PRACTICAL REPORT

TECHNIQUES AND MANAGEMENT OF SCIENCE LABORATORY

PREPARATION TECHNIQUE OF PLANT TISSUE

By :

NATURAL SCIENCE EDUCATION STUDY PROGRAM

FACULTY OF MATHEMATICS AND NATURAL SCIENCES
YOGYAKARTA STATE UNIVERSITY
2015

A. TITLE
Preparation Technique of Plant Tissue

B. OBJECTIVE
1.

Knowing how to manufacture and capable of making simple preparations of fresh

plants and examined using a microscope.

2.

Knowing the anatomical structures in plants the roots, stems and leaves.

C. THEORY
1. Microtechnic
Microtechnic or histological techniques is the science or art of preparing organs, tissues
or parts of the network to be observed and analyzed. The review is generally performed with the
aid of a microscope, because the network structure in detail was the norm too small to be seen
with the naked eye. The scope of which includes Microtechnic material can be obtained from a
number of definitions and terminology that can be used, just that we should bear in mind that a
specimen Microtechnic may constitute a part or a whole of the structure of the set. Besides being
equipped with a glass slide, the specimen was generally protected by a glass cover, which is a
very thin piece of glass or a transparent plastic that is glued on the specimen (Gunarso, 1989).
Microtechnic is the science which will study the methods / procedures of making microscopic
preparations. There are various preparations; based on the level of durability is divided into
three, namely: preparation of temporary / fresh, semi-permanent preparations, and preparations
preserved. Based on the method of manufacture is divided into Whole mount / intact, Smear /
lear, squads, Section and maceration (Ekosari R, et al. 2015). To make preparations used
technique is a way of making preparations microscopis called Microtechnic.
According to (Kentjananingsih, S. 1989), Microtechnic is a science which will study the
methods / procedures of making microscopic preparations. Microtechnic is a technique of
making preparations or preparations microscopically, of course, the theoretical approach is not
sufficient to understand thoroughly about Microtechnic, because the name of the technique
emphasizes understanding of the region use despite basically theoretical foundation is also
necessary in order to provide some guidelines that must be passed in order that the
manufacturing process preparation according to the procedural work and reason for the use or
selection of materials to be used in the manufacture of microscopic preparations.
2. Methods Microtechnic

1. Method Whole Mounts

Whole mounts method is often called the perfomed method intact. This is because the use
of this method, prepared preparations consisting of whole organisms (both animals and plants) in
their entirety. culture specimens, organs, or parts of organs, embryos, egg, sperm, pieces of
nerves, blood vessels, the types of thin film and so on. Through this method we get the
impression sought to be original form by maintaining taga-dimensional formats. Which becomes
the limiting factor is the size, thickness and transparency of the preparations that we make with
regard to observation through a microscope magnification factor later (Volk, Wheeler. 1988)
According to (Bloom and Fawcet.1994). whole mount is a method of making
preparations that will be observed with a microscope without preceded the cutting process. So in
this method, the observed preparations are complete preparations either in the form of cells,
tissues, organs and individuals. Images produced by the whole mounth preparations is visible in
the form intact as when the organism is still alive so that observations can be made is limited to
general morphology alone. Method for making preparations used for observation as a whole,
Meaning the study of vegetative and reproductive structures without making an incision on the
plant because this method uses all parts of the plant as preparations. Of course the plants were
observed to be small so as to fit on the glass objects. While on a rather large plant pruning can be
done to become more neat and small.
With this method prepared preparations consisting of whole organisms (both animals and
plants) in their entirety. culture specimens, organs, or parts of organs, embryos, egg, sperm,
pieces of nerves, blood vessels, the types of thin film and so on. Through this method we get the
impression sought to be original form by maintaining the three-dimensional formats. Which
becomes the limiting factor is the size, thickness and transparency of the preparations that we
make with regard to observation through a microscope magnification factor later. Preparations
with a thickness of 2 mm and transparent would allow it to be observed until the magnification
level of no more than 30 times. Preparations with a thickness of 0.5 mm may only reach the level
of magnification of 100 times (Kentjananingsih, S. 1989)
Permanent preparations with a thickness of 0.2 mm or more that have been dehydrated
and were given a complementary media requires the existence of a supporting glass cover so that

