Histopathology techniques

2.1 Introduction
 Once tissues are removed from the body, they undergo a process of self-destruction
or autolysis.
 By light microscopy, autolysed tissue presents a 'washed-out' appearance with
swelling of cytoplasm.
 Bacterial decomposition can also produce changes in tissues that mimic those of
autolysis and are brought about by bacterial proliferation in the dead tissue.
2.1.1 Specimen collection and handling
 In histopathology laboratory, proper biopsy preparation and correct diagnosis
depend on proper tissue collection and handling.
 Proper tissue handling avoids the grave mistakes committed by incorrect labeling
and mixed up of the tissue biopsy and request form.
 Another problem is the tissue placed in narrow mouthed bottle that necessitates
breaking of the container in order to take out the tissue.
 Sometimes the tissue biopsy may be placed in inadequate amount of formalin.
 Laboratory personnel must have appropriate knowledge of proper tissue handling.
 Collection and…
 Upon reception of tissue specimens, the following points should be noted: -
1. Request form submitted should be matched with the tissue specimen.
2. Tissue and organ specimens should be submitted in a wide- mouthed
capacious container in order to avoid distortion.
3. The tissue or organ submitted should be immersed with adequate amount of
fixative.
4. Label the tissue or organ specimen properly.
5. Record all the relevant information on the laboratory record book.
2.1.2 Tissue marking substances
 Tissue marking substances are used to prevent lose of small tissues or cells during
tissue processing.
 Criteria for the selection of suitable tissue markers are:
1. The marking substance must be relatively insoluble in fixative, processing reagents and
embedding medium.
2. It must survive fixation and processing and not result in unacceptable contamination of the
reagents and other tissues processed simultaneously.
3. It must remain on the surface of the specimen and not penetrate tissue.
4. It should not react unfavorably with histological stains and must be clearly identifiable both
macroscopically and microscopically.
 Collection and …
 Tissue markers are applied to the surface of the specimen using disposable swabs
and allowed to dry.
 Some of the tissues marking substances are
 India ink
 Silver nitrate,
 Alcian blue
  Eosin
 India ink provides good black macroscopic and microscopic marking. It is resistant
to processing, but takes 15-30 minutes to dry, and may spread beyond the marked
area.
 Silver nitrate provides a brown-black mark resistant to processing.
 Collection and …
 Alcian blue, 1% aqueous solution, is a rapid and reliable stain for marking
resection margins of fixed breast and other biopsies.
 The specimen is dipped into the stain for a few seconds then blotted dry.
 Eosin, Erythrosin and Rose Bengal, 1-2% aqueous solutions, are used to stain
small translucent specimens.
Tissue marking
 Tissue marking facilitates identification and correct orientation of particular tissue
pieces or surfaces during embedding and subsequent microscopic examination.
 2.2 Fixation and Fixatives
2.2.1. Theoretical aspects of fixation
 Fixation is the process by which the constituents of the cells, and therefore of the
tissues, are fixed in a physical and partly also in a chemical state, so that they will
withstand subsequent treatment.
 In situation where, for various reasons, the primary fixation process may not be
optimal, the wet tissue may be subjected to post fixation or secondary fixation.
 Fixation facilitates coagulation of tissue proteins and their constituents and prevents
their loss or diffusion during tissue processing.
 The passage through hypertonic and hypotonic solutions during tissue processing
would otherwise disrupt the cell.
 Fixation is, therefore, the first step and the foundation in a sequence of events that
culminates in the final examination of a tissue section.
 Fixation and…
Purposes of fixation
 The purposes of fixation include: -
 Prevent autolysis and bacterial decomposition & putrefaction.

 Coagulate the tissue to prevent loss of easily diffusible substances.

 Fortify the tissue against the deleterious effect of the various stages in the

preparation of sections and tissue processing.

 To leave the tissue in a condition which facilitate differential staining with

dyes and other reagents.

 Fixation …
2.2.2 Fixatives
  Fixatives are substances that will preserve the shape, structure, relationship and
chemical constituents of tissue and cells after death.
 Fixatives may work by several means: formation of cross-linkages (e.g., aldehyde
such as glutaraldehyde or formalin);
 Protein denaturation by coagulation (e.g., acetone and methanol) or a combination
of the two.
 A large variety of fixatives are now available but no single substance or known
combination of substances has the ability to preserve and allow the demonstration
of every tissue component.
 It is for this reason that some fixatives have only special and limited applications.
 Fixation and .…
 The selection of an appropriate fixative is based on considerations such as the structure
and entities to be demonstrated and the effects of short-term and long-term storage.
Classification of fixatives
 Over the years, various classifications of fixatives have been proposed, with major
divisions according to function:
 Coagulant
 Non-coagulant

