages immediately preceding and
succeeding were frequently observed. Scarcely anything has been
described in the literature regarding formation of the quartet, but it is
known to exist; it is also known that the megaspore nearest the axis of
the strobilus becomes the functional one, the three others aborting.
Cases are on record in which only three megaspores were apparently
produced, the outer cell formed following the reduction division failing
to undergo the second mitosis.
The functional megaspore enlarges and, in all the Coniferales, embarks
on a period of free nuclear division. The extent of this period, and con-
sequently of the number depends upon both the
of nuclei produced,
size and the shape of the swollen In long and narrow cells
megaspore.
the period is much shorter than in those that are nearly spherical. Low
numbers of nuclei occur in Taxus (256) and high numbers in Juniperus
and Pinus (2000). The free-nucleate stage is comparatively easy to
obtain in but careful fixation is required. If entire strobili are
slides,
fixed for this stage, there wi,ll be a little plasmolysis, the term here
meaning that the thin layer of protoplasm with its nuclei is wrenched
away somewhat from the periphery of the megaspore. The ovules
should be carefully cut away from the ovulifprous bract and fixed sepa-
rately in a strong chrom-acetic fluid. Since \he tissues dry out rapidly,
one should work fast, and material not being worked upon should bo
kept between damp cloths. A great deal of the unpleasantness involved
in working with pitchy cones can be avoided if leather gloves are worn.
After embedding, section the ovules perpendicular to their flat sides; if
cut parallel to the flat sides, only oblique sections are produced.
Wall formation presently sets in, and when more or less completed,
the formation of archegonia commences at the apex of the megagameto-
phyte. There are two general types of archegonial plexus, and the
number of archegonia developed is directly related to the type of the
group. In one plexus the archegonia are separated by vegetative
tissue; in the other they are in contact with one another. In the first
 CONIFEROPHYTA 435

group the number of archegonia is small, and they are arranged more or
less ina circle around the center. Pinus is of this type, the number of
archegonia ranging from two to not more than six. In the species
(common in Taxodiaceae and Cupressaceae) with an archegonial complex,

Fia. 93. —
Pinua laricio: longitudinal section of an ovule with two archegonia; one
at left with neck cells, ventral canal cell and egg nucleus. Fixed with strong chrom-acetic;
stained with safranin and anilin blue.

the number of archegonia ranges from 6 to 200. Sequoia sempervirens

has both types of archegonial plexus, while in Torreya taxifolia only a
single archegonium usually occurs. The archegonia in their youngest
stages are extremely difficult to find in most species, and the sectioning
and staining of considerable material are generally required. Among the
 y

436 SPECIAL METHODS FOB THE VARIOUS PHYLA

pines, P. laricio is the most favored species. lAhocedrus decurrensy

Thuja and Juniperus are good, but the genus with the largest number of
archegonia (Widdringtonia) neither occurs nor is cultivated in the
United States.
Division of the nucleus of the archegonial initial occurs about a
week after the cell is first recognizable. This results in the central cell
and the primary neck cell. The latter soon divides, but the central
cell requires about a month in which to become very greatly enlarged,
and its nucleus then divides to form a ventral canal nucleus or cell and
the egg nucleus (Fig. 93). This division evidently proceeds rather
slowly since it is commonly found in slides. Beginning with this stage,
it will be found advisable to cut sections somewhat thicker than before;

14 m is about right. Material should have been collected daily as soon

as the archegonia are known to have begun growth, since development is
so rapid that important stages would be missed in case a critical study of
the life history was being undertaken. As all the ovules in a given
strobilus develop nearly simultaneously, the ovules from any one strobilus
should be kept separate.
Fertilization. —The term ^^fertilization’’ is here taken in its broad
sense, to include all phasesfrom the arrival of the microgametophyte
immediately above the megagametophyte, up to and including the first
zygotic mitosis. The actual union of the male and female nuclei, to
which the term has by some been restricted, is more properly called
^^syngamy.” The latter term, nevertheless, has in turn a fairly broad
meaning since it includes both cases of actual nuclear fusion (as more
commonly occurs in Angiosperms) and those in which there is no fusion
of chromatin.
The probable occurrence of fertilization may be determined by
the external appearance of the ovulate strobili (Buchholz 1938): this
stage occurs at about the time, or just shortly before, the strobili have
reached their maximum size, following the period of rapid enlargement.
It has been claimed that this general size rule is infallible, with the
exception of Taxus, in which fertilization occurs long before the ovule
is fully grown, and certain of the Podocarpaceae, in which fertilization

takes place before the ovules themselves have attained their full size.
The microgametophyte reaches the megagametophyte while the
latter is in various stages of development, which differ according to
the species. In Torreyay for example, the megagametophyte is still in the
early free-nucleate condition, while in Pinus, at the other extreme, the
archegonium has almost reached maturity. There is a direct relation
between the stage of megagametophytic development and the nature of
the microgametes: where the latter are highly organized cells, the earlier
the stage of development.
 CONIFEROPHYTA 437

Fertilizationphenomena differ according to the family:

Pinaceae: At the tip of the microgametophyte a pore is formed by
rupturing, and the contents are ejected with considerable force. Sperms,
stalk, and tube nuclei are all discharged into the egg.

Fig. 94. —Pinus laricio: longitudinal section ofan ovule with syngainy progressing
simultaneously in two adjoining arohegonia. Fixed with strong chrom-acetic, stained with
safranin and anilin blue.

Sciadopitaceae: In Sciadopitys (with scattered archegonia), both

sperms enter.
Taxodiaceae: In all genera (with archegonial complexes) only one
sperm enters.
Cupressaceae : In Juniperus only one sperm enters, but the second
may follow it.
438 SPECIAL METHODS FOR THE VARIOUS PHYLA

Araucariaceae : Both sperms and some of the smaller nuclei (stalk,

tube, and prothallial, which all look much alike at this time) enter the
egg.
Podocarpaceae Although both sperms and stalk and tube nuclei enter,
:

all save one sperm nucleus remain on top of the egg and disorganize.
Taxaceae: In Taxus baccata only one sperm enters, but in T, cana-
densiSf sperms and stalk and tube nuclei all enter. Torreya taxifolia
resembles T, baccata in behavior.
Cephalotaxaceae In Cephalotaxus both sperms enter, but one remains
:

at the top and there degenerates.

The interpretation of structures within the megagametophyte during
fertilization and syngamy is fraught with great danger. It is extremely
easy to make misinterpretations, especially as there are so many bodies
having the appearance of nuclei. If in doubt as to the identity of any
structure, the (ioverslip may be removed from the slide, the sections
bleached free of stains, and Fuelgen\s reaction may be applied. This
will generally give a specific reaction to the nuclei and chromosomes
directly concerned with fertilization and particularly with syngamy.
Accounts of syngamy are meager (Beal 1934, Hutchinson 1915).
Various stages in fertilization and syngamy may be found by chance.
Species of Finns appear to be most suitable, since fixation and staining
are not at all difficult (Fig. 94). If a stage close to syngamy is found in
any ovule, the other ovules from the same strobilus are very likely to
yield earlier or later stages. Longitudinal sc^ctions of the ovule should
be cut at about 14ju; safranin will give a clear chromosome stain and may
be followed with a counterstain of anilin blue. Since the spindles are
very prominent, especially during the postsyngamic phases, particular
attention should be paid to clear staining of these structures.
—
Embryogenesis. In all Coniferales, with the exception of Sequoia
sempervirens, free nuclei are formed immediately after the division of the
syngamic nucleus. In Sequoia walls are formed after the first and sub-
sequent mitoses. The free nuclei are produced in the center of the
archegonium; they then migrate to the bottom of the archegonium,
and wall formation commences at the conclusion of the last mitosis in
such a manner that there are two or more tiers of cells with an average
of four cells in each tier. The next mitosis occurs in the cells of the
upper tier, followed by nearly simultaneous divisions in the lowermost
tier. Depending upon the species, the lowest or the next to the lowest
tier of goes into the formation of the embryonal organs root,
cells —
stem, cotyledons, and leaves.
The mimber of free nuclei and also the number of tiers of cells is
somewhat variable:
Pinaceae: Usually 4 free nuclei and 4 tiers.
 CONIFEROPHYTA 439

Araucariaceae : 32 free nuclei in Araucaria braziliana, 32 and 64 in

Agathis australis; 3 tiers.

Podocarpaceae Phyllocladus has 8 free nuclei, Podocarpus 16.

:

Cephalotaxaeeae Cephalotaxus has 8 free nuclei and 3 tiers.

:

Taxaceae: Austrotaxus has 8 free nuclei, Taxus has 16 and occasionally

32, but Torreya has only 4.
Cupressaceae Usually 8 free nuclei and 3 tiers.
:

Taxodiaceae: Usually 8 free nuclei and 3 tiers.

These early stages are readily found if the material has been col-
lected at the opportune time. The ovules at this stage are broader in
one direction than in the opposite plane; they should be microtomed at
12/x parallel to the broader surface. Stain critically with safranin and
anilin blue. Mitotic figures are frequent. After the tiers of cells are

developed, it is very common to find more or

which isless plasmolysis,
probably due more to partial desiccation of the tissues than to bad
fixation. Some technicians claim that this difficulty can be remedied
by soaking either the cones or the ovules in water before fixing, but
from the writer’s experience the remedy seems to be worse than the
disease.
The most significant stages during embryogenesis are those occurring
after the organization of theproembryo and preceding the differentiation
of the embryo into the various tissues and organs. Sections of entire
gametophytes were formerly used for the study of these stages, but such
sections are inadequate in that they never present complete pictures and
many details are so obscured that their presence can hardly be detected.
It is, for example, impossible to make out much of the phenomenon of
cleavage polyembryony, or to differentiate between primary and second-
ary suspensors. For these stages whole mounts of the dissected sus-
pensor-embryo system are required. It takes but a little practice to
prepare relatively satisfactory mounts which are not only intrinsically
valuable in themselves but are very useful for purposes of instruction.
Living material is always preferable to preserved or fixed gameto-
phytes (Buchholz 1929, 1938); the writer has had extremely poor success
with fixed material. The dissections, in the case of live material, should
be made under water or preferably under a 15% sucrose solution, but
equally good results may be obtained if the dissection is done under
formalin-propiono-alcohol or under 6% formalin in 50% alcohol. The
beginner will find the chances of the cells rupturing to be less in a fixative
than in plain water. The ovules should be removed from the strobili;
if a number are being removed at one time, they should be placed between

damp cloths. In dissecting large, pitchy cones, such as those of Pinus,

it willbe less messy to wear a pair of stout leather gloves and to remove
60 or so ovules at one time, The hardened seed coats and membranes
440 SPECIAL METHODS FOR THE VARIOUS PHYLA

are next removed, and the gametophytes are placed in a watch glass or
either half of a small Petri dish containing the sugar solution or fixative.
The container is then placed on the stage of a binocular dissection

microscope; both reflected and transmitted illumination are ordinarily

necessary for clear observation.
Finns, since material of this genus is most easily collected and the
gametophytes are large and thus more easily manipulated, will be taken
as an example in the following discussion of the technique of removing

A B E F
—
Fia. 96. Dissection of Pinua gametophyte (based on P. poiideroad): A, external view
before operation is begun; B, optical section showing extent of corrosion cavity note—
that it is narrowest at the neck; C, optical view showing how deep the preliminary cuts
should extend; D, the operation nearly completed; E, cuts extended almost to corrosiofi
cavity and operation completed; F, remaining tissue broken and suspensors partially
withdrawn. (Drawing hy Mrs. Carl F. Janiah.)

the suspensors and embryos. shown the gametophyb^

In Fig. 95^4 is

removed from the testa and ready 955 is a median

for operation; Fig.
longitudinal section through the gametophyte, designed to show the
average extent of the corrosion cavity occupied by the suspensors and
embryos at the time; Fig. 95C is a combination of the two in optical
section and shows (by dotted lines) the points at which cuts are to be
made or portions of gametophytic tissue are to be removed. The anterior
end of the gametophyte appears more or less like a cap; consequently
the position of the transverse incision extending completely around the
gametophyte is easily determined. For making the incisions a very
small cutting instrument with narrow, sharp blade is required. Some
 CONIFEROPHYTA 441

technicians use a needle flattened and sharpened to give a spear-shaped

end; others use a scalpel with a spear-pointed end, of which only one
edge is sharpened. Hold the posterior half of the gametophyte, as
gently as possible, with weak-springed forceps, then, with the gameto-

—
Fig. 96. Pinua ponderoaa: dissected whole mount of proembryos and suspensors.
Although the preparation is brilliantly stained, this object does not photograph well.
Fixed with forraalin-propiono-alcohol; stained with Harris’ hematoxylin and fast green;
dehydrated with hygrobutol and infiltrated with balsam.

phyte submerged in the fluid and the operation carried out under the
microscope, cut away wedges of tissue at the anterior end, completely
—
around the gametophyte in somewhat the same way that one cuts a
log in two with an axe. The nearly completed operation is shown in
Fig. 962). Complete the lateral incision with great caution, lest the
suspensors be cut into, by making a short, straight cut into the corrosion
 442 SPECIAL METHODS FOR THE VARIOUS PHYLA

cavity, as shown in Fig. Grasp the anterior cap with another pair
95jS?. ‘

of weak-springed forceps, and pull gently by means of successive jerks.

The suspensor-embryo complex usually comes out in a few moments
(Fig. 95F). During later stages of embryo growth, some of the embryos
may become wedged into the gametophyte at various points and may
readily become broken off. If the suspensors pull straight without the

embryo becoming dislodged, make a longitudinal incision down the

center of the gametophyte. A succession of short, jerky pulls is generally
more effective than a steady pull, and embryos are less likely to become
broken off. If the embryo complex still does not come out without
breaking off, wedges of gametophytic tissue may be cut away from all

around until the complex has been sufficiently loosened, but this should
be done only as a last resort since there is danger of cutting off embryos.
If the embryos were dissected in sugar solution, they should be trans-
ferred immediately to a fixing fluid, in which they may remain inde-
finitely. The embryos should be washed with water before being stained.
As the basic stain, Harris’ hematoxylin diluted one-half with 50%
alcohol or safranin is equally satisfactory; for counterstaining, orange G
may be used after the hematoxylin, and fast green after the safranin.
The older technicians mounted the embryos in Venetian Turpentine,
a slow and erratic procedure; they later dehydrated through 25 and 50%
ethyl alcohol and then mounted in diaphane. By far the better method,
however, is employ the gradual hygrobutol method (Fig. 96).
to
For paraffin sections of the same stages, remove the gametophytcs
as before, then cut off slabs from opposite sides, taking care not to
expose the corrosion cavity. Place in either formalin-aceto-alcohol
or a medium ehrom-acetic Dehydrate slowly, and infiltrate with
fluid.

paraffin over a period of several days. Microtome parallel to one of the

cut surfaces at 10 or ll/x. The ribbons are likely to give some trouble,
as they have a tendency to cling to the knife and other objects. If
this happens, only the most careful handling will help matters. Serial
sections are more useful than the usual two or three to a slide. Staining
is precise with safranin and fast green or a triple combination, but iron

hematoxylin and orange G may be used if desired.

In the upper portion of the corrosion cavity oi Finns and some other
genera there will usually be found a number of rosette embryos. These,
as well as all save one of the primary embryos, become aborted.
When several archegonia are present and an embryo arises from
each whose egg is fertilized, simple polyembryony is the result. When
each fertilized archegonium gives rise to several embryos, the phe-
nomenon known as cleavage polyembryony” occurs. The embryos
resulting by cleavage from a single zygote constitute an embryo system;
jl^en several embryo systems are confusedly intermingled, the entire
 CONIFEBOPHYTA 443

mass is known as an '^embryo complex/^ Among all these embryos,

the one possessing the greatest growth vigor is the one that develops to
maturity; it can frequently be recognized during early stages by its
straighter, stouter, and more rigid suspensor. The salient features
of the embryology in the different families, as far as known, may be
summarized (Buchholz 1929) as follows:
Pinaceae: Indeterminate cleavage polyembryony in Pinas, Cedrus,
Tsnga; each rosette cell may put out a suspensor with a multicellular
embryo {Cedrus, Pinus), or the rosette cells abort early (Tsv^a). Abies,
PseudMsuga, Picea, and Larix form only one embryo from each arche-
gonium. Rosette cells are present, soon collapsing; early growth apical.
Abies undergoes cleavage in a few cases. No rosette cells in Pseudotsuga.
Araucariaceae: Highly specialized; probably a group of polyem-
bryonic initial cells rather than a single embryo, f.c., a compound embryo.
Sciadopitaceae : Cleavage polyembiyony. Rosette cells none, few
or many. Apical cell present.
Taxodiaceae: Cleavage polyembryony in Taxodium and Cryptonwria.
Rosette tier proV)ably absent; growth apical.
Cupressaceae Cleavage polyembryony; rosette embryos probably
:

present {Juniperus, Biota, Libocedrus). Determinate cleavage poly-

(*mbryony in Dacrydium, Primary and secondary embryos resulting
from cleavage polyembryony in Chamaecyparis. Single embryo in
Thuja; growth distinctly apical; apparently no rosette cells.
Podocarpaceae Lowest proembryonic cells binucleate in all species
:

investigated; several types of embryonic development; cleavage poly-

‘embiyony in some species; rosette cells present or absent; sometim6^s only
one embryo from each archegonium; apical growth occasional.
Cephalotaxaceae Considerable similarity to conditions in preceding
:

family, but proembryonic cells are uninucleate. Rosette embryos

present. Distinctive deciduous cap cells present.
Taxaceae: Simple polyembryony; rosette cells rare, absent in Torreya;
growth apical tendency toward cleavage.
;

For the later stages in the development of the single embryo, game-
tophytes may be treated as for the earlier stages up until such time as
the embryo fills most of the corrosion cavity. After this period it is
better to dissect out each embryo and to fix it separately. For mature
embryos of Pinus and certain other genera, seeds (which may be pur-
chased from a seedsman) may be soaked in water for two or three days to
restore turgor, then the embryos may be dissected out. Fix with form-
alin-aceto-alcohol, section at not over 12)u, and stain with either
safranin and fast green or a triple combination.
The order of app)earance of the various regions in the embryo is as
follows: plerome tip of root, calyptoperiblem, stem tip, cotyledons and
444 SPECIAL METHODS FOR THE VARIOUS PHYLA

hypocotyl, epidermis, procambium, resin passages, leaf primordia,

protoxylem elements in the cotyledons.

Gnetales
The order contains three genera. Ephedra^ Welwitschia, and Gnetum,
generally combined into the one family, Gnetaceae. The two last-
named genera do not occur naturally in the United States, but species
of Gnetum may rarely be found in cultivation in botanical gardens.
Ephedra, on the other hand, is one of the characteristic plants of the
Southwest, and it is widely distributed elsewhere.
It is so doubtful whether material of Gnetum and Welwitschia would
be available that no further description of these genera will be pre-
sented. In any event, the various structures will require similar treat-
ment to that described below for Ephedra.
Ephedra occurs abundantly in certain parts of Arizona, New Mexico,
and California; in the last state it reaches as far north as the Paiioche
Pass, west of Fresno. The plants are so characteristic that once some
have been seen, the genus can always be readily recognized thereafter.
They form straggling, rough, xerophytic shrubs rarely over 2 meters in
height. In color they are grayish most of the year, becoming a bright
green in the younger portions in late winter and early spring, but some
Asiatic species when brought into cultivation become mesophytic and
remain a vivid green. Anatomical material can usually be obtained
from the botanical supply concerns, but care should be taken to specify
that it is for slide-making purposes, since preserved materiar' is

worthless.
Contrary to the statements in some texts {e.g., Chamberlain 1932),
Ephedra is easy to cultivate, except where it might be snowed under
for long periods. The Asiatic species are more amenable than the North
American species; E. distachya and E. alata have flourished and produced
great quantities of staminate and pistillate strobili in central California.
Transplanted plants of the Californian species can be obtained from
nurserymen specializing in desert plants in that state.
—
Root. A persistent tap root is present, but the lateral roots furnish
more satisfactory material for Slides. Root hairs are easily obtained
from young cultivated plants but are hard to find on plants growing
in nature. In the root tip there is little differentiation into growth
regions; consequently slides of this part of the root are not of so much
usefulness as are sections of older regions.
Younger roots may be fixed in formalin-propiono-alcohol and micro-
tome easily. Both transverse and longitudinal sections should be made;
safranin and anilin blue is a satisfactory stain combination, but others
may be expeiimented with.
 —

CONIFEROPHYTA 445

Stem. —Young stems, a few months old, section easily in paraffin

and afford most instructive preparations (Fig. 97). They reveal clear
evidence that photosynthetic activities are carried on within them.
Longitudinal sections of the first two to four nodes will show the meri-
stematic region at the base of each internode, also the development
of the abscission layer. The beginning of the vascular system will bo

Fig. 97. Ephedra alata: cross section of a young stem, showing assimilating tissue,
dark tannin-containing cells and gaps in central region. Fixed with formalin-aceto-
alcohol; stained with safranin and fast green.

encountered at about the fourth internode. Microtome young stems

at not over 12/x, and stain with safranin and anilin blue (or fast green,

with caution, may be used).