the specimen is not to be damaged and the glass cover itself is not broken because the process of
drying and shrinkage of the media. Lately, commonly also used a plastic tube that is cut
transversely to the supporting rings produced with a thickness corresponding to the height and
thickness of the specimen. Cutting edge should be smoothed by using sandpaper (Gunarso,
1989).
Mounth whole method has advantages and disadvantages. The advantages of this method
is able to observe the entire plant clearly each of its parts. The disadvantage is that this method
can only be done on plants with a small size alone can not plant large so this method should be
developed to perform a variety of experiments.
2. Method Smear / Apus
Ie by scraping over the glass object so get thin membrane. Example: pollen, blood,
vaginal pillowcase (to determine the pregnant animals or not), sekulen plants.
In addition to the types of methods used materials suffered death and fixation. For
observation of blood cells that are still alive are generally used as Yanus vital dye Neutral green
or red, because the blood cells have the ability to suck the dye at the appropriate concentration.
When the dye is used together then allows us to observe the mitochondria. It just happens to
rapid changes in the cells, because the cells can be killed by both colors simultaneously before
(Gunarso, 1989).
Examples of the preparation of blood super vital preparation is:
a. 1 drop of blood dripped on glass slide
b. Similarly drops 1 drop dye (eg Yanus green) with a concentration of 0.25 in
physiological saline
c. Cover with a glass lid
d. Let stand for 5 minutes
e. Give lacquers petrolatum around the edge of the cover glass.
3. Method Squash

Methods for preparing stiffened squeeze many chromosomes observations both animals
and plants. With this method the material crushed or destroyed so that each cell will be released
that allows further observation. So the purpose of crushing this does not mean destroying the
cells, but each free cell separated from each other by retained its original form.
4. Method Section
How to progress through the incision or incisions is considered a routine technique or
techniques for preparing specimens histology and pathology. The thickness of the incision
depends on the experience and specimen preparation purposes. Thick slice commonly ranging
between 6-15 microns (1 micron = 0.001 mm). Ukura incision also vary widely, ranging from
incision is very small blood vessels to the brain slice. The size of the incision is usually limited to
a size of 2x3 cm length because of the size thus most suitable for glued on a glass slide that is
commonly used. Of course size is quite small specimens will produce incisions also are also
much smaller than the size of the incision.
Incision or incision is generally performed with the aid of microtome, though often done
slicing by hand only for the types of specimens such as bone, teeth or fossil objects are often
needed a chainsaw to cut it. Microtome is kind of a special machine designed and marketed for
the purpose Microtechnic. The machine is designed such that it is able to perform incision
something specimens with a thickness equal to or at least approximately equal (Gunarso, 1989).
5. The method of maceration
Maceration is a form of artificial decay, in which parts of certain organisms become soft and
eliminated, while other parts are resistant to maserator solution will remain intact. For example
maceration on the carrier network plant, which separates the cells of the network elements of the
carrier, so that the cells apart from one carriage to another (Bloom and Fawcet, 1994).
6. The preparation method Range
In this method, preparations have not been fixed, treated such that besides obviously also
approaching its natural state through stretching. The type of material that UUM preparation is
stretched when fixed muscles, nerves, kind of thin tissue (membrane that encloses the heart, liver
and others) (Gunarso, 1989).

7. The method of preparation Rub

Its kind of hard tissue, such as bone, teeth, nails and some may once very difficult to
make preparations incision (except when experiencing a variety of special treatment before). To
overcome the above said, it is also made preparations with the general method of rubbing. For
example femur bone, first cut to the size of a few milli up to 1-2 cm. Pieces are then rubbed on a
stone until thin enough to be observed in the microscope (Gunarso, 1989).
8. The preparation method Description (Teasing Preparations)
Understanding teasing is outlining. To be able to separate components of a particular type
of tissue or organ tissue or tissue decomposition described by using a needle. Thus the notion of
teasng this means also surgery on a small scale. Levels on a regular surgery and micro surgery
performed using a needle decomposers. Teasing is done on the type of fresh preparations that
have been fixed and experienced staining.
In general, the type of tissue that can be analyzed through the method of this cover is the
blood, spleen, bone marrow fluid behind, cement Janan, preparations urine, as well as several
others. Each of these techniques usually require separate treatment in conducting reviewing or
spread on glass slide. For this type of liquid containing high suspension density is generally
diluted with water or blood serum in the ratio 1: 5 or 1: 10 (Gunarso, 1989).
In making preparations or Microtechnic there are several steps that must be passed,
namely
1. Fixation (Fixation)
Fixation can be chemical compounds that use formaldehyde / glutardehide and
mechanical manner or boiling difreezing. Fixation goal is to maintain the shape of the cells or
tissues such as the original tissue, as well as to prevent damage to the sample due to autolysis or
because of bacterial decomposition (bacterial decomposition).
2. Dehydration and Clearing
Using alcohol given in stages from high concentration to low concentration. The purpose
of dehydration is to eliminate the remnants of the liquid / water present in the sample so that