 Fixation and…
 According to their chemical nature in to three (3) general categories, which
include:
 Aldehydic, alcoholic, heavy metal fixatives.
Fixatives can be classified as: -
1) Aldehydes
 Formaldehyde, Glutaraldehyde, Acrolein, Formol- saline, Neutral-
buffered formalin
2) Oxidizing agents
 Osmium tetroxide, Potassium permanganate, Potassium dichromate
3) Protein denaturing agents (coagulant)
 Acetic acid, Alcoholic fixatives (Methyl and Ethyl alcohols), Picric
acid, Mercuric chloride
 Fixation and…
4) Other cross- linking agents: Carbodiimides.
5) Physical: Heat, microwaves.
 In order to obtain a fixative, which will comply this, it is necessary to mix together for
obtaining the combined effect of the different types fixatives.
 Based on this, fixatives that contain the individual substance are said to be simple
fixative,
e.g., formaldehyde, mercuric chloride, and acetic acid.
 Fixative resulting from the mixing of two or more of them is referred to as
compound fixative.
.
Characteristics of a good fixative.
A good fixative should: -
1. Kill the cell quickly with out shrinkage, swelling, /other distortion/
2. Penetrate the tissue and cells rapidly and evenly.
3. Render insoluble the substance of the cell and give good optical differentiation.
4. Inhibit bacterial decay and autolysis.
5. Strengthen (harden) the tissue and render it insensitive to subsequent treatment.
6. Allow tissue to be stored for longer period of time.
7. Permit the restoration of natural color for photography and mounting as museum
specimens.
8. Be simple to prepare and economical to use.
Factors involved in fixation:
1.Tempertature
2. Size of specimens and penetration of fixative
3. Changes in volume
4. pH and buffers
5. Osmolality
6. Concentration of fixatives and additives
7. Duration of fixation
 Fixation and…
2.2.4. Fixation of specific substance
1. Glycogen
 The retention of glycogen is thought to be the result of trapping in a matrix or mesh of
fixed protein, or due to its covalent binding to protein, which renders it insoluble in
water.
 The use of alcohols has been the main method of fixing glycogen in tissues.
 Earlier fixatives included ice-cold picro-alcohol-formalin or cold alcohol.
 Chemical assays on rat liver have shown 100% ethanol to be clearly superior for the
fixation of glycogen.
 Bouin's fixative is also a useful fixative for glycogen.
 Fixation and…
2. Lipids
 With standard methods of fixation, lipids are largely lost from tissues during
processing and only two reagents fix lipids in the true sense of rendering them
insoluble.
  These are osmium tetroxide and chromic acid, both of which alter the chemical reactivity
of the lipid considerably.
 Various additives have been mixed with glutaraldehyde in order to demonstrate lipids in
electron microscopy.
3. Proteins
 The fixation of tissue proteins by aldehydes is largely through production of cross-
linkages between various reactive groups in proteins.
 Fixation and…
4 Mucosubstances
 The loss of mucosubstances from tissue during fixation is well recognized and many
fixatives have been suggested to prevent.
 Formalin has always been an essential component of whatever fixative used to ensure the
preservation of proteoglycans.
 An appreciable proportion of tissue hetero- and proteoglycans remains soluble unless
subject to further precipitation in 70-80% ethanol (for 3-6 days) before clearing and
embedding in paraffin.
 The most successful method for preserving all types of mucin is freeze drying followed
by hot formaldehyde vapor with the all necessary safety precautions.
 Fixation and …
5. Nucleic acids and nucleoproteins
 The nucleic acids exist in many different states of polymerization and any method of
fixation induces changes in their physical state.
 Formalin is not a particularly good fixative for nucleic acids and nucleoproteins as it
blocks a large number of reactive groups reducing their subsequent staining by both basic
and acid dyes.
 This can be improved by adding mercury or chromium salts.
 Precipitant fixatives like alcohol, acetic acid, and Carnoy's fluid are preferable
 these agents precipitate nuclear proteins and at the same time progressively break
the bonds between nucleic acids and proteins, thereby increasing the number of acid
groups available for staining.
6 Enzymes
 Enzyme activity is best demonstrated histochemically in fresh frozen sections.
 The most common methods of preserving enzymes for paraffin embedding are fixation
in alcohol or acetone, usually at 4°C.
 The most significant problem in enzyme histochemistry is false localization due to
diffusion of the enzyme.
 Formalin-sucrose-ammonia and 1-4% glutaraldehyde have been used for cholinesterase.
 The fixation of acid phosphatase can be achieved with formaldehyde, and formalin
containing 0.1% chloral hydrate will preserve ß-glucuronidase

2.3 Decalcification
 Some tissues contain calcium deposits which are extremely firm and which will not
section properly with paraffin embedding owing to the difference in densities between
calcium and parffin.
  Bone specimens are the most likely type here, but other tissues may contain calcified
areas as well.
 A variety of agents or techniques have been used to decalcify tissue and none of them
work perfectly.
 Mineral acids, organic acids, ethylenediamine tetra acetic acid (EDTA), and electrolysis
have all been used.
 Strong mineral acids such as nitric and hydrochloric acids are used with dense cortical
bone because they will remove large quantities of calcium at a rapid rate.
 strong acids also damage cellular morphology, so are not recommended for delicate
tissues such as bone marrow.
 Organic acids such as acetic and formic acid are better suited to bone marrow, since they
are not as harsh.
 However, they act more slowly on dense cortical bone.
 Formic acid in a 10% concentration is the best all-around decalcifier.
 EDTA can remove calcium and is not harsh (it is not an acid) but it penetrates tissue
poorly and works slowly and is expensive in large amounts.
 Electrolysis has been tried in experimental situations where calcium had to be removed
with the least tissue damage.
 It is slow and not suited for routine daily use.
Techniques of decalcification
 The following technique is employed in decalcification: -
1. Selection of tissues
2. Fixation
3. Decalcification
4. Neutralization of acids
5. Thorough washing
 Decalci…
Selection of tissue
 Bone: 4-5mm slice of bone with a saw
 Calcified tissue: thin slices with a knife
Fixation
 General rules of fixation can also apply here.
Decalcification
 Decalcification can be done by:
1. Simple solutions usually acid reagents
2. Ion exchange resin
3. Chelating agents
4. Electrophoresis
 Decalci…
 Criteria of a good decalcifying agent are:
 Complete removal of calcium
  Absence of damage to tissue cell or fibers
 Non-impairment of subsequent staining techniques
 Reasonable speed of decalcification
 Neutralization of acids
 Treatment with alkali
 Washing
 To remove acid or alkalis wash with alcohol for 3-4 hours
 Decalci…
Decalcifying fluids
Solution A
8% Hydrochloric acid (HCl
1. Stock solution (98%)HCl -------------------------- 80 ml
2. Distilled water----------------------------------------- 920 ml
Solution B
8% Formic acid
1. Stock solution (40%) Formic acid----------------- 80 ml
2. Distilled water------------------------------------------ 920 ml
HCl / Formic acid working solution
 Equal parts of Solution A and Solution B are mixed.
 Decal..
Solution C
5-10% Nitric acid
1. Stock solution Nitric acid ---------------------------------------- 5 -10 ml
2. Distilled water ------------------------------------------------------ 100 ml
Procedure
1. Specimen should be decalcified in a solution 20 times their volume

2. Change to fresh solution each day until decalcification is complete

3. Wash specimen

 Agonal changes and artifacts

 As most tissues are removed surgically, the following agonal changes may occur.
 Organ or tissue become anoxic when tissue placed on fixatives;
 Anoxic changes are noticed;
 Enzymes such as those concerned with oxidative phosphorylation are lost
within 10 minutes;
 There will be tissue variation;
 Some degree of autolysis will also occur.
 Agonal changes…
Artifacts caused by fixatives include:
 Both large and small molecules diffuse from the tissue in to fixation solution;
 Chemical changes caused by fixation may give false histochemical reactions in tissue;
 While removing excess mercuric chloride after fixation, substances such as histidine and
tyrosine, may also be removed;
 Using substances specific fixatives results loss of many other cellular substances.
 Formaldehyde at pH 7.0 causes the loss of about 60% of catecholamines from chromaffin
granules.
 Agonal changes…
 Formalin is an inadequate fixative for the proteoglycans of many pituitary glands and
many neuropeptides such as luteinizing hormone-releasing hormones are ethanol soluble
and may be lost during dehydration.
 Enzymes may be released during fixation and ions may be lost from tissues.
 Prolonged fixation in formaldehyde results in the loss of water-soluble materials
particularly when fixation exceeds 6 hours.
 Proteins are degraded by osmium tetroxide.