Older stems become hard and rigid, but those of many species can
be sectioned in paraffin following prolonged softening under water of the
embedded material. The tissues are so compact that sections thinner
than 12/1 are necessary. Very old wood is not required to show the
characteristic structure elements; stems two to three years old show
everything clearly. Older wood requires treatment with 50% hydro-
446 SPECIAL METHODS FOR THE VARIOUS PHYLA

fluoric acid for two months, following which dehydration should extend
over a period of about two weeks, and the time in the paraflB.n oven should
take a week. Since the wood exhibits both angiosperm and gymnosperm
characters, sectioning and staining should be carried out with this feature
in mind.
Leaf. —
Most species possess only small scale-like leaves, but E,
foUaia from India has distinct leaves 1 cm. long (Fig. 98). In Gnetum
the leaves are broad and externally greatly resemble those of Angiosperms.
The leaves of Welwitschia are the most characteristic feature of that
genus.

Fig. 98 , —Ephedra foliata: cross section of loaf. Fixed with formalin-aceto-alcohol; stained
with saf ratlin and fast green.

—
Staminate Strobilus. Exceptions may occur with cultivated speci-
mens, but in most species the staminate strObili originate in December,
the microsporocytes are to be found early in February, and meiosis
occurs early in March (or a little later if the weather has been unusually
cold). At the time of shedding in mid- April, the microgametophyte
contains two prothallial cells, one of which is naked and the other is
cut off by a wall, a stalk cell, a body cell, and tube nucleus (Land 1904).
The staminate strobilus at all stages is well fixed with formalin-
aceto-alcohol, but for the meiotic figures the strpbili should be dissected
somewhat and fixed in a strong chrom-acetic fluid. Longitudinal sections
at 10m are most satisfactory. Staining may be in safranin with a suitable
counterstain; iron hematoxylin may be used on thin sections for cytologi-
cal details.
Germination of pollen grains may be observed by sowing them in
10% saccharose. In the ovules the grains come to rest at the bottom
of the pollen chamber very close to the archegonia, into which the
microgametophyte grows directly (Land 1907).
 CONIFEROPHYTA 447

—
Ovulate Strobilus* The ovulate strobilus originates at about the
same time as the staminate strobilus but does not grow with equal
rapidity. The two may be distinguished in that the staminate strobilus
is shorter and broader. The integuments and archesporial cell appear
about the first of March, and the reduction divisions occur within a few
days. Free-nuclear divisions then take place in the functioning basal
megaspore, covering a period of about 20 days. Wall formation next
sets in, and the archegonial initials appear about Apr. 1. The average
number of archegonia is two, but there may be three in some species
(Maheshwari 1935).
Excellent fixation of all stages in the growth of the ovulate strobilus
is given with formalin-propiono-alcohol.The bracts should always be
removed, and from the older flowe^rs the outer integument should be
dissected away. The material at all times should be handled very
carefully to avoid crushing. Sections should be microtomed rather thin,
lOju being the optimum thickness Safranin and fast green may give
good staining, but it appears that iron hematoxylin differentiated with
picric acid is superior for nuclear details.
Fertilization. — Pollination occurs about Apr. 1, but the exact date
is greatly dependent upon climatic conditions. Fertilization may occur
within 10 hours after pollination; consequently it is a difficult matter to
catch this stage.
—
Embryogenesis. Eight free nuclei arise following fertilization; of
these, from three to five develop into proembryos. Only one embryo
attains maturity.
For the earlier stages in (mibryogenesis prepare the material as for
older stages in archegonial development. For the later stages remove all

superfluous tissue possible, and cut slabs from opposite sides of the
gametophyte. The earlier stages may be sectioned at 12jLt, but later
stages should not be over lOju. Safranin and fast green have given excel-
lent staining of all stages.
 CHAPTER XXXI
ANTHOPHYTA
To the technician there is little difference between the dicotyledons
and monocotyledons; consequently the Angiosperms will be dealt with
by structures rather than by the phylogenetic sequencer which has bec^n
followed thus far. The dicotyledons, on the whole, are more difficult
than the monocotyledons by reason of their greattu* structural com-
plexities, but the technical methods nevertheless are essentially similar
for the two groups. If materials intended for research purposes an^

disregarded, the problem is one of finding the plant whos(^ root tips,
leaves, or whatever the structure may be, are most suitable from a
particular standpoint. It is possible in most cases to find equally satis-
factory species from both groups.

ROOT TIPS
The manner of treatment of root tips depends entirely upon the
purpose for which they may be wanted. The method to be followed if

the somatic complement of chromosomes is to be counted is entirely

different, for example, from the one to be used if it is merely the general
structural details that are wanted.
Methods of obtaining tips, however, are the same, no matter what
the ultimate purpose for which they are to be used. It is not a simple
matter of going out into the garden and digging up plants, for, as a
matter of fact, it is a very rare occasion indeed that any can be secured
in this way. The tips should be specially grown according to the method
most suitable for the plant concerned.
—
Securing Root Tips. Bulbs which produce roots quickly, as of the
Allium cepa and Hyacinthus type, may be germinated as follows: Bottles,
such as milk or cream bottles, with mouths of a diameter just wide enough
to hold the bulbs by their bases are the most convenient. Otherwise
obtain some wide-mouthed jars of about 1-quart capacity. If a suitable
place in total darkness is not available, paint the jars on the outside with
black paint. Fillthe jars to the top with a suitable nutrient solution
(Knop's or Pfeiffer’s); the solution must be thoroughly aerated, otherwise
the roots will bend upward. For the wide-mouthed jars suitable holders
be provided by cutting cardboard into squares wide enough to
stiff

project about 2 cm. beyond the mouths, then cut circular holes from the
centers of the cards of a size sufficient to allow the root ends of the bulbs
I 448
 y

ANTHOPHYTA 449

to project to a depth of about 2 cm. into the nutrient solution. To

prevent the cards from rotting, dip them into hot paraffin until they are
saturated, then remove, and cool. Insert the bulbs into the jars, then
set the whole in a dark place at ordinary room temperature. The tips
of Allium cepa are sufficiently long for removal in four days; those of
Hydcinthus require about 10 days. The type of hyacinth used should be
selected with care because most of the varieties on the market are either
triploids or have irregular chromosome numbers. The diploid variety
Yellow Hammer is best of all, with Gertrude as second choice. Other
diploid varieties of H. orientalis include: Garibaldi, Cardinal Manning,
Flora, King of the Yellows, and Roi des Beiges. Excellent triploid
(3n-24) varieties include: King of the Blues, Lady Derby, Grand Maitre,
and Lord Balfour.
All other bulbous plants should be started in clay pots they require ;

a longer time than the onion and hyacinth to develop roots and do not
produce so satisfactory tips in aqueous media as in soil. Moreover, the
longer period requin^d for development is prone to allow excessive
growth of bacteria and aquatic molds which penetrate the tips and spoil
them. Peat Is sometimes used in the pots and is excellent for all bulbs
and tubers wliose roots arc not covered with slime: Tulipa, Erythronium,
HyacinthuSf Crocus Narcissus Liliumy Gladiolus and tuberous species of
y y

Anemone and Ranunculus may be planted in peat. Shred the peat

finely and pack tightly into 6-inch pots. In order to allow as much room
as possible for the roots, insert the bulbs or tubers so that the crown is
just below the surface. Allow a single large bulb (as of Tulipa or
Lilium) or two or three smaller bulbs {Crocus or Anemone) to each pot.
Finely sifted garden loam, free from sand, may also be used. Place*,
a piece of broken pottery over the hole in the bottom of the pot, and
insert the bulbs with the crown barely emerging from the surface of the
soil. Soak the pots thoroughly with water, and set aside in a cool,
dark place for at least two weeks. Some bulbs will require about a
month before the root tips are large enough for removal. Take care
not to let the pots become dry. To remove the roots, knock the entire
mass out, wash quickly but thoroughly under running water to remove all
soil and other debris, then cut ofif the tips with a scalpel, and place in the
killing fluidwith the least possible lapse of time.
From annual species which grow very rapidly in the seedling stage,
root tips may be secured by germinating the seed in fine soil in any
suitable container; as soon as the first leaves are well developed, remove
the seedlings from the soil, insert individually in paraffined cardboard
squares, using nonabsorbent cotton to hold the stem in position, and
place the cards over the mouths of jars filled with nutrient solution.
Great quantities of fine tips should be available in about a week's time.
 :

460 SPECIAL METHODS FOE THE VARIOUS PHYLA

If it is desired to grow the plants to maturity, they may then be replanted

in clay pots and later transferred to the open garden.
Seeds of perennials and slow-growing annuals should be planted
in suitable seedbeds (pots or boxes), transplanted to 2-inch pots (which
should then be placed in a large shallow box with the interstices packed
with damp moss) if the mature plants are of small stature, or into 6-inch
pots if they grow to a large size. Fine soil without sand or organic
fertilizer should be used as the medium. In from two weeks to a month
the space between the soil and the pot becomes filled with roots and root

tips; few tips, if any, will be found within the soil. Place the base of the
stem between the index and middle fingers of the left hand, invert
the pot, and knock the edge against some solid object in order to cause the
mass of soil and roots to come free. The tips may then be snipped off
by means and placed in the killing fluid. The
of fine-pointed forceps
plant may be and grown on until buds are produced
reinserted in the pot
in this way both the monoploid and diploid chromosomal complements
of the same plant may be investigated.
—
Treatment of Tips for Cjrtological Purposes. This subject is dis-
cussed in Chap. XIV. In many species there are definite periods when
mitoses occur with maximum frequency and other periods when they
are at the minimum. In Allium cepa the maximum periods are between
11:30 p.M. and 12:30 a.m., and 12:30 ^.m. and 1:30 p.m. the minimal ;

periods are around 7 a.m. and 3 p.m. In Viciafaha the maximum period
is from 7 p.m. to 1 a.m.; in Pisum sativum from 9:30 p.m. to 1:30 a.m.

However, in most plants mitoses can be found in material collected at

any time if a large number of sections from a good many roots are
searched.
Treatment for Anatomical Purposes. —The tips should be fixed as
for cytological purposes, i.e., preferably with N avashin\s flui d. Sections
may be cut in any desired plane ;
since the chromosomes are of no particu-
lar significance, the thickness should be uniformly 10^ for all species.
If a single stain is desired, Harris’ hematoxylin is superb; safranin and
fast green give an excellent double stain, and any triple combination
should be useful.

ROOT HAIRS
Root tips grown in water or in nutrient solutions rarely have root
hairs; those taken from plants grown in pots sometimes have root hairs,
but these are usually not in a very satisfactory condition. A few plants
have roots that are abundantly covered with root hairs that are well
shoiVR in sections of the roots. Among such plants are nearly all species
of th« Poaceae and TriUium, Vagnera, Raphanus, Brassica, Solidago,
 ANTHOFHYTA 451

Aster Eupdtoriunij and Gleditsia,

^ Roots which soon become woody or
fleshy are ordinarily poor for root hairs.
Some roots are better sectioned longitudinally for the hairs, par-
ticularly if the hairs are turned toward the tip, but others should be
microtomed transversely. In either case the sections should be some-
what thicker than usual: from 12 to 16/i should be about right. A
triple combination will give the most satisfactory staining.
By far the best method of demonstrating the origin and growth of
root hairs is to prepare whole mounts of root tips. The following species
are excellent: Raphanus sativus 'knd other members of the Brassicaceae,
Hordeum vulgare, Secale cerealcy Avena sativay and other cereals and
grasses. Sow the seeds thinly on damp filter paper in large Petri dishes
or similar moist chambers. In three or four days the roots will be
between 1 and 1.5 cm. in length. Cut off the roots at this time, if they
are abundantly covered with hairs, but avoid removing with older
portions that are over 1 mm. in diameter. Fix with Navashin^s fluid
or with a fluid giving a basic fixation image, wash thoroughly, then place
in either Mayer’s carmalum or Grenadier’s borax carmin for 24 hours.
Wash dehydrate with hygrobutol or dioxan,
briefly with distilled water,
infiltrate with highly diluted balsam, and evaporate the latter to a
mounting consistency. If the roots are over 0.5 mm. thick, they should
be mounted in depression slides. The hairs are well shown by this
method, as are any incipient lateral roots.

ROOTS
An amazing variety of roots are to be found in the Anthophyta:
fleshy, woody, tuberous, fibrous, etc., which may be subterranean,
aerial, or aquatic in habitat. The structure, however, is relatively
uniform, and the vascular anatomy is of the same general type. Some
roots are easy from the technical standpoint, but most of them arc
beset with difficulties of one sort or another.
In digging up plants in order to obtain the roots, great care needs
to be exercised to avoid strains, since it is extremely easy to cause separa-
tion of the tissues in different regions, as between xylem and cortex.
The roots should be washed thoroughly under running water before being
cut into sections about 1 cm. in length and placed in the killing fluid.

Grit that lodges in wounds and other breaks, in the exfoliating epidermis
and in other places, is a very cause of torn ribbons and bad
common
nicks in the cnicrotome knife. Use a sharp scalpel for cutting the roots
into portions. Formalin-aceto-alcohol and formalin-propiono-alcohol
fix nearly all roots perfectly, but a strong chrom-acetic fluid may also

be used. Small fibrous roots should be embedded in bunches it is j

difficult to locate the tiny sections of single very small roots in the com-
452 SPECIAL METHODS FOR THE VARIOUS PHYLA

pleted preparations. If a few trial sectionings on the microtome reveal

that some elements in the root have become too hard to cut, the material
should be soaked under water for several days to a month or longer.
Roots which contain much starch should be watched and sectioned as
soon as they acquire a whitish-opaque appearance; if left under water
too long, it becomes impossible to microtome them.
—
Origin of the Root. The origin, structure, and development of
the young root must be sought during the growth of the embryo. This
can be found in any suitable plant, preferably one from the monocotyle-
dons. Zea mays is most extensively used: obtain young pistillate
inflorescences whose stage of development is about a week or 10 days
after fertilization. Remove the ovules individually. The embryo is
readily recognizable; cut slabs from the ovule on each embryo
side of the
perpendicular to the flattened surfaces, fix with formalin-aceto-aclohol,
and microtome at 10/x in the longitudinal plane perpendicular to the
flat sides. The young seed sections easily at this time, but afterward
soaking under water will be necessary. Safranin and fast green or a
triple combination both stain sharply, but the latter should be employed
only if it is desired to color the starch grains. Canna indica has a large
embryo which is also good for root origin. Among the dicotyledons,
Capsella has been most commonly employed, but
it is a poor example.

The whole easy to follow out in the Onagraceae;

process of root origin is

Epilobium may be recommended, as it grows wild almost everywhere.

One might try soaking seeds in water overnight, then removing the
testa, and killing the seeds before the roots have emerged.
—
Secondary Roots. The origin of secondary (lateral) roots occurs
about 1 cm. back from the tip. The best material for this is to be found
on perennial plants or trees growing with their bases submerged in water:
Salix pendula and other willows, Sagittaria and related genera, Typha
(the rhizome is even better than the root), and Pistia are all excellent,
but the roots of completely submerged plants are poor. Seeds of Rapha--
nus and Vicia faba germinated on filter paper quickly produce roots
which should show the origin of the lateral roots at a distance of from
1.6 to 2 cm. back from the tip. Kill and fix in a strong chrom-acetic
fluid or in formalin-propiono-alcohol, section at % to 10/i in either plane,
and stain with safranin and a suitable counterstain.

TUBERS, RHIZOMES
The majority of tubers are comparatively easy from the technical
standpoint. The main precaution to be observed is with regard to the
mucilaginous nature of the cell contents of many tubers and rhizomes.
A fixative which will not cause excessive cytoplasmic shrinkage should
be^lised; if acetic acid is an ingredient, the least possible amount should
 ANTHOPHYTA 453

be used* Most tubers and rhizomes contain starch, some (such as those
of Solarium tuberosum) in great abundance. For this reason a triple or
quadruple stain combination is indicated.
Mature tubers are less useful, except possibly under certain circum-
stances, as when the origin and development of the phellogen are particu-
larly wanted, rather than younger examples. The latter possess most
of the structures of the mature tuber and reveal more detail in a much
smaller extent of tissue. Tubers of the type of Beta vulgaris^ Daucus
carota, and Pastinaca saliva are in the optimum condition when about
15 mm. in diameter, and it is easy to cut sections across the entire struc-
ture at this stage. If sufficiently young tul>ers of the t 3rpe of I pomoea
batalas or Solarium tuberosum are unavailable, cut off triangular wedges
from large specimens, which should preferably have been freshly dug.
Specimens from grocery stores are likely to have been so vigorously
scrubbed that the epidermis is badly broken.
Rhizomes in general are less troublesome than roots, but those of
certain types may possess heavily lignified tissues which will require
softening under water. That of /m, which has an unusually prominent
endodermis, is an example. Large, fleshy rhizomes, like those of Zingiber
or NymphaeOj should be treated like tubers.

STEM APICES
Longitudinal and even transverse sections of stem apices are useful
for a variety of purposes: such sections generally show how the cells
which make up the structure of the stem originate; the development of
leaves; the origin of lateral branches; the protective covering of the
delicate meristematic tissues; and other features. The cells are gener-
ally small and isodiametric (probably 14-sided) and have delicate walls,
large nuclei, and tiny vacuoles. Mitochondria are occasionally present,
but most other cell inclusions are absent. Mitoses are abundant,
but, because of crowding, the chromosomes usually cannot be counted. In
most woody plants the stem tips are covered Mrith bud scales; these
scales are sometimes permeated with resinous or sticky substances which
interfere with staining.
Stem apices, on the whole, are easy subjects technically; the one
outstanding difficulty is to orient them for sectioning in such a plane
that perfectly median longitudinal sections are obtained. Nonmedian
sections are of little or no value. Hard scales and excess leaves should
always first be trimmed away, then the apices may be cut off from the
stem about 8 to 10 mm. back from the tip, save that greatly elongated
types (as those of Lonicera and Veronica) should be about 12 to 15 mm.
in length.
454 SPECIAL METHODS FOR THE VARIOUS PHYLA

Fixing fluids of strong penetrating power are necessary. Eormalin-

propiono-alcohol has given perfect results, but those who so prefer may
use a strong chrom-acetic fluid. Air should be exhausted under a
suction pump, otherwise the material is certain to float while in the
paraflSin oven.

Fig. 99 .
—
HydrUla verticillaia: perfectly median longitudinal section of the stem apex,
with meristematic region and developing leaves. Fixed with formalin-aceto-alcohol;
stained with safranin and fast green.

Elodea stem apices are more generally used than any other type;
the related and much largen HydriUa from the tropics is even better and
is also likely to have either staminate or pistillate flowers present (Fig.

99). Some people prefer Hippurus or Myriophyllunij but both are

rather difficult to stain sharply. These plants are employed primarily
for the origin of the leaves., Among herbaceous and semi woody plants,
vines and scandent species are particularly useful. Among them may
 y

ANTHOPHYTA 455

be mentioned Aristolochia, Loniceray Ampelopsisy Parthenocissusy VitiSy

Clematis Wisteriay Passifloray DolichoSy Asparagus and Cobaea,
y The
majority of the species in these genera have terminal buds that are
laterally flattened, which makes for greater ease in orienting for micro-
toming, since it is merely necessary to cut parallel to either flat surface.
Other herbaceous plants which have broad apical meristems are preferable
to those in which this region is narrow and of slight extent. Excellent
types are to be found in ColeuSy Syringaj RicinuSy Rosay and Veronica,
It will be noted that most of these genera have opposite leaves; plants
with alternate leaves usually are not very satisfactory for the apical
meristems. These materials should all be collected in early spring when
growth is most rapid. The terminal buds of woody plants, on the other
hand, should be collected in late winter or in early spring before they
have expanded. The outer scale leaves, which are generally too hard to
be sectioned easily, should first be removed. Practically any woody
plant will provide suitable material; those that are deciduous are some-
what better than those that are evergreen. Certain groups present
peculiar difficulties which require considerable ingenuity on the part of
the technician to circumvent. The buds of the Ericaceae, for instance,
are saturated with phlobaphene compounds which interfere with both
fixation and staining. The xylem in a few other types becomes so heavily
lignified at an early stage that soaking of the embedded material under
water becomes necessary.
Single and double staining methods should be employed. Triple
and quadruple combinations are of little service; there is, for example,
generally nothing for the violet to stain and the orange G of quadruple
combinations completely overshadows the green. Either Harris’ or
Delafield’s hematoxylin used alone is frequently excellent, as are safranin
and fast green. Foster’s tannic acid method, when correctly used, is
superb for differentiation of the embryonic cells.