when the next process is not formed ice in the sample. After the dehydration process in clearing
the xyline solution to clean the remnants of alcohol.
3. Embedding
Samples hardened using wax / waxes, resins to form a block parapin. Toughened so easy
to do the cutting sample.
4. Sectioning
Samples are cut by using a cutting tool for example microtome. Or if the earlier samples
were fixed by means of freezing can be further cut with a cryostat method (cutting frozen).
5. Staining
Further samples of colored, usually with hymatoksilin-eosin staining, or of other
derivates are used. The coloring to color the nucleus and cytoplasm.
6. Smoothing
After further colored sample can be directly observed under a microscope or in the
process of mouthing a sample for the purpose of maintaining the sample if stored longer,
coloring and the sample is not damaged. Typically using canada balsam.
3. Wide Mixture
Before observing the object by using a microscope, need to be prepared in advance of
preparations or preparations object to be observed. Mixture is an object that is placed on the table
objects that will be observed by using the objective lens and eyepiece on the microscope.
Mixture there are 2 kinds of preparations preserved and wet mount. Preservation preparations
done at the time to do a practicum Microtechnic plants and preparations produced can be stored
for a long time. Mixture wet lab work done at the time the structure of microorganisms and
preparations produced can not be stored long (Volk and Margareth, 1998). Making preparations
can be done by slicing vertically (upright) and horizontal (landscape). Slicing through the
vertical and horizontal directions to be obtained objects with cross-sectional and longitudinal. In
principle, there are three kinds of slices by cutting field, namely:

1. Sliced transverse (cross section, usually abbreviated to cs or xs) is sliced in the direction
perpendicular to the horizontal axis of the object.
2. Sliced lengthwise (longitudinal section, usually abbreviated to ls) is sliced parallel to the
horizontal axis of the object.
radial longitudinal slices: if the preparation is sliced perpendicular to the axis of the organ
Sliced longitudinal tangential: if the direction of the cut does not pass through the axis of the
organs but only parallel to the axis of the organ. Actually understanding is parallel to the outer
surface of the plant body.
To stem plants that square-shaped cross section for instance the cat's whiskers when sliced
lengthwise axis through the so-called diagonal oblong cross-section.
For materials such as castor beans that have symmetric field, the cross-section of sliced oblong
cross-section passing through the axis called median longitude.
3. Sliced middle (median section, usually abbreviated med.atau m.) Is sliced parallel with or
perpendicular to the central part of an object.
Mixture wet lab work done at the time the plant structure and the resulting preparations can not
be stored for long. Done by conventional incision, which is thinly sliced by a razor blade or
cutter new thin and sharp.
a. Slicing ways:
1) The glass objects that have been cleared of fat drops (1 drop) reagent middle - the middle of
the glass object.
2) Hold the material preparations perpendicular to the body, when very thin material can be aided
by slip material on the stem pith or cork manihot
3) Place the razor blade / knife / cutter on the material at an angle <30 degrees in order to get thin
slices. Navigate the knife towards the body.
4) Make slices 3-4 slices.

5) Put the slices in a glass that has been spilled reagents.

6) Close the cover glass that has been cleaned carefully and slowly.
7) Avoid the formation of bubbles of water, avoid assisted by placing a glass cover over the filter
paper and is closed slowly.
8) Excess water is sucked with a tissue.
b. Observations way
1) Prepare the microscope known apparatus and a clean state.
2) Place the microscope on the table without a mat books.
3) Adjust the lighting by using the objective lens magnification of 10x.
4) Place the preparation under magnification 10x objective lens with the correct position.
5) Look for shadows on preparations, by rotating the objective lens approaching makrometer up
preparations approximately 2mm above (look from the side), then turn back makrometer up
slowly, until you see the shadow.
6) If have seen the shadow of the object, act on the part to be observed, Clarify by rotating the
micrometer slowly and rotate the objective lens magnification to 40x. and clarify lai by turning
the micrometer slowly.
7) Determine the objects observed with the actual size can be done by using a micrometer with a
calculation:
Y=d/a
Y = length of cell in microns.
a = the number of cells along the broad view.
d = diameter of view in microns wide.
Plant parts to cite Mixture