Review questions
1. What are fixation and fixatives?
2. Explain the characteristics of a good fixative.
3. Discuss factors affecting quality of fixation.
4. Discuss about agonal changes and artifacts.

2.4 Tissue processing

 Tissue processing refers to any treatment of tissue necessary to impregnate them with a
solid medium to facilitate the production of sections for microscopy.
 There are two types of tissue processing methods: conventional and frozen.
 Conventional method of tissue processing is preferred than frozen method of producing
tissue sections because of the following advantages.
 Helps to section large number of tissues routinely;
 Suitable for impregnating tissues with a solid medium easy to store;
 Further section can be readily produced at a later date;
 Requires paraffin wax, which is the most suitable for routine microtomy.
 Tissue pro…
Completion of fixation before processing
 Before processing tissues, it is important to verify that fixation is complete.
 Completion of fixation can be achieved by the use of:
O
 Heat (if a rapid fixation is important); Buffered formaline raised to 60 C
(effective in fixing of 5 mm slice of most tissues within one hour);
 Dehydrating alcohols (for small fragments of tissue);
 Microwave irradiation of biopsy specimen in normal saline.
 Tissue pro…
Special treatment of tissues after fixation
  Following the use of some fixative solutions, tissues may need treatment before
processing.
 The following reagents are especially employed:
Potassium dichromate containing fixatives
 thorough washing in running tap water to remove traces of dichromate;
Picric acid containing fixatives
 tissue should not be in contact with water or aqueous solution prior to dehydration; some
of the protein-picrate complexes are slightly water-soluble.
 For precise studies, artifacts may occur unless tissues are transferred directly to 80 %
alcohol.
 Ø
 Tissue pro…
 Carnoy’s fluid
 Fixed tissue should be transferred to 95% or absolute alcohol, being already partially
dehydrated due to the alcoholic content of the fixatives
Principles of tissue processing
 The aim of tissue processing is to embed the tissue in a solid medium firm enough to
support the tissue and give it sufficient rigidity to enable thin sections to be cut.
 When the tissue is received it is usually partly or completely fixed in a suitable fixative,
nearly always-aqueous fixatives.
 Before the tissue can be embedded in paraffin wax, the tissue must be subjected to:
 completion of fixation;
 dehydration;
 clearing;
 impregnation;
 Once the tissue has been fixed, it must be processed into a form in which it can be
made into thin microscopic sections.
 Tissues embedded in paraffin, which is similar in density to tissue, can be sectioned
at anywhere from 3 to 10μ routinely.
 Tissue pro…
 Wet fixed tissues (in aqueous solutions) cannot be directly infiltrated with paraffin.
 First, the water from the tissues must be removed by dehydration.
 This is usually done with a series of alcohol; such as 70% to 95% to 100%.
 Acetone is a very fast dehydrant, but it causes a fire hazard.
 So it is safe only for small, hand-processed sets of tissues. Dioxane can be used
without clearing, but has toxic fumes.
 Tissue pro…
 The next step is called "clearing" and consists of removal of the dehydrants with a
substance that will be miscible with the embedding medium (paraffin).
 The commonest clearing agent is xylene.
 Toluene works well, and is more tolerant of small amounts of water left in the
tissues, but is three times more expensive than xylene.
 Chloroform was used before, but it is a health hazard, and has slow action
 Although they represent less of a health hazard, they are less forgiving with poorly
fixed, dehydrated, or sectioned tissues.
  Tissue pro…
 Finally, the tissue is infiltrated with the embedding agent, usually with molten
paraffin wax.
 Paraffin can be purchased that differ in melting point, for various hardnesses,
depending upon the way the histotechnologist likes them and upon the climate
(warm versus cold).
 A product called paraplast contains added plasticizers that make the paraffin blocks
easier for some technicians to cut.
 Tissue pro…
Dehydration
 Dehydration is the first step in tissue processing and is done by alcohol of various
types to remove fixative and water from tissue and replace them with dehydrating
fluids.
 Dehydration is achieved by immersing tissues in increasing strength of alcohol
(usually starting with 70% alcohol).
 The time of dehydration depends on the type of tissue to be processed.
 Generally, dehydration is done sequentially; first in 70% alcohol for three changes
then 95% alcohol for three changes and then absolute alcohol for three changes.
 Tissue pro…
Dehydration is effected as follows:
 Dilution dehydration, the most commonly used method.
 Specimens are transferred through increasing concentrations of hydrophilic or
water miscible fluids.
 Chemical dehydration, where the dehydrant, acidified dimethoxypropane or
diethoxypropane, is hydrolysed by free water present in tissues to form acetone and
methanol 43-50 % in an endothermic reaction.
 The dehydrant concentration at which processing is initiated depends largely upon
the fixative employed.
 Following fixation in anhydrous fixatives such as Carnoy's fluid, for example
dehydration is initiated in 100% ethanol.
 Tissue pro…
 To minimize tissue distortion from diffusion currents, delicate specimens are
dehydrated in a graded series from water through 10%, 20%, 50%, 95%, and 100%
ethanol.
 Dehydration is necessary in all infiltration methods, except where tissues are simply
externally supported by an aqueous embedding medium.
 Choice of a dehydrant is determined by
 The nature of the task
 The embedding medium
 Processing method
 Economic factors.
 Dehydrants differ in their capacity to cause tissue shrinkage
 Tissue pro…
 Duration of dehydration should be kept to the minimum consistent with the tissues
being processed.
  Tissue blocks one mm thick should receive up to 30 minutes in each alcohol, block
five mm thick require up to 90 minutes or longer in each change.
 Tissues may be held and stored indefinitely in 70% ethanol without harm.
Dehydrating fluids
 Water is present in tissues in free and bound (molecular) forms.
 Tissues are processed to the embedding medium by removing some or all of the
free water from the tissues.
 Tissue pro…
 During this procedure various cellular components are dissolved by dehydrating
fluids.
For example,
 certain lipids are extracted by anhydrous alcohols, and water-soluble proteins are
dissolved in the lower aqueous alcohols.
 The commonly used dehydrating fluids include:
Ethanol
 Ethanol is a clear, colorless, flammable liquid. It is probably the most commonly
used dehydrant in histology.
 It is supplied as 99.85% ethanol and as special Methylated Spirits (99.85% ethanol
denatured with 2% methanol)..
 Tissue pro…
 Processing times in absolute ethanol should be minimal.
 Progressive removal of bound water from carbohydrates and proteins during
prolonged immersion in absolute ethanol causes tissues to harden excessively and
become brittle.
 Ethanol is hydrophilic, miscible with water and many organic solvents and ensures
total dehydration.
 Alcohols are generally used in increasing order of strength.
Example, 1st; 70% ethanol;
2nd; 95% ethanol;
3rd; 100% ethanol.
 This sequence allows all the aqueous tissue fluids to be removed.
 Tissue pro…
Methanol
 Methanol is a clear, colorless, flammable fluid, highly toxic, miscible with water,
ethanol and most organic solvents.
 It is a poor lipid solvent, and will not dissolve nitrocellulose unless mixed with
acetone.
Isopropyl alcohol
 Isopropyl alcohol is miscible with water, ethanol and most organic solvent.
 Isopropanol shrinks and hardens tissues less than ethanol and is used to dehydrate
hard, dense tissues, which can remain in the solvent for extended periods without
harm.
 Tissue pro…
  To minimize shrinkage, fixed tissues are transferred via 60 % -70 % isopropanol or
ethanol to absolute isopropanol.