STEMS
Freehand Sections. — All stems which are sufficiently rigid may be
cut either freehand or in a sliding microtome in the fresh condition.
The older generations of botanists employed the freehand method almost
exclusively, then graduated to celloidin embedding. The celloidin
method still remains supreme, but the newer methods of embedding in
parafiBin plus soaking under water have enabled technicians possessed of
patience and a high degree of manipulative skill to produce thin, perfect
sections which can be mounted on slides for staining and do not require
handling as loose sections.
If the sections are cut freehand, fix them for 24 hours in formalin-
ac6tO“alcohol, then stain with safranin. The time in the safranin depends
456 SPtlClAL METHODS FOR THE VARIOUS PHYLA

upon the amount of lignification present.If scanty, the optimum time

is 24 hours or longer; abundant, shorten the period to 2 hours. It is
if

diflScult to differentiate heavily lignified tissues that have been over-

stained. If an aqueous counterstain is to follow, wash the sections
thoroughly with water, then differentiate with water slightly acidulated
with hydrochloric acid until the sections have a deep pink color generally,
with the lignified portions a bright red. Again wash thoroughly with
water to remove all acid, then apply the counterstain. Harris' or
Delafield's hematoxylin is excellent, or a 1% aqueous solution of fast
green may be substituted. After excess counterstain has been washed
out, or the hematoxylin differentiated in the usual fashion, dehydrate by
the gradual addition of hygrobutol or dioxan over a period of about
45 minutes, then infiltrate with dilute balsam, which should then be
concentrated quickly. Mount the sections as soon as practicable, as
they will become brittle in time and may crack when flattened.
If an alcoholic counterstain is to follow the safranin, wash the latter
out with water, then cover with 70% ethyl alcohol nearly saturated with
picric acid for about 10 seconds. Remove, and replace with a change of
85% ethyl alcohol, then apply the counterstain. Anilin blue and fast
green are the dyes most commonly used, and these work better if first
dissolved in methyl cellosolve and mixed with an equal volume of 95%
alcohol. The period depends on the material: some types stain quickly,
—
others slowly the sections should be allowed to remain until thoroughly
stained since there is little chance of overstaining. Wash with two
changes of 95% alcohol, then commence adding hygrobutol gradually,
and finally infiltrate with dilute balsam. Xylol should always be avoided
since it hardens sections excessively and causes them to curl up tightly.
Paraffin sections of woody materials may be placed in xylol to dissolve
the paraffin, then brought down to alcohol or water for staining, later
treated as if they were freehand sections. Materials which are difficult
to retain on the slides should be treated in this fashion.
Celloidin Sections. — Directions for manipulating celloidin sections
are given in the chapter describing the Celloidin Method.
Paraflba Sections. —All meristematic, herbaceous, and young woody
stems can, and should, be sectioned in paraflin. Fix with either formalin-
aceto-alcohol or formalin-propiono-alcohol, and follow preferably the
tertiary butyl alcohol method. Infiltration with paraflSn should be
thorough; some materials may need to be left in the oven for a week or
longer. Embed in a hard paraffin or in Parlax. After cooling, cut the
required pieces out of the block, trim roughly, expose the end which is
to be microtomed, then place under distilled water plus a small crystal
of phenol in a small bottle. The important point is to judge the period
of immersion accurately; this requires some practice. The optimum
 —

ANTHOPHYTA 457

time for young stems is overnight; for most herbaceous stems, the same
length of time suffices; for harder and older stems a few days are required;
for woody stems a week to two months may be necessary. As the tissues
absorb water and become softenc^d, they acquire a whitish-opaque appear-
ance. The stems are ready for sectioning as soon as this appearance
extends for their entire length. There is great danger in overimmersion;
stems of the Pelargonium type, which have an extensive pith, should not
be left in th(‘. water for more than 36 hours. If stems are immersed too
long, it becomes impossible to section them, since the tissues are torn

. vis,'

Fig. 100. Caasytha JUiformis: cross section of the parasitic with the haustoria
penetrating the stem of Paamfiora foetiiia. Fixed with fonnalin-at'eto-aleohol; stained with
safranin and fast green.

rather than cut by the knife. The thickiK^ss at which the s(‘ctions arv.

cut depends upon the nature of the tissues. V The tissues of some stems
are composed of huge numbers of rather small cells; such stems should
be sectioned at 10 to: 12/i. Other stems are made up of relatively few,
large cells and shoul^ fhet^iore be cut at thicknesses of from 14 to 24^.
A few trial sections, examined microscopically with or without staining,
should reveal the optimum thickness. The one great difficulty in
microtoming stems is to cut the cambium region smoothly and without
.wrinkling; the cambiaT cells are the weakest structurally, and if proper
care not taken, there will be more or less buckling at one or more
is

points. Another source of trouble is tearing of sclerenchyma aud ool-

lench 3nna cells, which may be in small isolated groups in some stems
(as in Aridolochia, Fig. 101), or in localized regions (as at the ooruors
of the square stems of the l/abiatae),
458 SPECIAL METHODS FOR THE VARIOUS PHYLA

Sections of many ste^ls come off the slides easily during the staining
or dehydration. For stem sections should always be taken
this reason,
through celloidin during the deparaffining. As a further precaution in
obstinate cases the sections on the dry slides may be coated with a 1 %
celloidin solution with a camers-hair brush, and this allowed to dry.

Fig. 101 . —
ArUtolochia hrazilienais: cross section of an older stem. The breaks in the
cortex at the left are caused by release of torsion and are faults that can not be entirely
avoided. Fixed with formalin-aoeto-alcohol; stained with safranin and anilin blue.

Thereafter it is necessary to avoid any fluid that is a celloidin solvent:

deparaflining may be with carbol-xylol, thence to 96% ethyl alcohol
and down to water, and dehydration may be done in the reverse order,,
except that the slides are passed through pure xylol immediately before
mounting in balsam.
Staining of stem sections is to a considerable extent a matter of
individual preference. A great variety of combinations is available.
The simplest is safranin and fast green. Others include safranin and
 ANTHOPHYTA 459

Harris’ or Delafield’s hematoxylin; malachite green, methyl green or

crystal violet and erythrosin or acid fuchsih; or safranin and anilin blue
or crystal violet. Quadruple combinations are more useful than triple
combinations since the orange G of the latter tends to overstain. The
basic stain should be selected with regard to the structures which are
particularly wanted to be shown to the best advantage.

Fiq. 102. —
Sedum praealtum: cross section of succulent stem with leaf trace. Fixed
with Sass’s acetone mixture, dehydrated with acetone and tertiary butyl alcohol; stained
with safranin and fast green.

Herbarium or other dried material of small stems, leaves, etc., may

be restored to a reasonably satisfactory condition for embedding and
sectioning by being cut into 4-mm. lengths, placed in absolute alcohol
for 24 hours, then taken through graded alcohols down to distilled water.

%
Next transfer to an 8 aqueous solution of potassium hydroxide for a week
at room temperature, then wash in 15% aqueous glacial acetic acid, using
several changes. Wash thoroughly with water, dehydrate, and embed.
460 SPECIAL METHODS FOR THE VARIOUS PHYLA

—
Vascular System. The ramifications of the vascular system may
be followed out by perfusing the living plant with basic (not acid) fuchsin
(Gourley 1930). Prepare the staining solution by dissolving 50 mg.
basic fuchsin in 2 cc. of 95% alcohol and diluting this with 100 cc. tap
water. Remove the plants from the soil (the plants should be of a
somewhat succulent type), wash the roots free of adhering debris,
immerse the roots in the staining solution, and cut off part of them.
If the stain does not show signs of penetrating within 12 hours, recut
the roots. The vascular system, even to the minute veins in the leaves,
should be well stained in from 24 to 48 hours. The plants may then be
removed from the solution and the roots washed thoroughly with water,
followed by weak alcohol to remove all excess stain.
The plants or portions thereof may
then be boiled in water or a very
dilute solution of potassium hydroxide in order to secure partial dis-
sociation. The parts are next placed under a binocular microscope and
dissected to the desired extent.
Or the plants may be cleared by placing them in a large test tube or
similar container and passed successively through 50, 70, 85, 95%, and
absolute alcohol, then cleared through 3 parts absolute alcohol and 1
part xylol, equal parts absolute alcohol and xylol, 1 part alcohol and 3
parts xylol, and two changes of pure xylol. The material should remain
in each change for about half a day.
Other methods have also been suggested (Camp and Liming 1931,
Simpson 1929, Stebbins 1938, Varrelman 1938). They are essentially
similar to the one described above but do not include prestaining of the
vascular system.
Fresh, dried, or preserved material may be boiled in water for 2 to 3
minutes, then bleached for one to three days in a mixture of equal parts
of concentrated ammonia and hydrogen peroxide, or 2 parts of the former
to 1 part of the latter (Stebbins 1938). The higher the degree of oxida-
tion, the more peroxide is used. Next transfer the material to 95%
alcohol,and harden for from 1 to 12 hours. Although staining is unneces-
sary, thismay be done at this juncture with 1% aqueous crystal violet.
The original schedule called for passing through three changes of normal
butyl alcohol (allowing about 2 hours in each), passing through equal
parts of normal butyl alcohol and xylol, next through pure xylol, then to
mount in balsam; this long procedure may be simplified by the use of
hygrobutol or dioxan. All cell contents are removed; the walls appear
transparent (without staining), and lignified xylem strands stand out
sharply.
The transpiration stream may also be traced with Ught green, usipg
the dye, which is nontoxic, in the proportion of 1 g. to each liter of
distilled water (Harvey 1930).
 ANTHOPHYTA 461

—
Macerated Stem Tissues. All stems may, by proper maceration
methods, be dissociated into the component cells. The term macera-
tion” unfortunately connotes, in the minds of many people, an entirely
erroneous conception. It is taken to mean that the tissues are so
altered that nothing can really be recognized. All that actually happens
is that the middle lamellae, which bind the cells together, are dissolved

and the cells are freed more or less completely from each other. If the
material has not previously been fixed, there may be some plasmolysis
of the cytoplasm of the more delicate cells, but this is not a matter of
any great importance.
Jeffrey’s method is recommended. In order to master the method
correctly, a semi woody stem should first be experimented with. Young
stems of Tilia^ Aristolochia^ Liriodendron^ Pyrus, or similar plants with
soft wood may be cut into portions about 2 cm. in length, then split
into slivers about the size of toothpicks. Stain with safranin after
maceration and subsequent thorough washing. It is advisable to use a
centrifuge cautiously in making changes of fluids. The stem of Pelar-
gonium is probably the best of all herbaceous types for maceration;
it affords a great variety of interesting cell types.

Slices of macerated tissues are very useful and furnish the only means
by which the entire topography of complete, uncut cells may be followed
out. A conception of the nature of cells quite different from that given
by sections is obtained.
Sectioning Hard Woods, —Special methods have been devised for
the easy sectioning of mature woody stems, which have long been a
nightmare to the average technician (page 104).
—
Abscission Layer. Abscission layers occur at the junction of the
stem with petioles and pedicels. Some of the more suitable plants in
which to demonstrate the abscission layer include petioles of Coleus,
SaliXy and Populus and pedicels of Lycopersicum,
—
Lactiferous Ducts. Stems which exude a white or yellowish fluid
when cut open may be presumed to contain either latex vessels or
latex cells. The following families contain numerous species with either
or both types of lactiferous ducts: Asclepiadaceae, Euphorbiaceae,
Papaveraceae, Caricaceae, Musaceae, and Moraceae.
Starch grains are frequently abundant in latex. For this reason a
triple or quadruple stain combination is indicated. Formalin-propiono-
alcohol is an excellent fixing fluid; the sections should be between 12
and 15m in thickness.
Resin Ducts. —Ducts in which resins, oils, gums, and other sub-
stances are secreted are present in many families. They occasionally
form extensive branching systems, but in certain families (as in the
fruits of the Umbelliferae) they are localized. Most fixing fluids dis-
 —

462 SPECIAL METHODS FOR THE VARIOUS PHYlfA

solve the secretory substtoces; consequently the presence of such sub-

stances cannot be employed to indicate the presence of resin ducts.
It would be better to cut freehand sections of fresh material and to employ
microchemical tests.
—
Internal Glands. These structures are of rather rare occurrence
in the Anthophyta. Young stems of any species of Citrus, particularly
of lemons, are the most useful. The lysigenous cavities are very promi-
nent and readily recognized following the customary methods. Bud
scales that are sticky on the external surface should, after being sectioned,
have glands present.
Lenticels. —Species of Sambucus are commonly used for the demon-
stration of lenticels in woody stems, but the most satisfactory stem

Fig. 103. Viacwn articulatum: crotss section of the stem of a parasitic dicotyledon.
Fixed with formalin-aceto-alcohol; stained with crystal violet and erythrosin. {From a
preparation by Dr. Panchanan Maheahwari.)

for the purpose has probably not yet been found. Menispermum,
Forsythia, Aesculus, and Pyrus and many other plants have also been
used.
Selected portions of the stem or cortex containing the lenticekmay
be removed and fixed with formalin-aceto-alcohol. The sections should
be cut in the transverse plane of the stem and should not be over 12/x
in thickness. The cell walls are thick in the outer regions and take
stains intensely because of their heavy suberization.
—
Tyloses. Many semiwoody and woody stems contain tyloses in
their vessels. They are conspicuous in certain species of Vitis (Fig.
104), Aristolochia, Menispermum, Aesculus, Rohinia, Juglans, Sassafras,
Rhus, Catalpa, Quercus, and Populus. They are less common and more
difficult to find inherbaceous plants, having been reported from Coleus,
Ritmex, Asarum, Convolvulus, and Cucurhita, Formation of tyloses
may be induced by wounding.
 ANTHOPHYTA 463

The walls of the tyloses are generally so thin

and delicate that some
plasmolysis must be expected. In rare instances the walls may become
thick and lignified. Methods commonly used on stems will reveal the
tyloses with sufficient clarity.
Cladodia. —These structures, variously also called “phylloclads”
and “cladophylls,” are stems, usually more or less flattened, which are
adapted for carrying on photosynthetic activities in the absence of the
usual type of leaf. All species in the family Ruscaceae and the genera
PhyUanlhus and Asparagus poss«>ss cladodia. Semele androgyna, a

Fia. 104. — longitudinal section of a tylose through the stalk.

Vitiit vinifera: Fixed with
fornialin-aceto-alcohol; stained with safranin and anilin blue.

member of the Ruscaceae, is widely cultivated in greenhouses and

furnishes excellent material.
Material should be treated as were thick, leathery leaves; it is
if it

difficult to section cladodia easily. The

tissues are compact; hence thin
sections are desirable; 10^ is about right. Safranin and fast green or a
quadruple combination are both excellent for staining.

LEAVES
The and earliest stages of leaf development are to be found in
origin
sections of leaf buds or stem apices. The cells which become the leaf
initials are usually several cell generations removed from the initials
of the main axis. The leaf initials are first recognizable as meristematic
protuberances. Growth is at first apical, but this phase is of relatively
short duration, and growth thereafter is general throughout the leaf
tissues. In the Poaceae and in certain other monocotyledons with long
linear leaves, such as Iris and Allium^ intercalary meristems are of com-
mon occurrence but of brief duration.
464 SPECIAL METHODS FOR THE VARIOUS PHYLA

Leaves of all types are rather erratic in their reaction to, technical
methods. The underlying reason is undoubtedly that of photosynthetic
activity, less so to minor structural variations. The marked differences
between the leaves of monocotyledons and dicotyledons are also a
frequent source of technical troubles. The leaves of most monocotyle-
dons possess parallel veins and have been considered to be phyllodia
by many botanists. The majority of dicotyledonous leaves, on the

Fio. 106 . —Atriplex hymendytra: cross section of portion of a densely trichophyllous

isobilateral leaf. Fixed with formalin-propiono-aloohol; stained with safranin and fast
green.

other hand, are dorsiventral. Isobilateral leaves are not common among
dicotyledons and are generally correlated with habitat factors. Those
of plants growing in regions of intense light and heat, as in Death Valley,
for example, are usually isobilateral in structure even though they appear
externally to be dorsiventral (Fig. 106). The most prominent technical
difficulty with leaves is to get sufficient sharpness and contrast with
stains. It may be recalled that it is practically impossible to obtain
differential staining with the leaves of many Pteridophyta; the situation
is not quite so anno3ring with the Anthophyta, but it is sometimes bad
 ANTHOPHYTA 465

enough. The chloroplasts are the structures most difficult to stain

adequately in the majority of cases. The blame for poor differentiation
does not always rest upon the staining procedure employed; sometimes
it belongs to the fixing fluid, occasionally to the dehydration method.

Rosa leaves are among the best for the chloroplasts; the more commonly
used Syringa is poor.
For some reason, possibly because the older botanists used the
handiest plant in their gardens, the leaf of the lilac, Syringa vulgaris^
is more universally employed than any other to demonstrate the struc-

ture of a ‘Hypical” dicotyledonous leaf. Objections on one ground or

another that it is not entirely a suitable type nevertheless do not prevent
it from being recommended as a test object with which the technician

can gain (ixpc^rience in th(i manipulation of leaves. Similarly, leaves

of the corn, Zea mays, may be utilized as a representative of mono-
cotyledonous leaves.
—
Manipulation. Perfect examples should be selected for fixation.
Diseased, damaged, senescent, or wilted specimens should be avoided.
Kvergreen leaves may b(^ collected at any time, but deciduous types an*
at their prime in spring and early summer.
There is considerable difference of opinion among technicians as to
th(*, most satisfactory killing fluid for leaves. Many claim that strong
chrom-acetic or medium chrom-osmo-acetic fluids are the only oik^s
which give completely adequate fixation.These claims were made
previous to the introduction of the newer methods of dehydration and
infiltration and can no longer be wholly justified. Chromic fluids an^
objectionable mainly on the ground that leaf portions over 3 or 4 mm.
in any direction are nc'ver completely and evenly fixed throughout.
Formalin-propiono-alcohol, if followed by tertiary butyl alcohol dehydra-
tion, gives almost perfect preservation of all elements except mitochondria
and permits sharper staining than has been obtainable after chromic
fluids. This method also has the somewhat strange virtue of not harden-
ing the leaves to the extent that soaking under water is required; sections
as thin as 8M:are easily micro tomed.
If the leaves are fixed in the middle of a sunny day, most of th(‘
stomata will be open; in late afternoon or after dark, they are mostly
closed. Most mature leaves have the stomata permanently open. Very
few leaves contain starch grains; if present, they are usually in or close
to the vascular bundles, and are more likely to be found in leaves fixed
just before daybreak. Pellionia is the most conspicuous example of a
plant whose leaves contain starch.
Leaves, no matter how small, should never be fixed entire but should
always ‘"have opposite sides or ends cut oi>en. This also applies to sub-
merged aquatic le?iv^s other than those of the dimensions of Elodca
466 SPECIAL METHODS FOR THE VARIOUS PHYLA

leaves. The epidermal layers of the leaves of allbut a few species are
cutinized, some weakly, others rather heavily. This prevents the pene-
tration of all liquids, hence the necessity for open areas.
The majority of leaves are sectioned transversely, across the veins.
If the leaf is not over 18 mm.
in width, it should be transected into
portions about 5 mm. Leaves over 18 mm. wide should be cut
deep.
into strips lengthwise about 12 mm. wide and the strips next cut into
transverse portions 5 mm. deep. The strips should always include the
midvein or in the case of unusually large leaves one of the principal
secondary veins. It will be understood that portions prepared as
specified are to be microtomed vertically along the longer edge. If

longitudinal sections are to be cut, i.e., parallel to the midvein, then the
portions should be longer in the lengthwise direction of the leaf. The
two types of leaf portions should never be mixed together but be embed-
ded separately, and record of their nature should be kept; it is virtually
impossible to determine their type from embedded material.
Scissors should not be used for cutting leaves since they compress
the tissues. Place each leaf on a hardwood board or piece of stiff,
smooth cardboard and make quick, straight cuts with a sharp scalpel.
As soon as the portions have been placed in the killing fluid, exhaust
the air under a water suction pump, but take care not to overdo the
process. There will be less danger of plasmolysis if only part of the air
is first exhausted and the remainder removed the next day, after con-

siderable hardening of the cytoplasm has occurred.

As stated above, the tertiary butyl alcohol method has given perfect
results. Some technicians carry out the preliminary dehydration with
glycerin: if an aqueous killing fluid was used, first wash out the fluid
thoroughly, then place in 10%
aqueous glycerin in a flat dish, and let
remain until the water has evaporated completely and the solution has
the consistency of pure glycerin. Wash out the glycerin thoroughly with
95% ethyl alcohol, and complete dehydration with absolute alcohol.
Pass successively through mixtures of absolute alcohol and xylol in the
following proportions 3 :1, 1 :1, and 1 :3 (about 4 hours in each).
: Finally
give two changes of pure xylol, and embed in paraffin as usual. Or the
glycerin may be washed out with tertiary butyl alcohol and then infil-
trated with paraffin. If an alcoholic killing fluid was used, wash out
with plain alcohol of the same percentage as that in the fluid, place the
material in aqueous glycerin in which the percentage of water equals
that of the alcohol in the killing fluid, and thicken the glycerin by evapora-
tion. Wash out, and proceed as usual.
Very succulent leaves, such as those of Sedum^ Bryophyllum, Aloe^
and Echetjeria, are troublesome because of the very thin cell walls and
d35;^essive water content. If they show no shrinkage during fixation,
 ANTHOPHYTA 467

they may be brought into paraffin by the regular tertiary butyl alcohol
schedule, provided the changes are made over extended periods. Acetone
dehydration is often successful (Sass 1932).
Most leaves should be embedded singly, but some types may be
embedded in bunches, one leaf portion being laid on top of another.
The tougher and more difficult types should always be treated singly.
Many types of leaves become hardened during the dehydration and
infiltration and consequently require a brief soaking under water previous
to microtoming. The process should never be prolonged since the leaves
have a tendency to shrink sufficiently to become loose in the paraffin
matrix, and the sections then fall out as each section is cut. However,
the greatest source of trouble in microtoming is the midvein. Some
midveins contain as great a quantity of lignified tissue as a small stem
and are even more rigid; hence particular attention should be paid to
this aspect at all times.
The stain combination most generally employed is safranin and fast
green. Aquadruple combination is frequently very good, but there is
rarely any structure with a special affinity for the violet part. Many
people prefer to use either Harris^ hematoxylin or Mayer's haemalum
alone; these stain practically all structures sharply in various shades of
purple and do not give color differentiation between diverse structures;
consequently they should be used only if one is familiar with the anatomy
of leaves.
—
Leaf Types. Leaves are usually classified according to the familiar
taxonomic types, but this classification is wholly useless to technicians
since they are primarily interested in anatomical structure. The follow-
ing rough classification into anatomical types has been devised in order
to assist the technician in selecting species to show particular features
of the anatomy:
Typical dicotyledonous leaves, with one palisade layer (the preferred
types for elementary instruction): Ligustrum ovalifolium, Syringa.
Typical dicotyledonous leaves, with two or more palisade layt'rs:
Quercus, PyruSy CitruSy PolygonuMy HederUy Rosa.
Malacophyllous types: Begonia Tradescantia Jiuminensis, Scoliopus.
j

SclerOphyllous types: Banksiay Hakea.