1. Root
By origin, the root of the plant is divided into two categories, namely primary root and
wild roots. AKR primer began to emerge from the plant is still in the embryonic phase and the
roots of the plants that remain for life. The primary function is to enforce the roots of plants that
can stand upright on the ground, absorb water and inorganic materials from the soil and store
food (Iskandar, S.M, et.al. 1977)
Wild roots emerge from the stem, leaves, and other tissues that can be permanent or
temporary. Wild roots have a variety of functions. No one has wild roots reach the ground will
serve as the primary root, da tones that are modified as a medium to shore, crawling, or attached
(haustaoria).
Roots growing from the root apical meristem at the tip of the protected kaliptra (hood
roots). Apical meristem always divide generating new cells. New cells form on the hood or
inside the root apical meristem. Cleavage apical meristem elongation forming region, called the
zone of cell elongation. On the back there is the zone of cell differentiation and cell maturation
zone. In the zone of cell differentiation, root cells develop into multiple cell permanently. For
example, some cells differentiated into the xylem, phloem, parenchyma, and sklerenkim (Estiti
B.Hidayat. 1995)
2. Trunk
Stem a plant organ that serves to straighten up and connecting the stems and leaves.
Activities related to the growing point, there are two theories about it (Gunarso, Wisnu. 1989).
a. Theory Histogen
According to him, the growing point consists of three layers, namely:
1) The outer layer forming the epidermis, called dermatogen,
2) mid-forming layer of the cortex, called periblem
3) The mid-forming layer stele, called plerom
b. Theory tunica corpus

According to the theory turnika corpus, the growing point consists of two layers, namely:
1) layer edge; of cells that are actively dividing thus extending portion growing point called the
tunica
2) The inner layer; made up of cells that divide and differentiate into all directions, called the
corpus. The corpus is located inside the tunica.
3) the anatomical structure of the stem is sebgai following:
The epidermis
The cortex
Stele
In the anatomical structure of the stem is very different from the root, because a large part stems
not have endodermis.
3. Leaves
The leaves are the site of photosynthesis. Photosynthesis can take place because the
leaves have parenchymal tissue containing chloroplasts, chlorophyll, the epidermis, and the
carrier beam.
Complete the leaf has sections such as upih leaf or stem of the leaf, petiole and leaf blade.
However, many plants that do not have all the parts are complete. For example, spiny (Calotropis
gigantean) only had leaves, acacia (Acacia auriculiformis Acunn) leaves the form of the
widening of the stalk (filodia). Leaves are usually arranged by various network as follows
(Dellmann, H. Dieter and Esther M. Brown. 1989)
a. Leaf epidermis
Contained in the leaf epidermis upper surface and the underside of leaves, generally
consisting of a single layer of cells that undergo cell wall thickening of the chitin (cuticle) or
lignin. In the epidermis there is a gap that is flanked by two cell cover, this gap is called stomata.

There stomata on both surfaces of the leaf, but there also have it only on the lower leaf surface
(Rudyatmi, Ely. 2012)
b. Mesophyll
Mesophyll is a parenchyma tissue in the leaves. Mesophyll epidermis located between the
upper and lower epidermis. In most plants Dicotyledoneae, mesophyll differentiate into tissue
poles (palisade) and the spongy tissue (spongy). While on grasses and other Monocotyledoneae
undifferentiated mesophyll.
c. Network carrier
File carrier on the leaves forming a complex of buildings called bone leaves. Plants have
a maternal bone Dicotyledoneae leaves and branches that form the mesh, while the leaves of
plants Monocotyledoneae bones lined up parallel to the axis of leaves and connected by bundles
of small transporters (Campbell, N. A. 2004)
Bone leaves serves to transport water and nutrients from the soil and yield of
photosynthesis from the leaves to other parts of the body.
d. Network skretoris
In plants, there are certain specialized cells, such as lymph channels, crystal cells and
glands, which are generally found in the leaf mesophyll.
4. Flowers
Flowers are the reproductive organs of generative that appears only when the plant has
reached a certain age. A perfect flower structure composed ats bottom interest (reseptakel),
perianth (periantum) covering lids (calyx) and crown (corolla), and Sari thread.
Stamens Angiospermae wall consists of two layers: eksin (outer layer) and intin (inner
layer). Eksin composed of sporopolenin, while intin composed of cellulose. Further eksin
divided into two layers, namely seksin and neksin. Seksin is the layer that has the ornament,
while neksin not. Wall structure stamens, particularly the eksin, is one of the characters used in

identification. Eksin fine structure can be divided into three tire, namely: tektat, semitektat, and
intektat (Bloom and Fawcet.1994).
Description of the stamens and the spores were made in the form of description
sentences, ranging from the general to the specific nature, or of the most easily observed toward
properties that require detailed observation. Pollen and spores seen based on morphological
traits, which include Unit, Forms (polar and equatorial view), ukura, Apertura (type, number and
position), ornamentation. The properties mentioned above are the minimum necessary to
delineate, and which allows it to be observed using a light microscope.( Sugiharto,1989).