Acetone
 Acetone is a colorless flammable liquid with sharp characteristic kenotic odor, low
toxicity and is freely miscible with water and organic solvents.
 It is a fast, effective dehydrant though it may cause tissue shrinkage; it may also act
as a coagulant secondary fixative.
 Acetone is the best dehydrant for processing fatty specimens.
 Tissues are dehydrated through four changes of acetone, the last of which should
always be fresh.
 Tissue pro…
Additives to dehydrating agents
Phenol
 Phenol can act as a softening agent for hard tissues such as tendon, nail, dense
fibrous tissue and keratin masses.
Glycerol/Alcohol mixture
 Act as a softening agent
Anhydrous copper sulfate
 Layer 1-2 cm of anhydrous copper sulfate in the final dehydrating bath and cover
with a filter paper.
 If there is any water, the anhydrous copper sulfate will turn blue,
 Tissue pro…
Clearing
 Clearing is the transition step between dehydration and infiltration with the
embeddingmedium.
 A solvent miscible with both the dehydrant and the embedding medium is used to
facilitate the transition between dehydration and infiltration steps.
 Shrinkage occurs when tissues are transferred from the dehydrant to the transition
solvent, and from transition solvent to wax.
 In the final stage shrinkage may result from the extraction of fat by the transition
solvent.
 Tissue pro..
 The term clearing indicates the fact that because some solvents have high refractive
indices.
 This property is used to ascertain the endpoint and duration of the clearing step.
 Prolonged exposure to most clearing agents causes the tissue to become brittle and
difficult for sectioning.
 Viscosity influences the speed of penetration of clearing agent.
 Transition solvents extract certain tissue substances such as lipids, but otherwise do
not alter tissue reactivity nor behave as secondary fixatives during processing.
 Tissue pro…
Criteria for choosing clearing agent
  The following issues should be considered when choosing a clearing agent:
 Type of tissues to be processed, and type of processing to be undertaken;
 Processor system to be used;
 Intended processing conditions such as temperature, vacuum and pressure;
 Safety factors;
 Cost and conveniences.
 Tissue pro..
 The following are routinely used clearing agents.
Xylene
 Xylene is generally used for routine paraffin embedding because of its
compatibility with many types and size of tissue specimen.
 It’s suitable for schedules of less than 24 hours and used when the block is less than
5mm in thickness and immersion must not be prolonged.
 Xylene two changes (20minutes to 1hour) will be employed.
Toluene
 Toluene has similar properties to xylene.
 It produces less damage with prolonged Immersion.
 Tissue pro..
Chloroform
 Chloroform is an expensive, heavy, highly volatile, slowly penetrating transition
solvent.
 It causes less brittleness than xylene and is often used on dense tissues such as
uterus and muscle that can be cleared overnight without undue hardening.
 Since chloroform attacks some plastics and sealants its use may be restricted in
certain closed system processors.
 Tissue pro..
Esters
 These are colorless flammable solvents miscible with most organic solvents and
with paraffin wax.
 n -Butyl acetate is used as a xylene substitute and nitrocellulose solvent.
 Amyl acetate, Methyl benzoate & methyl salicylate are chiefly used as
nitrocellulose solvents in double embedding techniques.
 They have low toxicity, but their strong penetrating odors necessitate good
laboratory ventilation.
 Tissue pro..
 They are ideal for manual processing as tissues may be left in them for extended
periods without affecting the tissue or organ.
 These esters are difficult to eliminate from paraffin wax and should be extracted
from tissues with one or two brief changes of toluene or similar solvent.
 Methyl salicylate clears tissues from 96% ethanol, hardens less and has a more
pleasant odor than methyl benzoate.
 It causes minimal tissue shrinkage and hardening and tissues can remain in it
indefinitely without harm but expensive transition solvents.
 Tissue pro..
Chlorinated hydrocarbons
 Chlorinated hydrocarbons are colorless solvents with sweet odors and are miscible
with most organic solvents and with paraffin wax.
 Members of this group clear more slowly but harden far less than xylene.
 Although non-flammable, solvents in this group decompose in the presence of heat
to form phosgene and hydrochloric acid.
 Tissue pro..
 They are all narcotic and toxic to varying degrees.
 Chlorinated hydrocarbons are ozone depleting chemicals, and from January 1996,
1, 1, 1- trichloro ethane and carbon tetrachloride are banned from use .
Citrus fruit oils
 Citrus fruit oils are extracted from orange and lemon.
 On sections prior to mounting the use of these clearing agents leaches out dyes.
 Tissue pro..
Impregnation
 Impregnation is the saturation of tissue cavities and cells by a supporting substance,
which is generally, the medium in which they are finally embedded.
 Tissues are infiltrated by immersion in a substance such as a wax, which is fluid
when hot and solid when cold.
 Alternatively, tissues can be infiltrated with a solution of a substance dissolved in a
solvent, for example nitrocellulose in alcohol-ether,
 which solidifies on evaporation of the solvent to provide a firm mass suitable for
sectioning.
 Tissue pro..
O
 Impregnation with paraffin wax takes place in an oven heated to 54-60 C.
 The temperature is depends on the melting point of the wax.
 The paraffin wax should be free from dust and other foreign matter and all wax
should be filtered.
 Tissue pro…
Procedure
1. The tissue is transferred from the clearing agents to molten paraffin wax.
0
2. The wax is kept in approximately 56 C The volume of wax should be 25-30 times the
volume of the tissue.
3. It should be changed 2-3 times by lifting the tissue from one pot to the next with warmed
forceps.
Vacuum impregnation
 Vacuum impregnation is the impregnation of tissues by a molten medium under
reduced pressure.
 Tissue pro…
 The procedure helps to:
 Assist complete and rapid impregnation of tissues with wax;
  Reduce the time tissues are subjected to high temperatures thus minimizing heat
induced tissue hardening;
 Facilitate complete removal of transition solvents;
 Prolong the life of wax by reducing solvent contamination
 Tissue proc…
 Vacuum infiltration requires a vacuum infiltrator or embedding oven, consisting of
wax baths, fluid trap and vacuum gauge.
 Modern tissue processors are equipped to deliver vacuum, or vacuum and pressure,
to all or some reagent stations during the processing cycle.
 The aim of vacuum impregnation is to:
 Avoid air bubbles in the tissue as may occur in porous tissue such as lung;
 Remove clearing agents more rapidly by increasing its vaporization.
 Tissue pro…
 Ideally, an impregnating and embedding medium should be: -
 Soluble in processing fluids;
 Suitable for sectioning and ribboning;
 Molten between 30°C and 60°C;
 Translucent or transparent and colorless;
 Stable;
 Homogeneous;
 Capable of flattening after ribboning;
 Non-toxic;
 Odorless;
 Easy to handle;
 Inexpensive
 Tissue pro…
Paraffin wax
 Paraffin wax is the commonly used embedding media due to the following features.
 Coast effective (cheap);
 Easy to handle;
 Enables section production with few difficulty;
 Has wide range of melting points.
Properties of paraffin wax
 Paraffin wax is a mixture of hydrocarbon produced during the refining of coal and
mineral oil;
0
 Its melting point ranges between 40 and 70 C; High Melting Point harder than low
Melting point paraffin waxes;