Typical, with large hypodermal cells below the upper epidermis:
Piper nigruniy Peperomia reflexay Ficus elasiicay Rosa. Arackis has large
hypodermar cells above the lower epidermis.
Little differentiation and poor internal organization not to be recom-
;

mended: Beta, Zantedeschiay Amsinckiay Montiay Sedumy Chrysanthemum,

Pelargonium.
Dicotyledons with structure resembling that of typical monocotyle-
dons: EschschoUzia, Portulaca oleraceUy Eichhornia, Atriplex hymenelytra.
468 SPECIAL METHODS FOR THE VARIOUS PHYLA

Isobilateral dicotyledons: Ranunculus aquatilis (aerial), Apteniay

ProsopiSj Larrea^ Eucalyptus (adult), Encelia, Dianthus caryophyllus.
Typical trichophyllous dicotyledons: niost desert species belong in
this category, e,g.y Salvia apiana, Encelia farinosa^ Atriplex hymenelytra.
Typical monocotyledonous leaves, no differentiated mesophyli:
Zea, Avena saliva.
Xerophytic monocotyledons, inrolled type, heavily lignified and
cutinized: all bamboos, such as Phyllostachys; Ammophila, Spartina.

Monocotyledonous phyllodia: Amaryllis^ Typha, Semele.

Monocotyledons with structure resembling that of typical dicotyle-
dons: Anthurium andraeanuniy Alliumy Trillium.
Aquatic types, with aerenchyma: Trapa^ Castalia, Neptunia.
With conspicuous external glands: Potentilla glamiulosa^ DionaeUy
Drosera.
With conspicuous lactiferous du(‘ts: Ficus carica, Jatropha, Eu-
phorbia.
With conspicuous internal glands: Citrus, liuta, Ficus elastica,
Eucalyptus.
Phyllodia . — Petioles which become flattened to carry on photo-
synthesis are known as Most species of Acacia hav(‘
‘^phyllodia.”
phyllodia, at least in mature In Coccoloha the bas(i of the pctioh;
plants.
is dilated into a phyllodium. Treat phyllodia as if they were cladodia
(page 463). Considerable care must be taken with the differentiation
of stains, as the tissues very readily overstain.
—
Leaf Epidermis. In order to show the number, disposition, and
surface anatomy of stomata, the epidermis of the leaf may be peeled
off, stained, and mounted in balsam. The epidermis does not peel
equally well from all types of leaves; in fact, the number which can be
utilized is quite small.
The most useful type is Graptopetalum (Byrnesii) weinbergii, one
of the Crassulaceae. The are large, grayish-green, and the
leaves
entire epidermis from either side may be stripped in one operation.
Take fresh leaves, cut off both ends and a narrow strip along each side,
then insert the edge of a scalpel under one lower end, and peel off th(^
epidermis slowly. It should not be stripped rapidly, or it will curl
tightly. Submerse immediately in formalin-aceto-alcohol, taking care
not to allow the edges to roll inward. After fixing for 24 hours, wash with
a change of 50% alcohol, then stain with Harris' hematoxylin.*' Differen-
tiate, wash with water, dehydrate with hygrobutol, infiltrate with
diluted balsam, then cut into portions about 5 mm. square for mounting.
Be sure to mount with the external side up.
If Graptopetalum is unavailable, most species of the related Sedum
are good, particularly those with broad and somewhat .flattened leaves,
 ANTHOPHYTA 469

The epidermis of Tradescantia virginiana is also excellent (Fig. 106).

Strip as large pieces as possible. Stain in safranin and anilin blue,
which gives a striking color contrast. The bulb scales of Allium are
modified leaves, and it is very easy to peel the epidermis from the inner

Fig. 106
. —
Tradescantia virginiana: portion of whole mount of leaf epidermis, with
stomata, hairs and secondary vein. Fixed with formalin-aceto-alcohol; stained with
safranin and fast green, dehydrated with hygrobutol.

surfaces of the scales. This type of epidermis is good only for simple
and their nuclei; there are no stomata.
cells
With other leaves which do not peel readily, the epidermis can be
more or less loosened by blanching with boiling water for a few minutes,
470 SPECIAL METHODS FOR THE VARIOUS PHYLA

then plunging into cold water, and peeling small portions. Fix as usual,
even if the boiling water is in the nature of a fixing fluid.
—
Whole Mounts of Small Leaves. To show the venation in suitable
small leaves (Ulmus and Fraxinus are excellent types), stain them in the
living condition by Gouiiey^s basic fuchsin method (page 460). Then
boil in slightly alkalinized 85% alcohol until all chlorophyll is removed,
pass through 95% and absolute ethyl alcohol, place in methyl salicylate
(synthetic oil remove the salicylate
of wintergreen) until cleared, then
with xylol, infiltrate with balsam, and mount.
Another method (McVeigh 1935), adaptable with either fresh, fixed,
or dried leaves, is to place them in any one of the common household
bleaching solutions (Chlorox, Sani-Chlor, or Purex) containing about 5%
sodium hypochlorite. The length of time required for bleaching depends
upon the thickness and texture of the leaves. Thin leaves and those of
most aquatic plants require 30 to 60 minutes, while thick ones, such as
those of Bryophyllum and Sedum^ require 24 to 96 hours. After bleach-
ing, wash thoroughly in running water for several hours to remove the
bleaching agent. Next the leaves may be dehydrated with ethyl alcohol
and stored in a mixture of 75 parts glycerin and 25 parts 95% alcohol
until they become impregnated with glycerin, whereupon they are
pliable, easy to handle, and the vascular system is clearly visible. If it is
desired to make starch tests, kill the fresh leaves with boiling water,
bleach, wash quite thoroughly, then apply the usual iodine-potassium
iodide solution.

FLOWERS
The subject of flowers and floral structure is so vast that it cannot be
treated at present in any great detail. In any event, the flowers of
almost each species present idiosyncrasies peculiar to each; consequently
more or less experimentation must be devoted to the one under investiga-
tion to determine the precise method of procedure. Reference should
first be made to a good general description of the family and order, to

the end that the salient structural features be understood (Hutchinson

1926, 1934), after which the general procedure becomes fairly clear in
most instances.
An enormous number of types of flowers exist, but a few general
features prevail.
Organogeny. —The
order of appearance of the different parts com-
posing the entire flcjwer is described as organogeny. At one end of a
hypothetical series of floral types are those lacking a perianth (naked or
apetalous), and at the other end are those with a perianth of sharply
differentiated calyx (sepals) and corolla (petals), with all sorts of modifica-
tions in between. The more primitive arrangement of the parts is the
 ANTHOPHYTA 471

spiral, such as found in the strobilus of more primitive plants, graduating

to the cyclic disposition among the more advanced dicotyledons.
In the spiral flower the order of succession is acropetal, meaning
that the parts arise successively toward the apex of the receptacle. The
succession is sepals, petals, stamens, carpels.
In a cyclic flower^ if the
acropetal successionmaintained, the order of appearance is centripetal.
is

However, the primitive type of succession may be disarranged. In

Capsella, for example, the order is sepals, stamens, carpels, petals; and
in the Asteraceae it becomes petals, stamens, carpels, sepals.
In cyclic
types there is frequently a definite number of organs in each cycle. In
cyclic monocotyledons, for example, the prevalent floral number is three,
although there are many spiral monocotyledons with no definite number.
Again, in cyclic dicotyledons, the floral number is usually four or five,
but there are innumerable spiral (li(*otyledons with indefinite numbers.
All parts of the flowc^r do not necessarily attain a simultaneous cyclic
condition, so that exceptions are common.
The first step in preparing slides to demonstrate the organogeny of
flowers is to examine the plants and to determine the time and place of
appearance of the youngest possible stages. The origin is generally in
embryonic regions. In some species an entire year may be occupied by
the initial stages of development this is particularly true of woody plants
;

and many semiwoody perennials. For such types, collections should be

begun in the summer preceding the unfolding of the flowers. Most
bulbous plants also first develop the flowers during the preceding summer
or early autumn. The apical flower buds are, as a rule, recognizabki
without difficulty but are sometimes not distinguishable from leaf buds
when the two types are distinct on the same plant, as in Syringa, Cut off
the buds, remove all overlapping scales or other protective coverings,
fix with formalin-propiono-alcohol, exhaust all air, microtome longi-
tudinally at lOg, and stain with safranin and fast green, Harris^ hema-
toxylin and erythrosin, or by Foster^s tannic acid-iron chloride method.
Most buds section without any except for tears occasioned by
difficulty,

raphides, but a few may need overnight soaking under water. If a

research paper is being prepared, one should, obviously, consult published

papers on related species or genera, using the indexes to Botanical
Abstracts (up to 1925), Biological Abstracts (after 1925), and the foreign
abstracting journals for the purpose.
Whole Mounts of Small Flowers. —Flowers
which are of a small
size and not too thick when pressed be may
fixed with formalin-
flat

aceto-alcohol, which generally clears them of pigments, washed with

50% ethyl alcohol, stained with Harris’ hematoxylin and erythrosin,
or with a carmin stain, dehydrated with hygrobutol or dioxan, infiltrated

with balsam, and mounted entire. Depression slides should be used if

 ;

472 SPECIAL METHODS FOR THE VARIOUS PHYLA

they are available, otherwise glass threads should be arranged beneath

the petals in order to hold the flower level.
The flowers of AnagalUs, Spiraea, Capsella, Erodium, various native
orchids with small flowers {Ihidium, Liparis, Piperia, Limnorchis),
CUntonia, and similar types provide very instructive preparations when
mounted so that they arc viewed from above. The flowers of various
Liliaceae may be cut open and spread out: AlWiim and Dichelostemma
are excellent. The petals of Calochortus may be mounted singly to show
the hairs and glands. The florets of the Asteraceae (e.g., Taraxacum,

Hieracium, or Erigeron) may be mounted flat for side views.

—
Vascular S3r8tem. The pedicel is structurally a stem and may be
treated like one. The which depart to the various organs start
traces
from the modified stele in the receptacle; they are similar to those of
leaves in origin, structure, and behavior. They pass off successively to
sepals, petals, stamens, and carpels according to the manner of arrange-
ment of these organs in the flower. Petals and stamens typically possess
a single trace, carpels three traces, and sepals generally the number of
traces found in the leaves of the same There are naturally
plant.
(jxceptions: in the Magnoliaceae there are three traces to each stamen;
petals that are more like sepals have traces comparable in number to
those normally possessed by the latter; and in some capsules the number
of traces is reduced to one. In most flowers the vascular system is
extremely complex, and confusion exists because of the fusion of adjacent
traces in the proximal parts.
Sections of buds and very young flowers show more or less of the
vascular system, but never in its entirety. To reveal the complete
system, the buds or flowers should be stained by Gourley^s or a similar
method (page 460), cleared, and mounted.
The flowers of the Ericaceae are especially desirable for demonstrating
the fusion of bundles in the proximal portions. Young flowers, selected
at about the time they have just begun to expand, of any species of
Vaccinium are excellent, but the much larger buds of Arbutus memiesii
are even better. Buds of the Ericaceae, unfortunately, are saturated
with phlobaphene compounds, which render staining extremely difficult
differential acidification might be attempted, followed by safranin and
fast green or a quadruple combination.
—
Petals and Sepals. Both of these floral organs are histologically
closely similar to leaves in structure, the sepals being the more so.
Sections may be prepared exactly as for leaves; the petals never give any
technical difficulties, but the sepals of some species may require treat-
n^ent similar to that needed by tough leaves.
The epidermal cells of petals, as seen in whole mounts, have an
e^remely interesting structure.
 ANTHOPHYTA 473

—
Stamens and Pistils. Sections of young buds of hermaphroditic
plants will reveal both the stamens and the pistils. In monosporangiate
flowers, the staminate and pistillate flowers may occur on the same plant
(the monoecious condition) or upon different plants (the dioecious condi-
tion). In the monoecious plants the nature of the buds cannot always
be determined, especially during the youngest developmental stages,
from external appearances. The preparation and examination of a
considerable number of buds will be nec(issary. It is usually possible to
distinguish the type of flowers of dioecious plants by crushing or dis-
secting few specimens and examining for anthers. Occasionally
a
pistillate flowers may contain aborted stamens (staminodia).
Longitudinal sections of the buds are better for the structure and
development of the stamens; transverse sections are principally useful
for determining the number. Both longitudinal and transverse sections
of the pistils will be required, as it is desirable to (‘orrelate observations
on the on(i with those on the other. For the later stages in the develop-
ment of the pistils, especially after the buds attain too large a size for
sections of the entire bud, the pistils sliould be removed from the buds
and treated independently. After tho, style commences rapid elongation,
itshould be cut off; if sections of the stigmatic surface and the develop-
ment of the rnicrogametophyte are desin^d, the styles sliould lie treated
individually.
Formalin-aceto-alcohol is an excellent fixing fluid for most plants,
but if plasmolysis or separation of should b(‘ had to a
cells occurs, resort

medium chroin-acetic fluid. Safraniii and fast green have always given
excellent results, but other combinations may also be tried. Special
methods for the treatment of styles will be described below under
microsporogenesis.

MICROSPOROGENESIS
For the earliest stages of the series of events leading up to the growth
of the rnicrogametophyte, very young buds are necessary. The anthers,
as a general rule, make their appearance long before the ovules originate
in the ovary, and meiosis is completed at about the time the megasporo-
cyte is differentiated in the ovule. The very young anther is a mass of
homogeneous cells. The sporogenous cells originate in the layer (hypo-
dermal layer) immediately beneath the epidermis; they are usually
recognizable in four distinct regions by their slightly larger size, larger
nuclei, and more densely staining cytoplasm. In transverse sections
each sporogenous region consists of from one to several cells; in longi-
tudinal section they may form a continuous row in most species (as in
the Liliaceae), but in others (as in the Onagraeeae) there are alter-
nating layers of sterile cells. Each band of initials divides periclinally
474 SPECIAL METHODS FOR THE VARIOUS PHYLA

to form two layers of cells. The outer layer is the primary wall layer
and the cells later divide to form several wall layers. The innermost
wall layer usually becomes part of the tapetum. The inner layer formed
after the first periclinal division becomes the primary sporogenous layer,
which next divides two or three times to produce the microsporocytes.
Meiosis then occurs in the microsporocytes, which process results in
tetrads that become microspores.
All these stages, as a rule, are readily obtained, but the reduction
divisions are difficult to secure in many species. In Tritoniaj for example,
meiosis will be encountered in only about one out of the hundreds of
flowers composing a single inflorescence, and in certain species of Godetia
thousands of flowers have been sectioned without any showing meiosis.
Lilium is probably the most favored genus, as it is always easy to secure
a complete series of well-fixed and stained stages. Tradescantia is also
excellent and has large nuclei. In these two genera and in most plants
the stage of development is approximately the same throughout all
anthers in the same flower at any given time. In a few plants, which
have a series of isolated loculi arranged lengthwise of the anthers instead
of two or four continuous loculi in each anther, a progressive series of
stages occurs, commencing at the distal end and extending downward
(as in the Onagraceae), but occasionally development may either proceed
simultaneously in all loculi or in a most irregular fashion.
The procedures that are customarily followed in producing slides
for all the phases of meiosis are described in the chapters on cytological
and smear methods. It is recommended that the beginner start with
Lilium, as there is practically no chance of failure with this genus. It
grows wild in profusion in many and is extensively planted in
localities
gardens. The most commonly found in
so-called Easter Lily, the one
greenhouses, should be avoided as the plants are likely to have been
subjected to treatments which produce abnormalities. Those species
which produce a number of flowers in an umbel are far more favorable
than those with very few flowers to the plant, and likewise those with
small flowers are somewhat better than those with enormous blossoms.
Dissect out the anthers, but do not cut them open, fix with NavasKin^s
fluid or a medium chrom-acetic or chrom-osmo-acetic fluid, and embed in
bunches of three or four. Up to the time the microsporocytes begin to
round up, sections may be cut in any plane, but after this period trans-
verse sections, at 12m, are better. If one is unable to collect material
locally, itmay be obtained from the botanical supply concerns. Blocks
tested for definite stages may be procured; they are well worth the high
price necessarily charged, since one avoids having to section a quantity
of material to find the desired stage.
 ANTHOPHYTA 476

In the case of other plants the fixing procedure depends upon the
nature of the flowers. Most of the Liliaceae and related families may be
treated like Lilium, but if the buds are too small to be easily handled
individually, all that is necessary is to cut off the distal tips of the buds
in order to allow fluids to penetrate readily. With compact inflorescences
usually only the tips of the larger buds are removed, but the suction
pump should always be used to exhaust air. If the buds are covered with
(jhaffy scales or very thick sepals, thes^'. should always be removed as
much Buds of the Asteraceae, which usually give a beautiful
as possible.
series ofdevelopmental stages, should be cut through between the apex
of the pedicel and the base of the receptacle and the outer overlapping
scales removed. If the buds (capitula) are over 1 cm. in diameter,

they should be bisected longitudinally. Short, broad capitula should be

raicrotomed transversely, other types longitudinally.
By far the best general stain for all stages up to, and including, meiosis
is iron hematoxylin; suitable counterstains, if one is desired, include
orange G and fast green. Cytologists rarely employ counterstains,
as they tend to obscure chromosomal details. For the meiotic chromo-
somes, a violet stain, used alone, is superb; the violets, however, rarely
stain the prophase stages adequately. Triple combinations are most
useful on materials difficult to stain with hematoxylin or the violets.
For the tetrad stage nothing is superior to certain genera in the
Asteraceae: Dahlia^ Coreopsis, Crepis, Erigeron, Cosmos, and annual
species of Chrysanthemum. This stage is best stained with safranin and
fast green, carefully controlling the safranin so as not to overstain the
cytoplasm, or with a triple combination.
For the maturing pollen grain, Lilium again is the most suitable
material, but Silphium is also good. Do not cut the anthers open, lest

the loose microsporcs float out during the dehydration. Microtome

at about S/x. Stain with a triple combination, which will differentiate

all structures; or with safranin and favst green, iron hematoxylin and

safranin, any preferred combination. The developing exine and

or
intine, as well as the germ pores, are well shown at this time. If it is

particularly desired to differentiate the exine and intine layers, stain the
sections for 6 to 10 minutes with 0.5% aqueous Bismarck brown, rinse

in water, dehydrate with 95% alcohol, then counterstain with fast green

in clove oil-absolute alcohol-methyl cellosolve— the exine is green and the

intine brown. The mitosis in the microspore which gives rise to the
tube cell and the generative cell, possessed by most species, is easily

found in a few species, but in the majority it is obscure and rarely encoun-
tered. The finest genus for the purpose is Trillium, with Tradescantia

irirginiana as second choice.

476 SPECIAL METHODS FOR THE VARIOUS PHYLA

In the genera Acacia^ AsclepiaH, Vincetoxicum, and in most orchids

the mature microspores are united into pollinia, with a common exine.
Sections are preferable to whole mounts or smears. The individual
microspores are rather small; consequently sections should not be over
10/i in thickness.
Smears. —Methods preparing smears microsporocytes are
of of
described in the chapter on Smear Methods.
Whole Mounts. — Mature pollen grains embrace a great variety of
forms and are particularly noteworthy for their sculpturing; these two
characters are of taxonomic importance (Wodehouse 1935).
The simplest method of making whole mounts of pollen grains is
to place them in a drop of melted glycerin jelly on a slip and add a cover-
slip. No staining is needed. Or one may place the pollen on a slide,
add 2 to 3 drops of anilin oil which has been tinted with crystal violet
only to a pale purple color (Wodehouse 1933). Heat gently over a
flame, but not beyond the point where the heated portion of the slide
becomes too hot to touch, until the grains become deeply stained. Cool
to room temperature, draw off excess oil with filter paper, wash by repeat-
edly adding xylol and absorbing it with filter paper until all the oil and
unabsorbed dye have been removed, then add a drop of balsam* and a
coverslip. The microspores may also be killed and fixed in any standard
fluid, washed, stained with safranin, a carmin stain, or by Fuelgen\s
reaction (in all cases with a counterstain if desired), dehydrated with
hygrobutol, and infiltrated with balsam.

MICROGAMETOPHYTE
Pollen grains may be germinated on glass slides appropriately coated
with an adhesive or solidified nutrient solution. The great difficulties
are (1) to retain the pollen tubes on the slides during the staining and
dehydrating processes and (2) to determine the optimum quantity of
.

nutrient to be added. Sucrose is the nutrient generally employed. The

amount varies from 0.5% to as much as 45%. A rough estimate can
be made by determining the relative stickiness of the stigmatic exudate
of the species whose pollen is to be germinated: the stickier, the larger
the amount of sugar required.
Various methods of producing whole mounts of germinating pollen
tubes have been proposed (Beatty 1937). One of the simplest methods
(Newcomber 1938) is to boil 0.5 g. agar and the optimum quantity of
sugar (1 g. may be tried) in 25 cc. tap water or any suitable nutrient
solution, cool to about 35°C., add 0.5
g. powdered gelatin, and stir until
jthe gelatin is Keep the solution at about 25®C. in an incubator
melted.
or on a warming plate. Smear a thin film on a clean slide with a finger,
and dust on the pollen. Next place in a suitable moist chamber for
 ^ .

ANTHOPHYTA 477

germination, removing occasionally to determine, under the microscope,

the progress of the germination.
The most suitable killing fluid is Navashin^s, especially since bleaching
isnot necessary after fixation. The slides should be left overnight for ade-
quate fixation. Stain the chromosomes by any preferred violet method,
and counterstain with gold orange in clove oil for about 2 to 4 minutes.
Another method is to fix stigmas on which the microspores have
germinated naturally, wash, stain with Mayer^s carmalum, d(*hydrate,
infiltrate with diluted balsam, and dissect and crush the stigmas on the
slide just before mounting. The stigmas of Nicoiiana and similar species
react well to this procedure.
Stigmas and styles may
be run into paraffin, sectioned in the longi-
tudinal plane of the style, and stained by any appropriate method.
The hollow styles of Lilium are commonly treated in this fashion, but
to make certain of^ the presence of microgametophytes, the stigmas
should be pollinated by hand and the pistils removed at the optimum
times. The period varies with the species; it may be 24 hours in some,
up to 96 hours in others (see also under Fertilization, below).
Numerous methods have been devised for treating styles to reveal
the presence, distribution, and rate of growth of pollen tubes (Buchholz
1931, Chandler 1931, Nebel 1931).

^
MEGAGAMETOGENESIS
In perhaps no other field of botanical investigation are there so many
making erroneous interpretations as during the develop-
opportunities for
ment of the megagametophyte. As an instance, there might be cited
the case of Lilium whose development was intensely investigated for
many decades, yet it was not until a few years ago that the correct
sequence of events was described. Insofar as the purely technical
aspects are concerned, sources of error may arise because of inadequate
fixation, poorly differentiated stains, but above allfrom using too thin
sections. The last fault is the most prevalent one; many embiyolo-
gists habitually cut sections as thin as 5/u and then attempt to follow
out the course of events by means of reconstructions. It is a far better
procedure to determine the approximate diameter of the megasporocytes,
megaspores, and megagametophytes at their various stages of growth
and to adjust the thickness of the sections to correspond to each stage.
The megagametophyte of Lilium during meiosis averages between 3(>
and 36iu in diameter; consequently the optimum thickness for sections ai
this stage is 24ju.
—
Megasporocyte. The megasporocyte (megaspore mother cell) orig
mates in most plants after the anthers are well along in development
This cell is usually just at the stage when it begins to expand preparatory
478 SPECIAL METHODS FOR THE VARIOUS PHYLA

to undergoing the first reduction division at the time that the anthers
are beginning to change color (if the mature anthers are other than white
in color) ;
the color change also indicates that meiosis has been completed
in the microsporocytes. The majority of plants have a single mega-
sporocyte, which one of two cells produced by a single hypodermal
is

archesporial cell; the other (tell produces the layers of cells between the
apex of the megaspore and that of the ovule. In a few plants there
may be either a group of archesporial cells (quite rare) or two to many
megasporocytes {Godetia, Fuchsia CalycanthuSy Erigeron). In most
^

plants very young buds are required for the archesporial cells; the mega-
sporocyte develops rapidly in some plants, but fairly slowly in others.
Unless one is working with a species whose course of development is
unknown, it is the better plan to start with a species with slowly develop-
ing buds: Lilium is excellent, as are Fritillaria, Erythroniurriy Trillium,
certain species of Erigeron, etc. The reason for selecting a slow-growing
species is that it is thereby possible to obtain a more complete series of
stages. Developmental stages are more or less simultaneous in all
ovules of a given ovary in most species, but in others, as in Lilium, there
is a progressive series of closely related stages, with the oldest stages

usually at the pedicel end of the ovary.

Itmust always be borne in mind that the megasporocyte is usually
a celldeeply embedded in the ovule, covered with a few {Lilium) to
many layers of nucellar cells (Vitaceae, Onagraceae), and in addition
the ovule may be invested by thin or thick integuments. Ovules of
some species have a single integument, others may have an inner and an
outer integument. The presence of all these protective coverings means
that, if it is not possible or entails too much tedious labor to remove the
individual ovules, the ovaries must be reduced to as small portions as
possible and that killing fluids of high penetrating power should be
employed. If there is considerable free space between the ovules and
the inner walls of the ovary, free circulation of the fixing and dehydrating
fluids within the ovaryis permitted. All superfluous ovarian tissues
shoiild be removed, not merely because they might inhibit penetration of
fluids, but because they may cause difficulties during the microtoming.
The ovarian tissues of the Onagraceae, for example, are full of raphides
which tear the ribbons badly, and the ovaries of other genera contain
vascular elements that are more or less lignified.
If the nucellar cells consist of only a few layers, a strong chrom-
acetic or Navashin^s fluid may be used. It has been found that the use of
Carnoy^s fluid for a few minutes preceding Navashin^s, as is commonly
done in the case of anthers, has a distinctly deleterious effect on the
subsequent staining. Many technicians have used Camoy's fluid alone,
but on the whole it does not appear to be recommendable. Others
 ANTHOPHYTA 479

use Bouin’s or Alienas B-15 modification thereof. If the nuccllus is of

considerable ’Extent, then a powerful fluid such as Gilson's or Petrunke-
vitsch's modification of Gilson's may, with caution in regard to over-
fixation, be utilized. The mercuric dc^posits whicdi such fluids k^ave in
the tissues should always be removed, pref(‘rably from the sections
after they have been brought down to watcu*. The ovules should always
be microtomed in the longitudinal plane; it is a simple matter to orient
the ovules if they are treated singly, but if entire ovaries or portions
thereof are being sectioned, care should be taken to microtome in such a
plane that as many ovules as possible are cut lengthwise. If the ovules
are oriented approximately parallel to the placenta or placentas (which
are usually more or less straight), then section the ovaric's longitudinally;
if they are attached perpendicularly to the placenta, cut th(‘ ovaries
transversely. The Liliaceae, for instance, should be microtomed trans-
versely, the Onagraceae longitudinally. If the ovules are inserted
haphazardly, one can only cut blindly in either direction, but transverse
sections should first be cut since one is more likely to get most of the
ovules in longitudinal section in this manner.
The sections of all stages of megagame togenesis and embryogenesis
should always be mounted in serial order, and care should be taken that
none is lost. (Mount on the slide so that a 24 X 50-mm. coverslip
can be used.) This is a most important point: one should make it kn
invariable practice to examine all the sections of any particular ovule

carefully in order that no nuclei or cells are overlooked. Commence

at the micropylar end and work toward the chalazal end: if the structure
under examination is the mature megagametophyte, for example, look for
the synergids, then the egg, next the polar nucleus or nuclei, and finally
the antipodal cells. In many species the antipodal cells are evanescent,
but vestiges nevertheless should be sought for. Haustoria may also be
present sometimes they are lateral, but they usually occur in the chalazal
;

region and may assume a variety of forms.

A variety of stain combinations have been suggested, most of them
being based upon individual preference. The writer's choice has long
been for safranin and fast green. Many ovules will take an excellent
iron hematoxylin stain, and a triple combination will frequently prove
valuable, as starch grains are common and a more precise differentiation
of obscure details is often secured. A number of investigators habitually
use Harris' or Delafield's hematoxylin or Mayer's haematein. However,
it is advisable to employ color-contrasting stains until one has acquired

sufficient experience in interpreting structures; then one should be able

CO use a single stain with confidence.
—
Megaspores. The megasporocyte undergoes a short period of rapid
•

growth before the first (reductional) mitosis occurs. This mitosis is

480 SPECIAL METHODS FOR THE VARIOUS PHYLA

always difficult to secure as it proceeds with comparative rapidity. A

wall is laid down in some types, but the nuclei remain free ih other types
(see below). If a wall is developed, the two cells thus resulting are
termed secondary megasporocytes.^' If no wall is formed, the mega-
sporocyte becomes metamorphosed directly into the megagametophyte.
The second division is equational (with the monoploid number of chromo-
somes); again, walls may or may not be established. Normally, there
is a linear row of four megaspores (the quartet), each of which is poten-
tially functional. However, depending upon the type, either the chalazal
—the one nearest the base of the ovule — or the micropylar —the outer-
—
most one, nearest the micropyle megaspore becomes functional and by
two or three further divisions becomes the megagametophyte.
The original megaspore cell enlarges but little during meiosis; con-
sequently methods are the same as described above for megasporocytes.
Chromosome staining, however, must be critically controlled; the
cjrtoplasm is dense and tends to overstain.
—
Megagametoph3rte. Four well-established types of megagameto-
phytic development may be recognized (Schnarf 1936), and in addition
there are minor variations under each type, together with a few prob-
lematical types. The principal types may be characterized as follows.
1. Normal Type —
This type is considered to be the original one and
.

as’typical for the Anthophyta for the following reasons: it involves the
largest number of mitoses; megasporogenesis and megagametogenesis
are separate processes within it; it is of general occurrence among the
Anthophyta, being absent in no group; and, finally, it is impossible to
regard any of the other types as the original one from which the normal
type may have been derived.
Two features characterize normal type: the megasporocyte
the
undergoes formation of the egg, and the mega-
five divisions before
gametophyte develops from a single megaspore. Furthermore, two
phases can be distinguished during the course of development. The
first leads to development of the megasporeand consists of two successive
divisions which involve conversion of the diploidy of the megasporocyte
into the haploidy of the megaspores; this is megasporogenesis. The
second phase, usually involving only one megaspore, embraces three
successive mitoses which produce the nuclei of the mature megagameto-
phyte: this is megagametogenesis and is characterized by considerable
growth and by establishment of the micropylar and chalazal poles.
Immediately after the first division of the nucleus in the functioning
megaspore, the daughter nuclei migrate to opposite ends of the cell,
and a large vacuole appears between them* Each nucleus divides twice,
to form a complex of four nuclei at each end. At the micropylar end
two of the nuclei (sisters) are enclosed by cell walls and become the syner-
 ANTHOPHYTA 481

gids; one of thetwo others becomes enclosed by a wall (the egg), and the
other remains free. At the chalazal end three nuclei become enclosed
by walls (or occasionally perish), and the other remains free. The two
free nuclei generally fuse more or less completely to form the polar
nucleus, which, after union with the secondary male nucleus, becomes
the primary endosperm nucleus.
2. Oenothera Type . —
This type is probably confined exclusively to
the Onagraceae. In contrast to the normal type, in which the chalazal
megaspore of the quartet is invariably the functional one, the functional
megaspore is the micropylar one. The megaspore next below sometimes
enlarges and rarely also develops into a megagametophyte. There arc
four divisions leading up to the organized megagametophyte: after the
first, secondary megasporocytes are formed, then a linear quartet of

megaspores. Next the nucleus of the functioning megaspore divides,

and both nuclei remain at the micropylar end, with a vacuole appearing
below them. The two nuclei are arranged one below the other: th(‘
upper one divides to produce the synergids, and the lower one produces
the egg and a single polar nucleus (which is haploid).
3. Scilla Type.
—
This type of megagametophytic development has
been described for a large number of species in widely scattered and
unrelated families. It has apparently been derived from the normal
type. Four successive mitoses are involved in the transition from
megasporocyte to megagametophyte; megasporogenesis and mega-
gametogenesis are not distinct but merged indistinguishably. After
the first division (meiosis) in the megasporocyte, a transverse wall is
formed, but after the second mitosis no wall is formed, although a purely
transitory one might appear. The four megaspore nuclei consequently
are situated in two adjacent cells, of which the micropylar one ordinarily
degenerates. The remnants of the degenerated cell remain visible for a
considerable period. The two megaspore nuclei in the chalazal cell
migrate to opposite poles: the micropylar one undergoes two successive
divisions which produce two synergids, an egg, and a single polar nucleus;
the chalazal nucleus degenerates, barely, however, the chalazal nucleus
may divide once, to produce a six-nucleate megagametophyte.
4. Peperomia Type . —
Following the first two mitoses in the mega-
sporocyte, no walls are formed. The four megaspore nuclei thus lie free
within the original megasporocyte cell. Two further divisions complete
the organization of the composite megagametophyte, which may assume
any one of six forms, depending upon the positions and behavior of the
four megaspore nuclei:
a. Penaea Form. —
This type is restricted to the Penaeaceae and to
certain species of Euphorbia. No species with this type is knowm to
occur in the United States.
482 SPECIAL METHODS FOR THE VARIOUS PHYLA

b. Peperomia Form. —
This type is restricted to the genus Peperomia,
which does not occur in the United States.

Fiq. 107 . —Lilium parryi: A, first division in the megasporocyte; B, binucleate mega*
gametophyte; C, second division; D, first four-nucleate stage.

c. Gunnera Form. —So as known,

far this form is restricted to the
tropical genus Gunnera.
d. Pyrethrum Form. —This form has been described in a single
species of Pyrethrum, P. parthenifolium, now presumed to be a form of
 —

ANTHOFHYTA 483

Chrysanthemum parthenium^ which occurs from New Brunswick to New

Jersey. As a result of meiosis, four free nuclei are produced, arranged
in a linear row in the cell, and separated from one another by vacuoles.

Fig. 108. LUium parryi: A, three nuclei have migrated to the chalazal end; B, third
division; C, second four-nucleate stage; Z), mature eight-nucleate megagametophyte.
(For figure of the fo\irth division, see Fig. 109.)

They then undergo two successive divisions. The four micropylar

nuclei produce the synergids, the egg, and the upper polar nucleus.
Of the remaining 12 nuclei, 1 remains free as the lower polar nucleus,
484 SPECIAL METHODS FOR THE VARIOUS PHYLA

4 become surrounded by a common wall, and each of the 7 other nuclei

has a cell wall developed around it.

e. —
Maianthemum Form. This type, again, characterizes a single
species, Maianthemum hifoUum, not found in the United States. There
are a number of closely related but as yet uninvestigated species.

Fia. 109 .
— Lilium parryi: fourth division in the megagametophyte. Fixed with strong
chrom-acetic; stained with safranin and fast green.

/. Fritillaria Form. —After meiosis, the four megaspore nuclei are

arranged in a linear row down the center of the original megasporocyte.
Presently three of the nuclei (the three lower ones) migrate to the chalazal
end of the cell. The third division next ensues, during which the spindles
of the three chalazal mitoses fuse. There thus result two micropylar
haploid nuclei and two triploid chalazal nuclei, and each pair becomes
separated by a vacuole. After the fourth division there are four haploid
nuclei at the micropylar pole and four triploid nuclei at the chalazal
end. The micropylar nuclei produce the two synergids, the egg, and the
 ANTHOPHYTA 485

upper polar nucleus. The chalazal nuclei form the three antipodal
cells and the lower polar nucleus. (The primary endosperm nucleus
thus becomes pentaploid after fusion with the secondary male nucleus.)
This form characterizes most of the Liliaceae but has been worked out
completely in comparatively few species in Lilium (Figs. 107, 108, 109),
Fritillariaj and Tulipa, It should be called the Lilium Form, but this
procedure is dangerous since it might be confused with the older and
erroneous so-called Lilium Type; consequently the name of the genus
in which it was first discovered is being used.
5. Adoxa Type ,
—
The older Lilium Type nevertheless has some
foundation in actuality and has been renamed the Adoxa Type. No
North American representatives of this type are easily available.
There may possibly be other types which should be recognized
as distinct (Schnarf 1936), but the descriptions for the most part have
been based upon investigations made many years ago and which should
be done all over again by modern methods and in the light of present
knowledge of megagametogenesis.
In preparing slides to show the development and organization of the
megagametophyte, the same procedure in preparing the material, fixing,
and staining as described above for the megasporocytes should be
followed. The thickness at which the sections are microtomed is a
matter requiring careful consideration and some experimentation; it
may vary anywhere from 12 to SOpi. The aim should be to obtain as
many ovules as possible in which all the nuclei of any particular stage
are present. If individual ovules are being worked with and they are
more or less flattened, they should be sectioned parallel to one flat face.
If haustoria are present, the ovules should always be so oriented that
the haustoria are cut longitudinally. Staining should always be criti-
cally controlled; the chances are that there will be either under- or over-
staining. It is occasionally difficult to get all regions of the mature
megagametophyte equally well differentiated. Contrasting stains are
always better than a single uniform stain, since they aid in identifying
the nuclei and other structures.

FERTILIZATION
Methods of preparing ovules for fertilization studies are the same
as for the megasporocyl/es. Sections should be a trifle thinner than for
the mature megagametophyte, and staining must be critically controlled.
Safranin and fast green are a good combination, but the best combination
is Fuelgen^s reaction and erythrosin. The microgametophyte commonly
brings in considerable quantities of foodstuffs, demolished cells, and
partially digested nuclei. All this may add confusion when it comes to
interpreting what is seen within the megagametophyte, which, upon the
 y y

486 SPECIAL METHODS FOR THE VARIOUS PHYLA

completion offertilization, becomes the embryo sac. In species in which

and tetraploidy are prevalent, the origin of these features should
triploidy
be watched for during fertilization.
one collects a complete series of stages in the life history, fertiliza-
If
tion certain to be found, but if the fertilization phenomena alone are
is

sought, it becomes necessary to know something about the time elapsing

between pollination and fertilization. This period differs widely among
different plants and is conditional upon so many different factors that
it is impossible to cite definite hours or days. It is therefore wise to make
a series of collections until dissections reveal the presence of young
embryos.
The periods intervening between pollination and the time the micro-
gametophyte reaches the megagametophyte are given for some common
plants; Betulay 1 month; CarpinuSy 2 months; AlnuSy 3 months; CoryluSy
over 4 months. QuercuSy 2 months in some species to 1 year in others;
FaguSy about 6 days; Hicoriay 5-7 weeks. Polygonurrij about a week;
FagopyrunXy 18 hours for self-fertilization or more than 72 hours for cross-
fertilization; PyruSy 2-4 days; Phaseolus vulgaris 8-9 hours; Trifolium
18 hours in summer to 35-50 hours in autumn. OenotherUy 36-72 hours
in most species; CitruSy 4 weeks; Convolvulus, only afew hours; Nicotiana,
about 2 days; Datura, in about 24 hours ;Ladwco, about 7 hours. Zostera
marina, 10 hours. Lilium, usually 60-72 hours; in L. auratum, 7 days,
24-36 hours in L. grandiflorum, 96 to 120 hours in L, martagon, 120 hours
in L. longiflorum, Tulipa, 8-10 days; Zea, 25 hours; Triticum, 32 hours
to 2 days; Secale, 7 hours. Various orchids require from 8 hours to
6 months.
The condition of the petals can sometimes be used as a criterion as
to what is transpiring within the ovule, but it is not a reliable one in

many plants. In Lilium, the Oenotheras, and many herbaceous plants,

one is likely to find fertilization if ovaries are removed from flowers whose
petals have withered but have not yet fallen.
There are not many plants which are ideal for fertilization studies,
and one is generally compelled to section a good many ovules or ovaries
before finding what is wanted. But when a good, well-stained prepara-
tion has been secured, it is so interesting and instructive that one feels
repaid for all the trouble.