 Tissue pro…
 To promote good ribbons of sections, wax of suitable hardness at room temp should
be chosen;
 Heating to a very high temperature alters the properties of the way by changing the
crystalline structure.
Modified paraffin waxes
 The properties of paraffin wax are improved for histological purposes by the
inclusion of substances added alone or in combination to the wax which will:
 Improve ribboning: prolong heating of paraffin wax at high temperatures or
use micro-crystalline wax;
 Increase hardness: add stearic acid;
 Tissue pro…
 Improve adhesion between specimen and wax (alter crystalline morphology): add
0.5% ceresin, 0.1-5% beeswax, rubber, asphalt, bayberry wax, or phenanthrene.
Microcrystalline wax
 Obtained from petroleum distillation;
 Has a much finer crystalline structure than paraffin wax with a melting point
between 62-88 0C ;
 Adding up to 5% to paraffin wax produces a hard medium.
 Tissue processing…
Paraffin wax additives
 Paraffin wax additives are substances like bee’s wax, ceresin rubber, dental wax
and diethylene glycol distearate, which are used alone or in combination.
 Such additives increase the hardness of paraffin wax thus enabling thin section to
be cut.
 Tissue pro…
 "Floaters" are small pieces of tissue that appear on a slide that do not belong there;
they have floated in during processing.
 Floaters may arise from sloppy procedure on the cutting bench dirty towels,
instruments, or gloves can have tissue that is carried over to the next case.
 It’s essential that you do only one specimen at a time & clean thoroughly before
opening the container of the next case.
 If re-usable cassettes are employed, you must be aware that tissue may potentially
be carried over and appear as "floaters" even several days later, when the cassette is
re-used.
 Tissue proce…
 The problem arises when, during embedding, not all the tissue is removed from the
cassette.
 In the cleaning process, not all of the wax is removed.
 The next person using the cassette does not pay attention to the fact that there is
tissue already in the cassette and puts his specimen in it.
 The floater that appears on the slide will look well preserved--it should, because it
was processed to paraffin.
 Tissue pro..
Factors influencing the rate of processing
Agitation
  Fluid interchange between processing reagents and tissues is promoted by exposure
of the maximum tissue surface area to reagents.
 Tissues should be loosely packed, suspended and agitated within the medium to
facilitate the exchange of dilute reagent from the tissues with the more concentrated
reagent replacing it.
 In automatic tissue processors, continual rotary or vertical motion of tissue
containers, or tidal action and flow of processing fluids ensures adequate fluid
exchange.
 Ideally, tissue cassettes should be placed in processors so that the cassette
perforations are perpendicular to the fluid flow.
 Tissue proc…
 For efficient and effective processing there should be a specimen volume to
processing fluid volume ratio of at least 1:50.
 Efficient agitation may reduce the overall processing time by up to 30%.
Heat
 Heat increases the kinetic energy of molecules and rate of diffusion, with a
corresponding decrease in solution viscosity.
 Mild heat within the range 37°C to 45°C, during the dehydration and clearing steps
considerably reduces processing times, but may increase shrinkage.
 Tissue pro…
 Tissue shrinkage during infiltration in paraffin wax results mainly from the effect of
heat on collagen.
 Heating increases the rate of penetration but higher temperature adversely affects
staining and immuno - cytochemistry.
 Heat is normally used only when urgent reports are required and temperature
0
limited to 45 C can be used effectively.
Viscosity
 Viscosity is the internal friction of a particular substance, which affects rate of flow
through tissues and is inversely proportional to temperature.
 Tissue pro…
 It is particularly important in the clearing and infiltration stages of processing.
 Substances with high molecular weight, such as some transition solvents and
waxes, have high viscosities and diffuse through tissues more slowly.
 If the viscosity difference between fluid inside and outside the tissue is too great,
shrinkage will result.
 Hence slow and gradual processing of tissues is necessary when viscous reagents
are used (for example in nitrocellulose embedding methods).
 Tissue pro…
Vacuum
 Vacuum impregnation is very important for tissues of lung, muscle, spleen,
decalcified bone, skin and tissue from CNS.
 It speeds up impregnation (reduce impregnation time by up to one half) by
removing any residual air bubble that indicates inadequate dehydration or clearing.
Processing methods and routine schedules
  Tissues are most conveniently processed through dehydration, clearing and
impregnation stages automatically by machine.
 There are two types of automatic tissue processors;
 tissue-transfer processors
 fluid-transfer processors.
 Tissue pro…
Tissue-transfer processors
 Characterized by the transfer of tissues, contained within a basket, through a series
of stationary reagents arranged in-line or in a circular carousel plane.
 The rotary or carousel is the most common model of automatic tissue processor.
 It is provided with 9-10 reagent and 2-3 wax positions, with a capacity of 30-110
cassettes depending upon the model.
 Fluid agitation is achieved by vertical oscillation or rotary motion of the tissue
basket.
 Processing schedules are cardnotched, pin or touch pad programmed.
 Tissue pro…
 allow maximum flexibility in the choice of reagents and schedule that can be run on
them, in particular, metal-corrosive fixatives, a wide range of solvents.
 These machines have a rapid turn-around time for day/night processing.
 In more recent models the tissue basket is enclosed within an integrated fume hood
during agitation and transfer cycles thus overcoming the disadvantages of earlier
styles.
 Tissue proce…
Fluid-transfer processors
 Processing fluids are pumped to and from a retort in which the tissues remain
stationary.
 There are 10-12 reagent stations with temperatures adjustable between 30- 45°C, 3-
4 paraffin wax stations with variable temperature settings between 48-68°C, and
vacuum-pressure options for each station.
 Depending upon the model, these machines can process 100-300 cassettes at any
one time.
 Agitation is achieved by tidal action.
 Schedules are microprocessor programmed and controlled.
 Tissue pro…
 Vacuum-pressure cycles coupled with heated reagents allow effective reductions in
processing times and improved infiltration of dense tissues.
 Fluid-transfer processors overcome the main drawbacks of the tissue-transfer
machines.
 Tissues are unable to dry out within the sealed retort and reagent vapors are vented
through filters or retained in a closed-loop system.
 Processors are provided with alert systems and diagnostic programs for
troubleshooting and maintenance.
 Representative schedules for rapid and overnight processing are provided in.
General consideration in automated tissue processing
 Baskets and metal cassettes should be clean and wax-free;
  Tissues should not be packed too tightly in baskets so as to impede fluid exchange;
 Processors must be free of split fluids and wax accumulation to reduce hazards and
to ensure mechanical reliability;
 Fluid level must be higher than the specimen containers;
 Timing and delay mechanism must be correctly set and checked against the
appropriate processing schedule;
 Tissue pro…
 A processor log should be kept in which:
 the number of speciemen processed
 processing reagent changes
 temperature checks on the wax baths
 the completion of the routine maintenance schedule is recorded as an integral part
of the laboratory quality assurance program.
Microwave-stimulated processing
 Rapid manual microwave-stimulated paraffin wax processing of small batches of
tissues gives excellent results.
 Processing is undertaken in a microwave oven, which is fitted with precise
temperature control and timer, and an interlocked fume extraction system to
preclude accidental solvent vapor ignition.
 Domestic microwave ovens with a temperature probe and timer accurate to seconds
are suitable for tissue processing.
 Toxic and flammable solvent vapors generated during processing cannot always be
adequately vented from these ovens and present an ignition hazard if the electrical
system is unprotected.
 Ovens should therefore be used within a fume cupboard to minimize this problem.
 Tissues are processed in conventional plastic cassettes.
 Picric acid fixed tissues should not be microwave processed, as there is an
explosion risk even in well-washed tissues.
 Ultrasound-stimulated processing
 Ultrasound-stimulated processing is used in histopathology to accelerate :
 Fixation
 tissue processing for electron microscopy
 the decalcification of bone
 improving the sensitivity of immunohistochemical reactions
 for conventional staining
 for accelerated tissue processing.
 Unfixed tissue blocks 1-2 mm thick can be fixed and processed to paraffin wax
using ultrasonic-stimulation in one hour and 45 minutes.
 Tissue pro…
 The most important effect of ultrasound at frequencies of 100 kHz-1 MHz is
agitation.
 Processing is performed in reagent containers suspended in a detergent solution
within the transducer tank of an ultrasonic cleaner operated at 50 watts.
 Tissues are placed in metal or plastic cassettes for processing.
 Coagulant fixatives provide optimal stabilization for ultrasonic-stimulated
processing.
  Tissues are dehydrated in ethanol and cleared in toluene, or preferably methyl
benzoate or methyl salicylate.
 2.5 Embedding
Embedding tissues in paraffin wax
 Embedding is the process by which tissues are surrounded by a medium such as
agar, gelatin, or wax which when solidified will provide sufficient external support
during sectioning.
 Tissues are embedded by placing them in a mould filled with molten embedding
medium, which is then allowed to solidify.
 Embedding requirements and procedures are essentially the same for all waxes.
 At the completion of processing, tissues are held in clean paraffin wax, which is
free of solvent and particulate matter.
 Embed…
 Requirements for embedding are as follows:
0
 A supply of clean filtered paraffin wax held at 2-4 C above its melting point;
 A cold plate to rapidly cool the wax;
 A supply of moulds in which to embed the tissues.
 Embed…
General Embedding Procedure
 The following general embedding procedure is employed: -
1. Open the tissue cassette; check against worksheet entry to ensure the correct number of
tissue pieces are present.
2. Select the mould, there should be sufficient room for the tissue with allowance for at least
a two mm surrounding margin of wax.
3. Fill the mould with paraffin wax.
4. Using warm forceps select the tissue, taking care that it does not cool in the air; at the
same time or pour some amount of paraffin wax to cover the base, place and orient the
tissue and then fill the mould with wax.
5. Chill the mould on the cold plate.
6. Insert the identifying label or place the labeled embedding ring or cassette base onto the
mould.
7. Cool the block on the cold plate, or carefully submerge it under water when a thin skin
has formed over the wax surface.
8. Remove the block from the mould.
9. Crosscheck label on block, and worksheet.
 Embed…
 During cooling, paraffin wax shrinks up to 15%, causing compression in tissues.
 This compression is almost fully recovered when sections are floated on a warm
waterbath.
 Embed…
Other embedding media and processing methods
 Alternative embedding media may provide optimum support for tissues in
applications for which paraffin waxes are unsuited, for example, when:
 Tissue components are heat or reagent labile;
  Hard or dense tissues are inadequately supported;
 Adhesion between specimen and wax is poor;
 Very thick or very thin sections are required;
 Sectioning whole organs such as lung or brain.
 Resin embedding methods are now used for many of these applications.
 Embed…
 Non-resinous embedding media include those listed below.
Aqueous media
 Agar has a high melting point and low gelling temperature.