EMBRYOGBNESIS
The polar nucleus, or nuclei, Which may be haploid, diploid, triploid
(prevalent condition), or tetraploid {Lilium, Friiillaria), is usually
^'fertilized'^ somewhat in advance of the egg nucleus and invariably
divides first.
 ANTHOPHYTA 487

—
Endosperm. The polar nucleus, or nuclei, which lies free in the
megagametophyte, customarily in the center or immediately below the
egg, is the original source of the endosperm, whether the latter is coeno-
cytic or becomes cellular. Fusion of the secondary male nucleus with
the polar nucleus (whether this is a single nucleus or two or more fused
nuclei) initiates endosperm growth. There is no definite rule governing
endosperm formation, although it is more or less constant for a given
species. In long, comparatively narrow embryo sacs, such as those of
Nicotiana, Solanum, and Verheruiy a cell wall is produced following the
first mitosis, and all subsequent mitoses result in walls. When the
megagametophyte is large and about as broad as long, the megagameto-
phytic cavity (t.e., the embryo sac) becomes almost filled with coenocytic
nuclei which then begin to develop walls, with a single nucleus to each
cell, but rarely with two or more nuclei in each cell. Lilium^ Reseda,
Capsella, and Ranunculus are characterized by this type of endosperm
growth. Intermediate types of megagametophytes generally contain
few coenocytic nuclei; sometimes walls may later be formed, but instances
are known where both cellular and coenocytic endosperm exist in different
ovules in the same ovary. Mitoses in the endosperm may be simul-
taneous throughout during the earlier stages in both coenocytic and
cellular types; in the latter type the later mitoses are sporadic, and in the
former type the mitoses later commence at the micropylar end and
proceed in waves toward the chalazal end.
Coenocytic endosperm is invariably evanescent, the substance being
absorbed by the developing embryo. Endosperm which is at first
coenocytic, then becoming cellular, generally shows some vestiges even
during the final stages of embryo maturation, but is not always of a
permanent nature. Endosperm which is cellular from the first mitosis
onward is of a permanent nature and may even acquire very thick walls
and become incredibly hard (Palmaceae, Diospyros, Coffea, etc.). In
general the endosperm type is uniform throughout entire orders among ihv,
dicotyledons but is not so uniformly typical among monocotyledons.
Among the monocotyledons, endosperm which is cellular from the
first mitosis on is very rare (Lemnaceae). In the Lilaeaceae and Orchi-
daceae endosperm is entirely absent. The other monocotyledonous
families are about equally divided into those which are coenocytic
throughout the life of the endosperm and those in which the so-called
Helobiales type prevails (this is essentially endosperm coenocytic at
first, then becoming cellular). Both types may be found within the
same family (Araceae, Potamogetonaceae, and Amaryllidaceae).
Preparations intended for the youngest stages in embryogenesis
will always reveal more or less of the endosperm development. Among
the more satisfactory types may be mentioned Nicotiana for the cellular
488 SPECIAL METHODS FOR THE VARIOUS PHYLA

type, Lilium or Capselld for the coenocytic-cellular type, and some

member of the Onagraceae for the coenocytic type. All these except
Lilium also show fine stages in the growth of the embryo. The same
fixing fluids as used for embryos will preserve the endosperm. Safranin
and fast green are recommended for staining.
Those endosperms that acquire thick walls and become very hard are
excellent for showing the nature of plasmodesma (protoplasmic con-
nections). "Nearly all palms (Palmaceae) have such an endosperm, as
do species of Diospyros. Material should not be hard to secure: dates

Fig. 110.— Dioepyros discolor: portion of a freehand transverse section of mature

endosperm, with prominent plasmodesma. Fixed with formalin-aceto-alcohol; stained
with iron hematoxylin and orange G,

{Phoenix dactylifera) can be purchased at a grocery store, or seeds of

related speciesand numerous other palms may be obtained from nursery-
men. The American persimmon, Diospyros virginiana^ is available at
fruit storesfrom late November into February, but not every fruit contains
seeds. The Philippine persimmon, D, discolor^ is better, but seeds are
difficult to obtain (Fig. 110). Fully mature endosperm has better
plasmodesma than does immature material. Remove the pulp and
testa, and fix the naked endosperm in formalin-aceto-alcohol for about a
week. If the endosperm is fixed whole, it is large and rigid enough to
be sectioned in a sliding microtome at 10^. Small portions are just as
good as complete sections; consequently it does not matter if the knife has
a tendency to spring up. Or the endosperm may be cut into small
 ANTHOPHYTA 489

portions (4 to 5 mm. cubes), fixed, dehydrated very slowly by the tertiary

butyl alcohol method, embedded, soaked under water for two weeks or
longer, and cut in a rotary microtome at 10^. The sections are easily
cut, but must be treated as freehand sections as it is impossible to retain
them on the slides because of their oily contents. It may be diflBcult
to get good staining unless the and fats are first removed. Sections
oils

cut either freehand or after embedding are therefore transferred to

chloroform (warmed slightly to dissolve paraffin from embedded sections)
or ether for a day or longer, then washed successively with absolute,
95, and 50% alcohols for hour in each. Wash with water, and place
in 4% ferric ammonium sulphate for 24 hours. Wash
thoroughly in
water, then stain with 0.5% hematoxylin Wash again
for 24 hours.
with water, then examine microscopically. If the plasmodesma appear
to be properly stained, dehydrate, and mount in balsam without differ-
entiating. Otherwises first differentiate cautiously, being careful not
to carry the destaining too far.
—
Young Embryo. In the majority of plants it is a troublesome matter
to study the young embryo. If the endosperm is cellular, the difficulty
is usually to distinguish the few-celled embryos from the surrounding
endosperm cells. Main reliance in such cases must be placed upon the
counterstain, which has a greater affinity for the cell walls of the embryo

than for those of the endosperm: fast green is usually good, but anilin
blue is sometimes even better. For later developmental stages the
difficulty is that of sectioning them in the correct plane. It should be
borne in mind that the cells of embryos are comparatively small and
filled with dense cytoplasm; conseciucntly sections should rarely be over
\2}x in thickness.
In many embryo grows slowly {Lilium^ Nicotiana, Oeno-
plants the
thera), in others itdevelops rapidly (Capsella, Zea). In any case, a long
series of preparations will be required in order to follow out the complete
sequence of events. In any one ovary the ovules will all show practically
the same stage of development. Fixation has usually been excellent
with formalin-propiono-alcohol, and staining is sharp with safranin and
fast green, but Harris’ hematoxylin, with or without a counterstain of
orange G, is also good.
—
Older Embryo. After cotyledon development has commenced, the
ovules should always be dissected out of the ovaries, unless the latter
themselves are too small for easy manipulation, and treated individually.
The ovarian walls by have become hardened, with extensive
this time
lignification and even and sectioning becomes increasingly
sclerization,
difficult. Formalin-aceto-alcohol may be used for fixation but should be
allowed to react for several days; also, the dehydration and infiltration
should b0 gradual. Ih species which have very large cotyledons or an
‘
 490 SPECIAL METHODS FOR THE VARIOUS PHYLA

extensive endosperm, these structures should be trimmed down as much

as possible before fixation; it is annoying to have to cut interminable
useless sections on the microtome until the embryo proper is reached.
Either safranin and fast green or a triple combination stain beautifully
during all the stages of embryo growth.

FRUITS
A fruit is essentially the matured ovary, accompanied or not by
various accessory organs, usually containing the seeds. The division
of fruits into those which are dry and those which are fleshy is excellent
in that it indicates the general technical treatment of each type. The
dry fruits are far more diflicult to manipulate than the fleshy ones.
Botanists apparently have never been much interested in the structure
of fruits, being content to leave the problem to pharmacologists. As a
matter of fact, one can secure more information regarding the micro-
scopical structure of fruits from a text on pharmaceutical botany than
frobfi the average botanical text.
The structure of dry, dehiscent fruits —legumes, capsules, and
follicles — is well shown, during at least the earlier stages of development,
in preparations of the ovary intended for megagainetogenesis and
embryogenesis. After the ovarian walls become hard and more or les^
desiccated, resortmust be had to celloidin embedding.
—
The dry, indehiscent fruits achenes, grains, nuts, schizocarps,
samaras— are even more difficult than the preceding group. The
younger stages are, of course, not very troublesome. The most interest-
ing of the indehiscent fruits is the schizocarp, characteristic of th(i

Umbelliferae, and featured by the presence of resin ducts and con-

siderable variation in general structure. The hard walls of nuts require
special methods; none of the ordinary sectioning methods gives even
passable results. The best procedure is to cut out very small portions,
treat with hydrofluoric acid, then embed in celloidin.
Amongthe fleshy fruits, entire longitudinal and transverse sectigns
of young pomes are readily made until they become about 18 mm. in
either length or diameter. The central part of the fruit, to include the
core, should be taken. Fix with formalin-propiono-alcohol, microtome
at about ll/z, stain with safranin and fast green or a triple combination.
Young drupes may be treated similarly until the endocarp becomes too
hard to cut with a scalpel.
The berry of Musa, Lycopersicum, and Solanum, the pepo of Ctumrbita
and CiicumiSf the syconiun of and the hesperidium of Citrus
are all easily fixed, sectioned and stained at all stages of growth up to the
time that the seed coats become too hard to cut readily. The young
fruits may be sectioned entire, but as they become larger, wedge-shaped
 ANTHOPHYTA 491

portions should be cut out. The berries of VitiSj the pepo of

CitrulluSf and similar fruits are composed of such thin walls and contain
80 much water that it is hard to fix and to dehydrate them. Aqueous
fluids should be used. The acetone or glycerin dehydration methods
afford excellent results; but whatever the schedule used, changes should
always be made over long periods.
Aggregate fruits of a type similar to those of Fragaria, RubuSy and
the Araceae, are easily manipulated until the walls of the carpels become
too hard. The walls of the carpels are thick from an early stage in
growth; consequently dehydration and infiltration should be prolonged
as much as possible. Embed in a hard paraffin or in celloidiii. Mul-
tiple fruits may be treated as a unit or cut into smaller portions; one
usually obtains the most satisfactory sections by microtoming per-
pendicular to the long axis of the entire fruit.

SEEDS
As a general rule, it is a nearly impossible feat to make sections of
entire mature seeds. The trouble is to get fluids and embedding media
to penetrate. Seed coats may be cut into small portions and treated
as if they were hard woody tissues.
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 INDEX
Numbers in boldface indicate pages on which special directions for treatment of the genus or species
concerned are given, or on which formulae, procedures, or methods are described in detail.

A Adhesive, Ullriches, 21
Adiantmn, 403, 412
A bies, 426, 443 Adoxa, 486
halsarninea, 432 Aesculus, 462
leaf, 199 buds, 185
AbsciB.sion layer, 461 leaf, 198
Acacta, pollinia, 476 Agardhiella, 292, 303
Acetabular ia, 249 coulteri,303
Acetone, 17 25 , tenera, 303
Achrochaetium, 296 Agaricaceae, 348-349
Achromatic figure, 64 Agaricales, 346-349
Achyla, 324 Agaricus (see Psalliota)
Acid, butyric, 36 Agathis australis, 428, 432, 439
c^rminic, 64 Agrimonia, 351
chromic, 36 Alarin, 379
citric, 200 Alariaceae, 279
formic, 199 Albuginaceae, 327
glacial acetic, 36-86 Albugo, 827
malic, 200 Alcohol, absolute ethyl, 15, 25, 34
nitric, 36 benzyl, 25
osmic, 7, 37 - 38 63 ,
ethyl, 16- 16 , 17
oxalic, 199 iso-propyl, 17 , 25
picric, 36 51, 62
,
methyl, 16
propionic, 36 95 % ethyl, 35
tartaric, 200 normal butyl, 18 25 ,

valeric, 36 normal propyl, 18

Acid fuchsin, 66 101 ,
propyl, 25
and fast green, 93 secondary butyl, 25
Acid green (see Light green SF) tertiary butyl, 18 , 25
Acidification, differential, 69 Alcoholic iron hematoxylin. 76
Acids, aromatic, 203 Aldehydes, 183
Acrasieae, 317 Aleuria, 840
Acrogynae, 370-378 Aleurone, 202
Actinomycetes, 226-226 318 ,
Algin, 270
cultivation, 226 Alizarin red S., 56
slides of, 226-226 Alkaloids, 63, 188-184
Actinostele, 381 Allium, 188, 468
AdinoBtrobus pyramidaliB, 432 cepa, 355
Adhesives, 20-21 bulb scale, 197
ceUoidin, 21 leaf, 198, 463

Hau|>t*s, 20 , 150 mitochondria, 100

Mayer's, 21 root tips, 173 448 , 460
,

503
 ,

504 PLANT MICROTECHNIQUE

Allornyces, 324 Anthophyta, megaspores, 479-480
Aloe, leaf, 466 inegasporocyte, 477479
Aniann’s iiiodium, 313 microgam etoph 3't;e, 476477
Anuiryllidacoae, 487 microsporogencsis, 473476
A maryllis, 468 organogen.y, 470471
Amines, 184r-186 origin of root, 462
Amino acids, 184-186 pt^tals and sepals, 472
Ammonium, 197 phyllodia, 468
Ammophila, 468 resin ducts,461462
AmpelopHs, 455 rhizomes, 462463
Amphipiiloic solenoslele, 381 root hairs, 460461
Anisinckia, 467 root tips, 448460
Amygdalin, 191 roots, 461462
Amygdalus, seeds, 192 secondary roots, 462
Amylodextrin, 188 seeds, 491
Amyloid, 194 stamens and pistils, 473
Anabaetia, 283, 286-287 stem apices, 463466
azollae, 287, 413 stems; 466463
cycadeue, 287 tubers, 462463
Anacrogynae, 372'* 373 tyloses, 462
Anagallis, 472 vascular system, 460
Ancylistales, 324-326 of flowt'f, 472
Andreaea, 379 whole mounts, of flower, 471
Andreaeobrya, 379 of leaf, 470
Anemone, 449 Anthracnose diseasi^s, 350
Angiopteris, 401 A nthurinm aruiraeanum, 468
Anilin blue WS, 66 Antithanmion, 306-307
Anilin 25
oil, Aphanocapsa, 285
Anilin water, 101 Aphanochaete 238
Anilin water-methyl violet, 216 Aphanothece, 285
Antherozoids, 109 Apparatus, 8-14
Anthoceros, 118, 148, 373-876 Aptenia, 468
enrolinianus, 287 Araban, 193
Anthocerotae, 373-376 Araceae, 287, 491
Anthocyanin, 192 Arachis, 467
Anthophyta, 448-491 Araucaria, 428
abscission layer, 461 bidwilli, 428
cladodia, 463 braziliarta, 432, 438
embryo, 489-490 Araucariaceae, 428, 431, 432, 438, 439,
embryogenesis, 486-490 443
endosperm, 487-489 Araucariales, 860
fertilization, 486-486 Arbutin, 192
flowers, 470^78 Arbutus menziesii, 472
fruits, 496-491 Arcularius’ solution, 108
lactiferous ducts, 461 Arginine, 184
leaf epidermis, 468-470 Aristolochia, 465, 461, 462
leaf types, 467-468 stem, 143, 467-468
leaves, 463-470 Aromatic acids, 208
lenticels, 462 Arthrospiray 286
macerated tissues, 461 Artifact, 180
tnegagametogenesis, 477-486 Asarum, 462
m^g^ametophyte, 480486 Asclepiadaoeae, 461
 ,

INDEX 505

AsclepiaSy pollinia, 476 Basidiomycetae, preservation, 346

AscocycluSy 271 sources of material, 346
AHcomycetaa, 330-846 Batrachospermaccae, 296
cultivation, 331 Batrachospermum, 291, 296
fixation, 381 Beech wood creosote, 18, 25
morphology, 331 Beggiaioa, 224
sexual re^produciion, 331-332 Begonia, 200, 467
sources of material, 331 Beijerinck’s medium, 238
staining, 331 Belling’s acetocarmin, 164
Asparagine, 184 Beiiecke's solution, 230
Asparagus, 455, 403 Bcfizol, 19, 25
Aspergillac(‘ae, 336-337 Berlx^ridaceae, 1 S3
Aspergill ales, 336-387 Berheris, h*af, 352
Aspergillus, 336-337 Bergamot oil, 25
herbariorum, 336 Berlin blue reaction, 191
Asperococcaceae, 277 Berry, 490-491
Asperococcus, 277 Beta vulgaris, 453, 467
Aspidiurn, 412 root, 199
Aster, 351, 450 tuber. 19fi
Ast(*rac(^ae, 475 Betula, 486
buds, 173 Biota, 443
Asterella, 367 Bisman^k bro\^•n Y, 67
A triplex hy nienelyt ra, 464, 467 Blastocladiales, 324
Au ran tin, 66, 101 Bleaching, 68-69
A ustrotax us439 reagents, 21
Avena sativa, 468 Boestroem’s stain, 226
root hairs, 451 Boletiis, 347-348
Azolla, 287, 401, 412-413
Borax carmin, alcoholic., 71
jiliculoides, 413
Lynch’s, 71
Bordeaux red, 67
H
Botrychiuni, buds, 144
irirginianutn, 381
Bacillariophycavie, 267-261
267 Botrydium, 245, 256, 398-401
collection,
mounting of, 260--261 Bawenia, 415, 416
preservation, 267 Bowen’s schedule, 97
taxonomic study 268-260 of, Brassica, 450
Hackman’s smear stain, 164 oleracea, 197, 321

Bacteria, 101 rapa, 197

Balsam, Canada, 23 Brazilin, 60
dammar, 23 Bryophyllum, leaf, 466
Bangia, 294 Bryophyta, 360-880
Bangiaceae, 294-296 collecting, 360
Bangiales, 293-296 embedding, 361-362
Bangioideae, 293-296 fixation, 361
Banksia, 467 staining, 362
Basic fuchsin (see Pararosanilin)
whole mounts, 863
Basic fuchsin-indigocarmin, 91
Bryopsidaceae, 247
Basidibolaceae, 830
Bryopsis, 247
Basidiomyoetae, 346-866
Bulbochaete, 242
cultivation, 846
Buxbaumia, 380
morphology 846 ,
606 PLANT MICROTECHNIQUE
C Cepkdlotaxusy 432, 438, 439
Ceramiaceae, 306-807
Caecoma (see Kunkelid) Ceramiales, 806-309
Cajal stain, 91 Ceramiuniy 291, 307
Calcium, 195 califomicuniy 306
oxalate, 196 Ceratiomyxay 316-317
CallistephuSj 351 porioideSy 315—316
Callithamnion, 807 412
CeratopteriSy 381, 402,
CaUophylliSj 302 Chaetangiaceae, 299-300
furcata, 302 ChaetoceroSj 258
Callose, 63, 64, 185-^186 Chaetomorphay 241
CaUymenia, 302 ChaetopeltiSy 238
Callymeniaceae, 302 Chaetophoraceae, 238
Calobryales, 373 ChamaecypariSy 443
472
CalochortitSj Chamaesiphonales, 286—286
CalothriXy289 Champiay 311
CalycanthuSj 478 Champiaceae, 310-311
Canna indica, 452 Chantransiaceae, 296
Capinpin's brazilin, 163 Ckaray 249-261 \
Capsellay 452, 472, 488 Charophyceae, 249-261
Capsules, staining of, 219-220 cultivation, 249
Carbohydrates, 186-189 fixation, 261
Carbol-methylene blue, 217 staining, 251
Carbol-methyl violet, 217 whole mounts, 249-261
Carbol-thionin, 217 Chemicals, 206
Carbon bisulphide, 25 Chenopodiunty 185, 198
Carbon tetrachloride, 25 China blue (see Anilin blue)
Carbonates, 199 Chitin, 189-190
Caricaceae, 461 Chlamydomonadaceae, 233
Carmalum, Mayer*s, 71 Chlamydomonas, 233
Carmin, 64 Chlorellay 247
alcoholic borax, 71 Chlorides, 199
Grenacher's alum, 70 Chlorococcaceae 246 ,

**rubrum opticum,'' 71 Chlorococcales, 244-247

Carminic acid, 64 Chloroform, 19 , 26
Carotin, 201 Chlorophyceae, 227-249
CarpinuSf 486 collection,227
Cassytha filiformiSy 467 cultivation, 228-231
CcLStalia, 468 fixation, 231-282
Catalase, 191 herbarium specimens, 231
CcttalpOy462 illumination of, 230-231
Cedar 24, 26
oil, occurrence, 227
CedruSy 426, 443 preservation, 231
deodaray 432 staining, 232
Cell walls, cutinized, 64 whole mounts, 232
lignified, 64 Chlorophyll, 201
suberized, 64 Chlorophyta* 227-261
Celloidin, 21, 22 Chodat-Grintzesco medium, 229
Celloidin methods, 121-126 ChondritSy 292 , 303
Cellulose, 63, 189 criapibSy 304
cell walls, 64 ChordarUiy 274
Cephalotaxaceae, 438, 439, 443 Chordariacoae, 274
 INDEX 507