 This property of agar makes it ideal for double embedding of multiple small tissue
fragments.
 Agar is generally unstained by overnight stains, but will stain with alcian blue.
 Embed…
Gelatine
 Gelatine is used for simple embedding in a similar manner to agar.
 However the low melting point of gelatine (35-40°C) makes it unsuitable for
double embedding.
 In phospholipid and enzyme studies tissues may be infiltrated and embedded in
gelatine and the resulting blocks sectioned on a freezing microtome.
Polyvinyl alcohol (PVA)
 Polyvinyl alcohol is a water-soluble medium suited to a variety of applications, in
particular Histochemical studies of lipids and enzymes.
 Embed…
 Tissues are infiltrated at elevated or room temperatures through an ascending series
of aqueous PVA-glycerol solutions and embedded in 15% aqueous PVA which is
slowly dried to produce a firm block.
 Sections are cut at 1-100 μm in the normal manner.
Water-miscible media
 Polyethylene glycols (PEG) are water-soluble media used for investigation of heat
and solvent-labile lipids and proteins.
 The polyethylene glycols are polymers of varying length.
 In general they are less elastic, denser and somewhat harder than paraffin wax.
 Embed…
Water - tolerant media
 Diethylene glycol distearate is a hard, brittle, water tolerant ester (melting point 47-
52°C).
 It has certain deficiencies when used for routine embedding, unless combined with
other substances as in ester waxes.
 However, it may be used unmodified for thin sectioning (0.5-2 μm) of freeze-dried
and osmium tetroxide fixed tissues for high-resolution light microscopy.
 Tissues are dehydrated and cleared as in the paraffin wax method.
 Embed…
Double embedding and double infiltration methods
 Tissues are first embedded or fully infiltrated with a supporting medium such as
agar or nitrocellulose then infiltrated a second time with wax in which they are also
embedded.
 Double embedding methods such as agar-paraffin embedding are used when tissues
require external support or particular pre-embedment orientation.
 Paraffin wax double infiltration methods provide hard tissues with additional
support provided by substances such as agar or nitrocellulose, with the convenience
and ease of wax microtomy.
 Embed…
Agar-paraffin wax double embedding
 is a reliable and convenient method of handling minute and friable tissue fragments
such as endoscopic biopsies, which can be lost during tissue processing.
 It also overcomes the difficulty of manipulating small tissue fragments during
embedding.
 Facilitates correct orientation and identification of tissues for histochemical and
immunohistochemical control tissues.
 Embed…
Nitrocellulose - paraffin wax double infiltration
 is mainly used for brain, friable tissues and decalcified bone .
 It combines the plasticity and support provided by nitrocellulose with convenient
handling and microtomy of the paraffin technique.
 Tissues may be :
(a) infiltrated with thick nitrocellulose solutions and the resulting block trimmed,
hardened in chloroform and infiltrated in paraffin wax,
(b) infiltrated with thin low viscosity nitrocellulose (LVN) solutions, which are
integrated into a normal processing schedule.
 Proprietary celloidin-ethanol-ether solutions provide a simple and convenient double-
embedding method.
 The principle drawbacks of this technique are prolonged exposure of tissues to absolute
ethanol and the high flammability and volatility of diethyl ether precluding machine
processing.
 Use of methyl benzoate or methyl salicylate as LVN solvents overcome these
deficiencies.
2.6. Microtomy(sectioning)
 Once the tissues have been embedded, they must be cut into sections that can be placed
on a slide.
 This is done with a microtome.
 The microtome is nothing more than a knife with a mechanism for advancing paraffin
block at regulated distances across it.
 Knives are either of the standard thick metal variety or thin disposable variety (like a
disposable razor blade).
 The former type allows custom sharpening to one's own satisfaction, but is expensive.
 Plastic blocks are sectioned with glass or diamond knives.
 A glass knife can section down to about one μm.
  Thin sections for electron microscopy (1/4 μm) are best done with a diamond knife,
which is very expensive.
 Microtomes have a mechanism for advancing the block across the knife.
 Usually this distance can be set, for most paraffin embedded tissues at 6 to 8 μm.
 It is important to have a properly fixed and embedded block or much artifact can be
introduced in the sectioning.
 Once sections are cut, they are floated on a warm water bath that helps remove wrinkles.
 Then they are picked up on a glass microscopic slide.
 The glass slides are then placed in a warm oven for about 15 minutes to help the section
adhere to the slide.
 If this heat might harm such things as antigens for immunostaining, then this step can be
bypassed and glue-coated slides used instead to pick up the sections.
Frozen sections
 Frozen sections are methods that produce sections without the use of
dehydrating, clearing and embedding media.
 Frozen sections used to demonstrate soluble substance and in the diagnosis of
urgent biopsy specimen.
Principle of frozen sectioning
 When the tissue is frozen, the water with in the tissue is changed to ice, in this state the
tissue is firm and the ice acts as the embedding medium.
 The frozen sections have many important applications in histopathology laboratory, such
as for:
 The rapid production of section for urgent diagnosis;
 Diagnostic and research enzyme histochemistry, when enzyme are liable;
 Diagnostic and research in non-enzyme histochemistry;
 Immunoflourescent methods;
 Immunocytochemical methods;
 Some silver method, e.g., in neuropathology study.
 Frozen sections are performed with an instrument called a cryostat.
 The cryostat is just a refrigerated box containing a microtome.
0
 The temperature inside the cryostat is about -20 to -30 C.
 The tissue sections are cut and picked up on a glass slide.
Freezing techniques
 Freezing techniques used to freeze tissue and organ employs the following techniques:
0
 Liquid nitrogen (-190 C);
0
 Isopentane cooled by liquid nitrogen (-150 C);
0
 Carbon dioxide gas (-70 C );
0
 Aerosol sprays (-50 C ).
 If sections were adequately dried the problem of sections floating off during staining
would not occur.
Occasions that make sections to float( Detach )
 Sections submitted to strong alkali solution during staining;
  Cryostat sections for immuno-fluorescence, immuno cytochemistry and urgent
diagnosis;
 Tissue from CNS;
 Section submitted to high temperature;
 Tissue containing blood clot;
 Tissue that have been decalcified.
…
 Equipment required for tissue sectioning
 Microtome and knife;
 Water bath;
 Drying oven or hot plate;
 Fine pointed and blunt forceps;
 Small squirrel hair brush;
 Clean slides;
 Slide rack;
 Scalpels;
 Ice tray.
 Section…
Microtome
 Microtomy is the means by which tissues can be sectioned and attached to a surface
so that examination by microscopy takes place.
 There are different types of microtomes based on their use.
Cambridge rocking microtome
 Consists of a heavy base and two arms (upper and lower arm);
 Hard tissues are difficult to section;
 Simple in operation and maintenance.