Chordariales, 274-275 Concerns, botanical supply, 205

Chromic acetate, 36 Congo red, 67
Chromic anhydride (see Acid, chromic) brilliant {see Vital red)
Chromosomes, 64 Coniferales, 425-444
Chroococcaceae, 285 embryogenesis, 438-444
Chroococcales, 285 fertilization, 436-438
Chrysanthemum, 467, 475 leaf, 427-428
parthenium, 483 megagam etogenesis, 433-436
Chrysophyceae, 256-257 microgametes, 432-433
Chrysophyta, 256-261 microgaraetophyte, 430-432
Chytridiales, 322-324 inicrosporogenesis, 429-430
Cilia, 109 root, 425-426
Citrullus, 491 stamina te strobilus, 428-429
Citrus, 467, 468, 486, 490 stem, 426-427
glands, 462 Coniferophyta, 193, 420-447
Cladochytriaceae, 324 Conocephalum, 367
Cladodia, 463 conicum, 367
Cladonia, 358 Convolvulus, 462, 486
dadophora, 241 Cooper's triple stain, 89
Cladophoraceae, 240-241 Coplin jar, 11
Cladophorales, 240-242 “Copliiis,” 11
Cladophylls (see Cladodia) Copper-lactophenol, 231
(Jlarite, 24 Coralliriaceao, 301
Clavaria, 347 Corallorhiza, 108
Clavariaceae, 347 Cordyceps, 343
Clanceps, 343 Coreopsis, 475
Clearing reagents, 18-20 Cork, 63
Clematis, 455 Corrosive-osmic, 98
Clintonia, 472 Corticium vagum solani, 347
Coal-tar dyes, 54-64 Corylus, 486
Cobaea, 455 Cosmos, 475
Cochineal, 53 Cotton blue (see Ariilin blue)
Mayer^s alum, 70 Counters taming, 152
Codiaceae, 247-248 Coverslips, 13, 206
Codium, 247 Craigie's stain, 86
Codoniaceae, 372-373 Crassulaceae, 193
Coffea, 487 buds, 85
Coleochaetaceae, 239 Crataegus, seeds, 192
Coleochaete, 239 Creosote, beechwood, 18
Coleosphaeriurn, 285 Creosote method, 114
Coleosporium solidaginis, 351 Crepis, 475
Coleus, 462 Crocus, 449
petiole, 461 root tips, 173 , 177
Coliodesmaceae, 274 Cronartium ribicola, 361
Coliodesme, 274 Cryptomeria, 443
Colloidin, 22 japonica, 432
Colour Index, 65 Cryptomitrium, 367
CompUctoria, 880 Cryptonemiales, 300-302
Compaonema, 275 Cryptopleura, 807
Conant^s cold pack method, 105 Cryptosiphonia woodii. 800
Conant's hot method, 105 Crystal violet, 67 , 216
Conant’s quadruple stain, 87 ^
and erythrosin, 89
508 PLANT MICROTECHNIQUE
Cucumis, 185, 462, 490 DaMiay 475
Cucurhila, 490 tuber, 184, 185, 188
pepo, 108 DanaeOj 401
Cumagloiaj 295 Dasycladaceae, 249
anderaoniij 296-299 Daiura, 486
Cunninghamiaj 432 Daucus carotOy 196, 453
Cupressaceae, 428, 431, 432, 437, 439, 443 root, 199
Cupressusy 430 Dehj^drating reagents, 16-18
arizonicay 432 of stained slides, 162-164
govenianay 432 Dehydration, 180-184
Cupric acetate, 36 with acetone, 184
Cutin, 63, 190 with benzol, 184
Cutinized cell walls, 64 with chloroform, 182
Cutleritty 273 with dioxan, 184
Cutleriales, 273 with essential oils, 182
Cyanophyceae (see Myxophyceae) with secondary butyl alcohol, 182
Cyanophyta, 106, 189, 288-290 with tertiary butyl alcohol, 180
Cyatheay 382, 403, 409 with xylol, 182
Cyatheaceae, 409 Delafield\s hematoxylin, 117
Cycadales, 414r-419 Delesseriay 807
leaf, 416-416 Delesseriaceae, 807
petiole (rachis), 416 Dennesiaedtia adiantoideSy 409
pistillate strobilus, 419 Derhesia, 248
root, 414 Derbesiaceae, 248
staminate strobilus, 416-419 Dernwcarpa f ucicolay 286-286
stem, 416 prassina, 286
Cycadophyta, 414-419 Dermocarpaceae, 286-286
Cycas, 109, 287, 417 Desmarestiay 268, 276
circinalis, 414 Desmarestiaceae, 276
revolutay 414 Desmarestiales, 276-276
Cyclosporeae, 280-282 Desmidiaceae, 248—244
CydoniGy seeds, 203 Detmer^s solution, 280
Cylindrocapsaceae, 238 Dianthus caryophylluSy 468
Cylindrospermum, 289 Diaphane, 24
Cyperaceae, 198 Diatom aceous earth, 268-261
Cyrtomiuniy 404 Diatoms, 267-261
Cystophyllumy 274 Dichothrix, 289
Cystopus (see Albugo) Dicksonia punctilohay 381
Cystoseira, 269, 275, 282 Dictyostele, 381
Cytological piethods, 170-181 polycyclic, 382
Dictyosteliuniy 317
Cytological staining, 178-180
Dictyotay 278—274
Cytoraixis, 181
Dictyotaceae, 978-274
Cytoplasm, 64
Dictyo tales, 278-274
Czapek’s medium, 818
Differential acidification, 69
Dinobryoriy 266
D Dinophyceae, 266
Jt>i(maeay 468
DficramyceSy 849 Diooriy 416
Dacromycetales, 849 Diospyrosy 487-488
D&crydiumj 443 discolor 488
y

in$ermediumy 432 virginianay 488

 J

INDEX 509

Dioxan, 17, 25 Entomophthorales, 330

method, 118 Eniylomaj 366
Dipteris, 381 Enzymes, 190-191
DolichoSy 455 f>)sin, bluish, 68
Dothidiales, 344 yellowish, 68
Double embedding, 124-126 Ephedra^ 444r447
Draparnaldia, 236 aUitay 444 44 6
-

Drosera, 468 distachya, 444

Dadleydj buds, 85 foliata, 446
Dumontiaeeae, 300 Epichloe typhina, 343
Dunaliella, 233 Epidermis, leaf, 468-470
Dyes, 207 Epilithon, 301
anthraquiiione, 54 Epilobiuniy 452
azo, 54 Equisetales, 392-397
coal-tar, 64-64, 79-93 aerial branches, 393
natural, 60-64, 70-79 gametophyte, 396-397
*

nitro, 54 leaf, 394

nomenclature, 55 rhizome, 393
phenyl methane, 54 root, 393
quinone-imid(j, 54 strobilus, 394-396
solubility of, 55 Equiset inae, 392-397
xantheue, 55 Equisetum, 119, 198, 392-397
arvense, 394, 396
E debile, 396-397
laevigatuni, 396
Eherthella iyphi, 224 Ericaceae, 192, 455, 472
Echeveria, leaf, 466 Erigeron, 472, 475, 478
Ectocarpaceae, 271 Erodium, 472
Ectoearpales, 271-272 Erwinia, 223
Ectocarpm, 265, 268, 271 Erysipluiles, 337-338
acutus, 272 Erysiphe aggregata, 337-338
Kctophloic solenostele, 38 dchoracearurn, 337
Eichhornia^ 467 grajninis, 338
Elaehisteaceae, 274-276 Erythroglossum, 307
Elodea, 464 Eryihroniunij 449, 478
leaf, 239 Erythrosin, bluish, 68
Embedding, double, 124-126 Erythrotrichidy 293-294
media, 22-23, 207 carnedy 294
ovens, 11 Erythrotrichiaceae, 293-294
in paraffin,134-138 Eschscholtzidy 467
tray, 136 Ether, 25
Embryo, 489-490 petroleum, 25
Erabryogenesis, 486-490 Ethereal oils, 202
Emerald green {see Malachite green) Euascomycetae, 334-346
Empusa (see Entomophthora) Eubasidii, 346-360
Encelidj 468 Eubrya, 379-380
EncephalartoSj 415 EucdlyptuSy 468
Endodadiaf 292 Eudorirtdy 234
lilndomycetales, 382-334 Eugleridy 262-264
Endosperm, 487-489 iriridisy 253

Enteromorphaf 240 EuglendnKyrphdy 262

Entomophthoraceae, 830 Euglenophyta, 262-264
510 PLANT MICROTECHNIQUE
Eumycetae {see Mycophyta) Frozen sections, 106
Euparal, 23 Fructose, 186
Eupatorium, 461 Fruits, 490-491
Euphorbia, 196, 468, 481 Fucaceae, 268, 280-282
Euphorbiaceae, 461 Fucales, 280-282
Eusporangiatae, 398-401 Fuchsia, 478
Exoascales, 341-342 Fuchsin, acid, 56^
Exoascus (see Taphrina) basic (see Pararosanilin)
Exobasidiaceae, 346 Fucus, 265, 280-282
Exohasidium, 346 furcatus, 281
Exosporeae, 316-317 vesiculosis, 281 282
,

Funaria, 360
F hygrometrica, 380
Fagopyrum, 486 sporophyte, 361
Fagus, 486 Fungi Imperfecti, 366-366
I'astgreen FCF, 69 118 , Fungus and host, staining of, 320-321
Fast red B, P (see Bordeaux red) Fusarium, 342
Fat ponceau R (see Sudan IV)
Fats, 191 G
Fern prothallia, 114
Ferric acetate, 36 Galactan, 194
Ferric ammonium sulphate, 51 Galactase, 193
Ferric chloride, 52 Galanthus, 188
Fertilization, 486 Galaxaura, 299-300
Feulgen’s nucleal reaction, 96-97 Gasteromycetes, 349
Ficus, 196 Gastrodonium, 292
carica, 468 couUeri, 310-311
elastica, 467, 468 Gelidiaceae, 300
Filament, 283 Gelidiales, 300
Filicales, 407-413 Gelidium cartilagineum, 300
Filix, 402 crinale, 300
fragilis, 381 Gemmae, 365, 370, 376
Fixation, 27-48 Gentian violet (see Crystal violet; Methyl
images, 33 violet)
acid, 39-47 Gigartina, 292, 304
basic, 47-48 binghamia^e, 304
Flagella, staining of, 220 canaliculata, 304
Flemming^s 84-87
triple stains, Gigartinaceae, 304-306
Floral organogeny, 470-471 Gigartinales, 302-306
Florideae, 295-311 Ginkgo biloha, 420-426
Plowers, 470-473 Ginkgoales, 420-426
FKickiger test, 186 embryogenesis, 424—426
Formaldehyde, 183 leaf, 421
Formalin, 16 , 35
megagametophyte, 423
Forsythia, 462
microgametophyte, 422
Fossombronia, 373
pistillate strobilus, 422
Foster's stain, 91-93
root, 420
Fragaria, 491
Fraxinus, 470 staminate strobilus, 421
Freehand sections, 102-104 stem, 420
FrUiUaria, 478, 486 Gladiolus, 449
'

mekagris, root tips, 177 Glands, internal, 482

 INDEX 511

Qleichenia, 382, 408 Hakeay 467

Gleicheniaceae, 408 Halimeday 247
Gleditsia^ 451 HcUoactcciony 291, 309
Oloeocapsa, 285 HcUymeniay 301
Gloeothece^ 285 Haplostele, 881
Gloeotrichiay 289 Haplostichineae, 274-276
Gloiophloeaj 299-800 Hapteres, 278
coTtfusa, 298“300 Hard woods, 104
Gloiostphoniaj 302 Harris' hematoxylin, 52, 76, 117
Gloiosiphoniaceae, 802 Haupt's adhesive, 21, 50
Glucose, 186 Hazen's cement, 120
Glucosides, 191-193 Hederay 467
Glutamine, 184 HelianthuSy 198
Glutathione, 185 tuherosus, 188
Glycerin-gelatin, 119 stem, 197
Glycerin 25
jelly, Helminthocladiaceae, 296-299
Glycerin method, 119-120 Helminthoray 296
Glycerin-xylol method, 114-115 Helminthostachysy 381, 398
Glycogen, 189 Helotiaceae, 339
Glycol stearate, 22 Helvelltty 341
Gne tales, 444-447 Helvellales, 341
embryogenesis, 447 Hematein, 53
fertilization, 447 Hematoxylin, 50-53
leaf, 446 alcoholic iron, 76
ovulate strobilus, 447 Delafield's, 52, 77, 117
root, 444 Ehrlich's, 58, 79
staminatc strobilus, 446 Harris', 52, 76, 117
stem, 445-446 Heidenhain's, 50, 72-76
GnetuTTij 444 Ide-Roza, 79
Godetia^ 478 Iron, 50, 72-76, 117
Gonidia, 286 and Bismarck brown, 74
Ooniuniy 234 and safranin, 75
Gowodkowa^s medium, 333 Weigert's variation, 79
Gradlariay 291, 303 Hemibasidii, 350-355
sjostedtiiy 304 Hemicelluloses, 189, 193-194
Gracilariaceae, 804 Hepaticae, 363-373
Gramms stain, 217 Herposiphoniay 308
Graptopetalum iveinbergii, 468 Hesperidium, 490
Grateloupuiy 300 HeaperophycuSy 265
Grateloupiaoeae, 300-301 Heterochordaria, 272
Grenacher^s alum carmin, 70 Heterochordariaceae, 272
Guignard^s test, 191 Heterogcneratae, 274-282
Gums, 203 Heterokontae, 256
Gunneray 482
Heteronemay 307
Gymnoascaceae, 335
Heterosiphonia, 308
Gyromitray 341
Hicoria, 486

H Hieradumy 472
Hillary's smear method, 169

Haemalum, Komhauset's, 78 UippuruBy 454

Mayer*B, 52, 78 Hiss stain, 220
Sass’s, T$ Histidine, 184
512 PLANT MICROTECHNIQUE
Hordeurn vulgar e, 354 Isoetes, 381, 390-392
root haiFB, 451 nutlaUii, 391
Hormogoiiales, 286-290 Isogen era tae, 271-274
Hyacinthus, 176, 449 IsraeFs stain, 226
rneiosis, 173
root tips, 173, 448 J
Hydnaceae, 347
Hydnum, 347 Janus green B, 69
Hydraj 247 Jairopha, 468
Hydrangea, 351 Jeffrey's method, 104
HydrilUi vertidllata, 464 Johansen's methyl violet stain, 168
Hydrodictyacoac, 246-246 Johansen's im'tliyl violet-erythrosii
Hydrodictyon, 246 stain, 90
Hydrogoii peroxide, 21 Johansen's quadruph^ stain, 88
Hydrurm, 267 Juglans, 462
Hygrobutol, 17 J ungerm an n i ales 370-373
,

method, 110-113 Acrogynae, 370-372

Hy 111 cnoph y 11 a cejio 408-409
,
Anacrogynae, 372-373
Hymenophyllurn, 381, 403, 408-409 Juniperus, 428, 434, 436, 437, 443
Hypochnaceae, 347 communis, 432, 433
Hypochnus, 347 virginiana, 432
ochrolmicus, 347
Hypocrcales, 342-343 K
Hypomyces, 343
Hyrax, %i, 208 Kaiser’s glycerin-gelatin, 119
Karo, 24
I
Kauffman's smear method, 161
Indigocariuin, 64 Keefe's pniserving fluid, 106
Tndulin black (nee Nigrosin) Killing and fixation, 27-48
Infiltration, cytological, 176 nature of, 27-32
Internal glands, 462 Killing and fixing fluids, 39-48
Inulin, 63, 188 Allen's B-15, 46
Iodine, 38, 63, 197 Benda's, Taylor’s modification, 43
Iodine green, 69 Bouin's, 46
and acid fiichsin, 93 Allen’s modified, 46
Iodine-potassium iodide, 183 Carnoy-Leb run's, 40
Iridaea, 291, 303, 304-306 Carnoy's, 40
Iridopkycus (see Iridaea) Champy's, 97
Iris, leaf, 463 chrom-acetic, medium, 42
rhizome, 463 stock, 42
Iron, 197 strong, 43
Iron acetocarmin, 164 weak, 42
Iron hematoxylin, 117 chrom-osmo-acetic, Chamberlain’s, 44
Iron propionocarmin, 166 strong, 43
468
Isobilateral leaf, Taylor's, 43
Isoetales, 390-392 weak, 43
gametophyte, 392 combining of reagents in, 32-33
leaf, 390 Craf, 46
rhizophore, 390 formalin-aceto-aloohol, 41
root, 390 formalin-propiono-alcohol, 41
sporangia, 99^92 Gilson's, 40
etem, 390 Karpeebenko, Taylor’s modified, 44
 7

INDEX 513

Killing and fixing fluids, LaCour^s 2BD, Lemnaceae, 287

46 Lenticels, 462
LaCour’s 2BE, 46 Leptomitaceae, 326
Lewitzky’s, 378 Leptosporangiatae, 401-413
Licent’s, 101 gametophytes, 406-407
Navashin, Belling’s modification, 44 leaf, 403

RandolphVs modification, 46 rhizome, 402

Petrunkevitsch’s, 41 root, 402
Schaudinn’s, 47 sporangia, 403-404
Sharp’s, 411 stem, 402-403
Tcllyosiiicky’s, 46 LessoniopHtSy 279
Worccat(ir’s, 47 Leucine, 185
Zenker’s, 216 Leucojum, 188
Zirkle-Erliki, 47 Ijewitzky’s fluid, 878
Zirkle’s reduced cliromic, 48 Liagora, 297
Knives, 9 LihocedruH, 443
sharpening of, 10 decurrens, 432, 436
Knop’s solution, 230 Lichenes, 367-369
Kolatchev method, 97 classification, 368
Kornhaiis(‘r’s haemalum, 78 collection, 367
Kunkeiia nit mu, 363 fixation, 367
microtoming, 367
L preservation, 367
staining, 368
Laboratory 6 7
rules, - whole; mounts, 368
Laboiilbeniales, 344-346 Light green (see Methyl green
LaCour’s snu'ar method, 160 Light green E (sec Malachite green)
Lactiferous ducts, 461 Light green SF, 69
liMctophenol, 24 Lignified cell walls, 64
copper, 231 Lignin, 194-196
Laciuca, 480 lAgustrmn ovalifolium, 4t>7
haminaria, 279 Lilaeaceae, 487
Iwaminariales, 209, 278-279 Liliaceae, 478-479 486 ,

blade, 279 Ldliurn, 176, 449, 486 488 ,

gametophytes, 279 meiosis, 474

holdfast, 278 in anthers, 126
sporophylls, 279 ovaries, 177
stipe, 278 Lilium auratunif 486
Laothoe, 192 grandiflirrum, 486
Larix, 426, 431, 443 Imigiflorurn^ 486
Larrea^ 408 martagoriy 486
Laurencia, 301, 309 parryi, 482-484
apectahilis, 295,310 Linurn, seeds, 203
virgata, 295, 309 311 ,
Lipoids, 196
L(;af epidermis, 468-470 Ldriodendron, 461
types, 467-468 Ldthotkamniorij 301
Leathesiaceae, 276 Loeffler’s methylene blue, 2 1

Leaves, 463-470 Lomentaria, 292, 310-311

Lecithin, 196 Lonicera, 453, 455
Leif son’s stain, 220 Lophosiphonia, 308
Lemanea, 296 Lunularia, 365, 367
Lemaneaceae, 296 Lupinus, seedlii^gs, 181, 18.^
514 PLANT MICROTECHNIQUE
Lycoperdales, 849 Marchantiaceae, 866-869
Lycoperstcumy 490 Marchantiales, 363-869
pedicel, 461 Maraileay 401, 409-412
Lycopodiales, 883—887 veatiia, 381, 411
gametophyte, 887 Marsileaceae, 409-412
leaf, 386 Martins yellow, 60
root, 888 Materials, sources of, 204-208
stem, 888 Maionia pectinatay 381, 382
strobilus, 386 Mayer’s adhesive, 21
Lycopodinae, 888-892 alum cochineal, 70
Lycopodium^ 888-887 carmalum, 71
adpressuiTij 381 haemalum, 78
cernuumy 381 Medicago aativOy 339
clcLvotumy 381, 384—386 Megagainetogenesis, 477—486
dendroideSj 386 Megagametophyte, 480-486
inundatumy 381, 386 Adoxa type, 486
obacurumy 386 Fritillaria form, 484-486
phlegmariGy 381 Gunnera form, 482
pithyoideSy 383 Maianthemum form, 484
aerratumy 383 normal type, 480-481
iriatachyumy 381 Oenothera type, 481
voluhilcy 381 Penaea form, 481
Lygodium, 408 Peperomia form, 482
palmatumy 381 type, 481—486
Lynch’s borax carmin, 71 Pyrethrum form, 482
LynghyOy 286 Sdlla type, 481
Megaspores, 479-480
M Megasporocyte, 477-479
Melampaoray 342, 361
McCallum’s propionocarmin, 166 Melampsoraceae, 361
McClintock’s acetocarmin, 166 Melanconiales, 366
Macerated stem tissues, 461 Melohesiay 301
Maceration, 104 Membranopteray 807
McLcrocyatiay 270, 275, 278 Meniapermumy 462
Macrozamia, 415, 416 Mercurialisy 198
deniaoniiy 416 Mercuric acetate, 36
Magdala red, 58, 60 Mercuric chloride, 88
Magnesium, 196 Meriamopediay 286
Magnoliaceae, 472 Meaemhryanthemumy 200
Mahoniay leaf, 352 Mesotaeniaceae, 243
Maianthemum hifoliumy 484 Metachromatic granules, 221
Malachite green, 60 Methods, whole mount, 110-118
Malacophyllous leaf, 467 Methyl benzoate, 25
Maltose, 187 Methyl green, 60 ^ *'

Manganese, 197 and acid fuchsin, 98

Mannan, 194 Methyl pentoses, 198
Mannite agar, 314 Methyl violet, 60, 216
Mann-Kopsch method, 98-99 Methyl violet-erythrosin, 90
Marattiay 381, 401 Methylal, 17
Marat tiales, 401 Methylated spirits, 16
I^archantiay 866-867 Methylene blue, 61
362 Unna’s, 102
 INDEX 515

Methylene green, 61 Mycophyta, 318-369

Metzgeria, S72 cultivation, 318-319
Microcachrys, 431 differential staining, 320-821
Microchemical methods, 182-208 fixation, 319
Microchemical reagents, 182-183 general staining, 320
MicrocoleuSj 286 Mycorrhiza, 107-108
Microcystis, 286, 288 Mycosphaerellaceae, 343-344
Microgametophyte, 476-477 Myriogloia, 268
Microscopes, 8 andersonii, 276
Microsphaera alni, 338 Myriogloiaceae, 276
Microspora, 238 Myrionema strangulans, 275
Microsporaceae, 238 Myrionemataceae, 276
Microsporogenesis, 473-476 M yriophyllum, 454
Microtomes, 9 Myxogastres, 317
freezing, 106 Myxornycetes, 312-317
sliding, 102 collection, 312-313
Microtorning, of paraffin, 138-146 cultivation, 314-316
of refractory materials, 146-146 fixation, 313
Middle lamellae, 64 occurrence, 312
Millon’s reagent, 183 preservation, 312-313
Milovidnov’s method, 101-102 sections, 813
Mineral substances, 196-199 staining, 314
Mitochondria, 64, 90-102 whole mounts, 318
Mniutn, 380 Myxophycoae, 283-290
affine, 376-377 cultivation, 283-284
Mollisiaceae, 339 fixation, 284
Monilia, 366 occurrence, 283
Moniliaceae, 366 preservation, 284
Moniliales, 366 staining,284-286
Monoblepharidales, 324 whole mounts, 286
Monostroma, 240 Myxothallophyta, 189, 812-317
Montia, 467
Moraocae, 461 N
Morchella, 341
Mordants, 67 Narcissus, 449
Motility, in bacteria, 221 Nectria, 342
Mounting media, 23-26, 207 cinnaharina, 342
Mounting of paraffin sections, 146-160 Neisseria gonorrheae, 223
Mucilages, 203 Nemalion, 114, 291, 295, 296-299
Mucin, 64 Nemalionales, 296-300
Mucor, 327, 335 Neomeris, 249
Mucoraceae, 327-329 Neptunia, 468
Mucorales, 327-380 Nereocystis, 278
Musa, 490 Neurocarpus, 273
Musaceae, 461 Neurospord, 842
Musci, 876-880 Neutral red, 61
antheridia, archegonia, 877-878 Nevillite V (see Clarite)

gemmae, 376-377 Newton’s gentian violet-iodine, 169

protonema, 876-876 Nicotiana, 184, 487
sporophyte, 878 alata, 157
Mushroom, 348 Nigrosin, 61
Mycelia Sterilia, 866 Nitella, 249
516 PLANT MICROTECHNIQUE
NitophyUnnij 807 Oxalis, 200
Nitrates, 198 Oxidases, 190
Norit, 96
Northen^s stain, 92 P
Nostoc, 58, 283, 286-289
Nostocaceac, 287-289 Padina, 274
Notothylas, 878 Palmaceae, 198, 487, 488
Nucleal reaction, P'culgen’s, 96-97 Pandorina, 234
Nucleoli, fixation and staining, 107 Papavcraceae, 183, 461
Nymphaea, 453 Papilionacoae, 185
Pappenheim-Saathof stain, 223
O Paraffin methods, 126-164
Paraflftn sections, mounting of, 146-160
Oedocladium, 242 Paramecium, 247
Oedogoniales, 242 Paramylum, 252
Oedogonium., 242 Pararosanilin, 61
Oenothera, 481, 486 Parkeriaceae, 412
ovaries, 177 Parlax, 22
Oidium, 866 Parlodion, 22
dermatitidis, 866 Parowax, 22
Icxtis, 366 Parthenodsaua, 455
Oil, bergamot, 19, 25 Passiflora, 455
cedar, 19, 24, 25 Pastinaca sativa, 453
clove, 19 Pec tic substances, 200-201
origanum, 25 Pectin, 63
Olpidiaceae, 323 Pediastrum, 246
Onagraceae, 478-479, 481 Pelargonium, stem, 467, 461, 467
Oocystaceae, 247 Pellia, 372
Ophioglossales, 898-401 epiphylla, 372-376
fertile spike, 400 neesiana, 372-373
gametophyte, 401 Pellionia, leaf, 466
leaf and petiole, 400 Pelvetia, 265
root, 400 faatigiata, 282
stem, 400 Penaeaceae, 481
Ophiogloasunij 381, 898-401 Penicillium, 318, 336-337
pendvlum, 399 glaucum, 336
reticulatum, 399 Peperomia, 481, 482
vidgatum, 399 reflexa, 467
Orange G, 61 Pepo, 490
Orcliidaceae, 198, 472, 487 Perispo rales (aee Erysiphales)
Organic acids, 199-200 Peronosporaceae, 326-327
Organogeny, 470-471 Peronosporales, 326-827
Osazone test, 186 Peroxidases, 191
OsciUatoria, 283, 286, 288 Peroxide-ammonia, 21
Oscillatoriaceae, 286 Petals, 472
Osmium impregnation methods, 97 Petri dish, square, 156
Osmium tetroxide (see Acid, osmic) Peyaaonnelia, 800
OsmundUi, 381, 404, 407-408 Peziza, 840
cinnamomea, 404, 408 Pezizaceae, 840
daytoniana, 404, 408 Pezizales, 888-340
408
reyatia, 404, Phacidiaceae, 888
Osmundaceae, 407-408 Phacidiales, 888
 INDEX 517

233
J*kac()tacea(‘, Pinus muricaiay 434
Phaeophyceae, 262-282 ponder osa, 440-441
collecting, 263-264 Piper nigrum, 467
cultivation, 266-268 Pipette, giant, 14
fixation and embedding, 268-269 Pistia, root, 452
rnicrotoming, 269 Pistils, 473
occurrence, 262 Pisum sativum, root tip, 460
preservation, 264 266 Pithyrogramnm, 403
staining, 27(^271 Plasmodesma, 108- 109 284 ,

taxonomy, 271 Plasmodiophora, 321

whole mounts, 268 brassicae, 321-322
Phaeophyt.a, 203-282 Plasmodiophorales, 321-322
Phaseolu^ vulgarifs, 486 Plastids, 64
Phloba phene, 85, 193 Plectostele, 381
(See also Tannin) Pleopeltis simplex, 381
Pidoxine, 58 Pleuridium, 380
Phloxine B, 61 Pleurococcus (see Protococcus)
Phoenix dactylifera^ 488 Plocamiaceae, 304
Phorniidiuniy 286 Plocamium, 304
Phosphates, 198 Plowrightia morbosa, 344
Phragmidium.^ 353 Poaceae, 198, 202, 450
Phy com gees, 327 Podocarpaceae, 428, 432, ^36, 438, 439,
Phycomyc<*tae, 321-330 443
Phyllachora graminis, 344 Podocarpus, 428, 431, 439
Phyllactinia, 332 Pogonaturn, 380
coryleae, 338 Pollen, germination of, 476-477
Phyllanthus, 463 whole mounts of, 476
Phylloclads (3adodia)
(see Polyblepharidaceae, 233
Phyllocladus, 426 439 ,
Polycyclic dictyostele, 382
Phyllodia, 468 solenostele, 382
Phylloglossum, 383 Polyem bryony, cleavage, 442
Phyllospadix, 301 simple, 442
Phyllostachys, 468 Polygonum, 467, 486
Physoderma, 324 Polyneura, 307
Phytomonas, 223 Polypodiatame, 412
Phytophthora, 326 Polypodium, 381, 403, 404, 412
Phytosterol, 196 lineare, 404
Picea, 443 Polyporaceae, 347-348
e.xcelsa, 432 Polyporus, 348
Picro-indigocarinin, 236 sulphur e us, 348
Pigments, 201-202 Polysiphonia, 307-308
Pilobolaceae, 329-330 Polys phondyli um ,317
Pilobolm, 329-330 Polystele, 381
Pilularia, 381, 409 Polystichineae, 276-279
Pinaceae, 432, 437, 438 , 443 Polytrichum, commune, 378-380
Pinus, 196, 426, 428 429, 431-436 , 440-
,
sporophyte, 361
443 Populus, 351, 462
root tips, 107 petiole, 461
stem, 351 Porella,360 371-372
,

Pinus contortaj434 navicularis,371-372

coulteri, 427, 433 Porphyra, 292, 294-296
laricio, 436-437 perforata, 295
518 PLANT MICROTECHNIQUE
Portidaca oleracea, 467 Pyrua, 461, 462, 467, 486
Postdsia^ 270 , 278, 279 seeds, 192
pahnaeformis, 279 Pythiaceae, 326
Potamogetonaceae, 487 Pythiuniy 324-326
Potassium, 196 graciUj 325
Potassium bichromate, 39
PotentiUa glanduloaay 468 Q
Praaiolaf 240
nbvadensiSj 241 Quadruple stains, 87-88
Preservation of plants, 106-106 Conant's, 87
Primvla ohconica, stem, 199 Johansen's, 88
Prionitia, 801 Quartet, 480
ProaopiSy 468 Quercua, 193, 462, 467, 486
Proteins, 63, 202 root tips, 85
Protoascomycetae, 332-334 Quince-seed jelly, 253
Protococcaceae, 288 Quinone, 101
Protococcua^ 238
Protonema, 376 R
Protoaiphon^ 246
Protosiphonaceae, 246 Ralfsiaceae, 272
Prunua lauroce.raaua^ 192 Rana, tadpoles, 252
peraica, 341 Ranunculua, 449
Paalliota campeatriSj 345, 348-349 aquatiliSf 468
Paeudopezizay 339 leaf, 198
medicaginia, 839 RaphanuSf 450
Paeudotattga, 431, 432, 443 root, 462
Psilophytinae, 382-383 aativuSj root hair, 451
Psilotales, 382-383 Reagents, 16-26 206 ,

Pailotunij 381, 382-388 clearing, 18-20

nvdumj 382 dehydrating, 16-18
Pteridiumj 382, 402, 403 Refractory materials, sectioning of, 146 -

aquiliniurrij 412 146

kUittaculunij 412 Resin ducts, 461-462
Pteridophyta, 381-413 Resins, 203
stelar types, 381-882 Resorcin blue, 62
Pteroaiphonia, 307-808 Rheum, 200
Pterygophora^ 279 Rhizidiaceae, 323
Pucdnia graminia, 361-363 Rhizobium, 222
Pucciniaceae, 361-863 leguminoaum, 222
Pucdniaatrum^ 851 radidcolum, 222
Rhizoclonium, 241
Punctariaceae, 277
Rhizoclonia, 347
Punctariales, 276-277
Rhizomes, 462-463
Pylatella, 268, 271
Rhizopua, 118, 827-828
P3rrenoids, staining of, 238
nigricansy 329
Pyrethrurrij 482
Rhododendron, 346
parthenifolium, 482 Rhodogloaaum, 304
Pyrolaceae, 192 Rhodomela, 277, 286, 292
Pyrommaj 332 larix, 309
confluena, 339-340 Rhodomelaceae, 30 7-309 |

Pyronemaceae, 339-840 Rhodophyceae, 291-811

Pyrrophyta, 266 collection, 298
 INDEX
Rhodophyceae, embedding, 292-298 Safety razor blades, 11
fixation, 292 holders for, 11
occurrence, 291-292 Safranin O, 62, 80-87
preservation, 292 and anilin blue, 82
whole mounts, 298 and crystal violet, 82
Rhodophyllidaceae, 808-804 and orange G, 84
RhodophylliSj 808 and fast green, 80, 118
Rhodophyta, 291-811 and Harris^ hematoxylin, 88
Rhodymenia, 810 and picro-anilin blue, 82
Rhodymeniaceae, 809-810 Sagittaria, 452
Rhodymeniales, 809-311 Salicornia, 200
Rhus, 462 Salix, 351
Rhytisma, 838 pendula, root, 452
Rihes, 491 petiole, 461
leaf,351 Salsola, 200
Ricardia saccataj 309 Salvia apiana, 468
Riccardia, 372 Salvinia, 412-413
Riccardiaceae, 372 natans, 412-413
Riccia, 364-366 Salviniaceae, 412-413
hischoffi, 364 Sambucus, stem, 462
curtisiij 364 Saponaria, root, 192
Jiuitans, 363-864 Saponin, 63, 192
glauca, 363, 364, 866 Saprolegnia, 324, 32&-826
sorocarpa, 366 Saprolegnialcs, 326-826
Ricciaceae, 368-366 Sarcodiotheca, 303
Ricciocarpus, 148 Sargassaceae, 282
natans, 360, 863-864 Sargassum, 282
Ricinus, 455 Sass^ haemalum, 78
Riella, 370 Sassafras, 462
Riellaceae, 370 Sax^s crystal violet-iodine, 160
Rivulariuj 289-290 Scenedesmaceae, 247
Rivulariaceae, 289-290 Scenedesmus, 244, 247
Robinia, 462 Scharlach R (see Sudan IV)
Root, origin of, 462 Schizaea, 381, 408
secondary, 462 Schizaeaceae, 408
Root 460-461
hairs, Schizocarp, 490
Root 449-460
tips, 172, Schizogoniales, 240
Roots, 461-462 Schizomycetes, 211-226
Rosa, 455, 467 capsules, 219
leaf, 353 cell structure, 212
Rubus, 491 culturing, 213
orange rusts of, 353 flagella, 220

Rumex, 200, 462 metachromatic granules, 221

Ruscaceae, 468 morphology, 211
Ruta, 468 motility, 221
smears, 216
S soil microflora, 221
sources of, 213
Sabouraud^s medium, 318 special methods for, 222-226
Sacckaromyces cerevisiae, 189, 832 spores, 219
eUipsoideus, 882 staining, 216-219
Saccharomycetaceae, 882-834 tissue fixation, 216
520 PLANT MICROTECHNIQUE
Schizophyta, 211-226 Slides, mounting of, 162-164
Schizosaccharomyces octoapm'ua, 332-333 Smear methods, 166-169
Schultz's Farhstofftabelln, 55 Sjhear technique, 166-168
Sciadopitaceae, 432, 437, 443 "Sme^, staining of, 168-169
Sciadopitys, 432, 437 ^cetocarmin, iron, 164
Scilla, 481 Backman's method, 164
Sciruiiaj 299 Belling 's acetocarmin, 164
Sclerophyllous leaf, 467 Capinpin's brazilin, 163
Hcleroiinia fructigenay 339 method, 169
Hillary’s
ScoliopuSf 467 Johansen’s methyl violet, 168
Scytonenuiy 289 Kauffman’s hematoxylin, 161
Scytoiiemataceae, 289 ^aCour's method, 160
Scytosiphon lornentariay 277 McCallum's propionocarrnin, 166
'''

Scytosiphoiiaceae, 277 McClintock’s acetocarmin, 166

Secaky 486 Newton's gentian violet-iodine, 169
cereale, root hairs, 451 propionocarinin, McCalliim’s, 166
•SfHJondary niegasporocytes, 480 Sax’s variation, 160
roots, 462 Tuan’s hematoxylin, 162
Secretions, 202-203 Warmkc’s method, 168
Sectioning of hard woods, 104 Zirkle’s methods, 167
Sections, freehand, 102-104 Smears versus sections, 171
frozen, 106 Smith's picric acid-Gram stain, 90
versus smears, 171 Sodium, 196
Seduniy 467 Soil microflora, 221
leaf, 466 Solanurn, 198, 199, 490
praealiuniy 459 tuberosum, 453
Seeds, 491 tuber, 185, 196, 197
Selaginella kraussiarui, 381 ,
388 Solenostele, amphiphloic,, 381
Selaginellalcs,388-390 ectophloic, 381
gametophyte, 389-390 polycyclic, 382
leaf, 388 Solidago, 351, 450
rhizophore, 388 Solieriaceae, 303
root, 388 Solvents, 65-66
stem, 388 Soranthera, 277
strobilus, 388-389 ulvoidea, 277
Sefnele, 468 Sorbosti, 187
androgynay 463 Sources of materials, 204 -208
Sempervivuniy 199 Spartiua, 468
Sepals, 472 Special methods, 96-109
Sequoia, 431 Spermothavinion, 807
aefnpervireris, 429 432, 436 , 438
,
Sphacelaria, 268, 273
Sharp's fluid, 411 Sphacelariaceae, 278
Silicon, 198 Sphacelariales, 278
Silphium, 475 Sphaerellaceae, 236
Sinapsis alba, seed, 197 Sphaeriales, 343-844
Siphonales, 247-248 Sphaerocarpaceae, 369-870
Siphonocladiales, 249 Sphaerocarpales; 869-370
Slide containers, 14 SphaerocarpoSy 369-370
Slides, 12, 206 criatatusy 368-369
cleaning, 13-14 Sphaeroplea, 241
culture, 12 Sphaeropleaceae, 241-242
depression, 12 Sphaeropsidales, 366-866
 1 )

INDEX 521

Sphaerotheca^ 332 Stems, 466-463

humuli, 837 Stender dish, 1
pannosa, 337 ‘‘Stenders,” 11
Sphagnobrya, 378-879 Stenogramme interrupta, 306
Sphagnum^ 378-379 Stentor, 247
Spinada oleracea, 197 Sticta, 235
Spiraea, 472 pultnonaria, 368
Spirogyra, 242-243 Stigeocloniuni, 238
Spirulina, 286 Stigonenia, 289
SpondyloiTioraceai^, 236 Stigonernataceae, 289
Spongoapora auhterranea, 321 Stilophora rkizoides, 276
Hpoms, staining of, 219 Stilophoraceae, 276
Spo roc, hna ao ,275 Stock wc'll’s stain, 85
Sporochnalos, 275 Strehhmema, 268, 271
Sporochn us ,275 Stypocaulon, 273
Sporodinin, 329 Sty rax, 24
Sqnainariacoa(^, 300 Suberin, 190
Stain, acid fuchsin and fast green, 93 Suberized cell walls, 64
Cajal’s basic fuchsin-indigocarinin, 91 Sucrose, 186
Cooper’s triple, 89 Suction pump, 128
Foster’s tannic acid-ir()n cl)lorido, 91 Sudan III, 62
Gram’s, 217 Sudan IV, 62
iodine green and acid fuchsin, 93 Sugars, 186
Joliansen’s methyl viohd-eryth rosin,' Sulphates, 198
90 Sulphur, 198
methyl green and acid fuchsin, 93 Supply concerns, 205
Northen’s, 92 Syconium, 490
Smith’s picric acid-Grarn, 90 Synchytriaceae, 323
Staiiung, of paraffin sections, 161 Synchytriuni, 323
procedures, 65-94 amsmeJdae, 323
progressive, 69 Synthetic resins, 24
regressive, 69 Syringa, 455
Staining dishes, 11 leaf, 338
Stains,49-64 vulgaris, leaf, 466
bulk, 64
chromatin, 64 T
effects of fixatives on, 67
general, 69 Tannic acid-iron chloride stain, 91
mordants for, 67 Tannin, 107 193 ,

nuclear, 64 {See also Phlobaphene

quadruple, 87-89 Taphrina, 331, 341-342
solvents for, 65 deformans, 341-342
specific;, 69 Taraxacum, 188, 472
strength of, 66 Targionia, 366
for whole mounts, 116-118 Targioniaceae, 366
Stamens, 473 Taxaceae, 428, 431, 432, 438, 439, 443
Stangeria, 415, 416 Taxodiaceae, 431, 432, 437, 439, 443
Starch, 63, 188 Taxodium, 443
Statoliths, 109 distichum, 432
Steles, 881-882 Taxus, 430, 432, 434, 439
Stem apices, 468-456 baccata, 432, 438
Stemonitis, 312 canadensis, 438
522 PLANT MICROTECHNIQUE
Terpineol, 19 Tyloses, 462
Tetrasporales, 289-287 TyphUy 462, 468
Tkafnniochaete, 238 Tyrosine, 186
Thekphoray 847
Thelephoraceae, 847 U
Tkielavia basicoUiy 885
Thionin, 68 Ullriches adhesive, 21
ThioihriXy224 UlmuSy 470
Thujay 436, 443 UlothriXy 287
Tiliay 461 Ulotrichales, 237-288
Tilletiay866 Ulvay 289
foetans, 866 Ulvaceae, 289-240
triiiciy 866 Ulvales, 289-240
Tilletiaceae, 864-866 Unna^s methylene blue, 102
Tissue maceration, 104 Uredinales, 860-864
Tmesipterisj 382 Urocystis cepulacy 866
Toluol, 19, 25 UromyceSy 364
TolypothriXy 289 UrophlyctUy 324
Torreyay 432, 436, 443 UrticOy 198, 199
iazifoliay 432, 436 , 438 UsneOy 245
Tonilaceae, 334 Ustilaginaceae, 864
Tradescantia, leaf, 197 Ustilaginales, 364-366
meiosis, 474 UstilagOy 364
Tradescantia fluminenaisy 467 avenaCy 364
virginianay 469 hordeiy 864
Trapay 468 leviSy 364

Trebouxiay 246 zea^iy 364

Tremellales, 349-860
Trentepohliay 239 V
Trentepohliaceae, 289
Treponema pallidum 224 y
Vacdniumy 346, 472 »

Tribonemay 266 Vagneray 450

Trichitty 313 Vnloniay 249
Trichloroethylene, 19 , 25 Valoniaceae, 249
TrichomaneSy 408-409 Vascular system of stem, 460
Trichome, 283 Vaucheriay 248 , 324
TrichophyllouK leaf, 468 terrestriSy 248, 325
Trifoliumy 486 Vaucheriaceae, 248
TrilUufriy 176, 4^, 468, 476, 478 Venetian turpentine, 24
chZoropetalumy 159, 161, 168 Venetian turpentine method, 116-^416
meiosis, 173 Venturiay 332
omtumy 156, 159, 162 inaequaliSy 848-844
Triticumy 486 Veronicay 453, 455
bunts, 366 Vida faha, 176
Teugoy 443 root, 452
canadenaiB, 432 root tip, 460
Tuan^s smear stain method,. 162 Vincetoxicum, pollinia, 476
Tuberales, 840-841 Vital red, 63
Tubers, 462-468 VitiBy 465, 462, 491
Ttdipay 449, 486 , 486 vinifera,463
THirpineol, 26 Volutin, 284
 INDEX

Xylan, 198
Volvocaceae, aSS-iSC
Xylene (see Xylol)
Volvocales, 288-286
Xylol, 20, 25
Vdvox, 28^86
W Y

Wiitmke’B smear
metbo^ W8 Yeasts, false, 884
true, 382
Washing of material,
Waaserbltithe, 288
Aniln. blue) Z
Water blue (see
Zamia ftoridana, 414-418
wSrtSthod,9»-99
Zantedeschin, 467
Welwitschia, 444
284 Zea, 486
Wettstein's solution,
mays, 176, 198, 354, 462, 468
Whidbeyella, 299 iid
1 leaf, 466
Whole-mount methods,
Ziehl-Neelson 's carbol-fuclisin, 217
Wisteria, 455
Zingiber, 453
Woods, hard, 104
Zirkle’s smear met hods, 167
Woodmrdia, 404
Zonaria, 274
Woroni'oa, 824
Zosiera, 301
Woroninaceae, 824
marina, 486
X Zygnema, 243
Zygnemataceae, 242-248
Zygnematales, 242-244
Xanthophyceae, 266
Zygorhynchus, 327
Xanthophyll, 202
Zygosaccharomyces, 332
Xenococcusj 286