Rotary microtome
 Also called as Minot microtome;
 Excellent for preparation of serial section;
 Successfully used in cryostat;
 Graduated in units of 1-30 micrometer;
 Suitable for very hard tissue;
 Produce good accurate sections.HistologyTissue Preparation for Light
Microscopy_x264.mp4

 Section…
Base sledge microtome
 Used for sectioning specimens embedded in all forms of media;
 Graduated in division of 1micrometer-20micrometer;
 Adopted for frozen section cutting by replacement of the paraffin wax object
holder with either a co freezing stage or a thermo module;
2
 Best for resin-embedded undecalcified bone.
Sliding microtome
 Sliding microtome is best for sections from tissue embedded in celloidin.
Rotary rocking microtome
 Rotary rocking microtome can be used for both paraffin wax work and in cryostat
sections.
Ultra microtome
 Ultra microtome is used exclusively for electron microscopy for sectioning of frozen
block at between 50-150 micrometer from either fixed or unfixed tissues.

Freezing microtome
 Freezing microtome is used for cutting sections of fixed tissues when speed is utmost
importance.Freezing the section_x264.mp4
Types of microtome Knives
 Microtome knives are classified according to their cross-sections (profiles) as follows:
1. Wedge-shaped: plane on both sides.
2. Planoconcave: hollow ground on one side.
3. Biconcave: hollow ground on both sides.
4. Tool-edge: plane on one side with a steep cutting edge.
Wedge-shaped knife
 Wedge-shaped knife is the most commonly used knife.
 It is recommended for the majority of sectioning such as frozen tissue, paraffin wax
embedded tissue and hard materials embedded with celloidin.
Biconcave knife
 Biconcave is used for sectioning of tissue embedded in paraffin wax.
Tool-edge knife
 Used for the sectioning of very hard materials like un decalcified bone;
 It is not recommended for routine sectioning of soft materials due to the shallow
rakeangle and the difficulty of re-sharpening.
 Section…
Knife sharpening
 Knife sharpening is carried out either by manual or automated means.
Manual knife sharpening
 Manual knife sharpening requires more skill, experience and time.
 practically it is done using naturally occurring slabs of stone which had a wide range of
abrasive properties or synthetic composite slabs of abrasive material particularly
carborandum.
 Section…
 These solid abrasive blocks are with the following disadvantages:
1. Its surface area is insufficient to allow more than about half of the knife to contact the
stone at any one time.
2. Large accurately flat stones are expensive.
 3. The range of abrasive properties is limited.
4. Surface wear causes concavities on the stone.
 Section…
Automated knife sharpening
Advantages of automated knife sharpening
 A damaged or dull cutting edge may be replaced by a new perfect edge within seconds
without time consuming.
Disposable blades
 Disposable blades are modified, thickened razor blades produced from high-quality
stainless steel and give reproducible, compression-free, good quality sections.
 The thin blade is held rigidly in its own special holder to minimize vibration during
microtomy.
 These blades appear to be the first choice, nowadays, for routine histopathology.
 Section…
Water bath
 The thermostatically controlled type is preferable.
0
 The temperature of the water bath should be about 10 C below the melting point of the
paraffin wax.
 Alcohol or small quantities of detergent are added to reduce surface tension and allowing
the section to flatten out with greater ease.
…
Drying oven or hot plate
 Designed for drying tissue sections on slides;
 Should be set at the melting point of the wax;
 Drying is complete in 30 minutes. For more delicate tissues example;
0
 Tissue from CNS 37 C for 24 hours or longer is recommended.
Brush and forceps
 Brush and forceps are necessary for the handling of sections during cutting and also for
the removal of folds and creases formed in the section during floating out
 Section…
Slides
 Clean, grease free slides are used.
 Labeling is done by;
 Diamond marker;
 Frost ended slides, using pencil;
 Automated slide-labeling machine.
Section adhesives
 Protein adhesives such as albumin, gelatin and starch are prone to bacterial overgrowth.
 They also stain heavily with dyes, hence are not recommended as section adhesive.
 The two best adhesives are:
1. Poly-L-lysine
  Available as a 0.1% solution
 Further diluted for use, 1 in 10 with distilled water.
 Coated slides should be used with in a few days.
2. 3 - Amino-propyl triethoxy saline (APES)
 The best section adhesive
 Coated slides can be stored for a long time
 The APES –coated slides have proved invaluable in cytology.
 Section…
 Adhesives can also be prepared from equal volume of glycerin, water and white of
egg.
Lubricants
 Lubricants are essentials in most sharpening techniques of microtome knives for the
following reasons.
 Lubricants act as coolants and prevent the extreme edge of knife;
 Fine metal particles are allowed to fall away from the knife and fresh abrasive
particles to contact the knife-edge;
 Reduce the tendency of the stones ‘pores’ to become blocked with finely Divided
metal particles.

Review questions:
1. Explain the different stages of tissue processing.
2. List the commonly used dehydrating fluids and clearing agents.
3. List the criteria in choosing clearing agents.
4. Describe the characteristics of good embedding media
5. Discuss the various factors that affect tissue processing.
6. Discus tissue sectioning and its importance.
7. Mention the various equipments used for tissue sectioning.
8. List freezing techniques used in tissue sectioning.
9. Explain the different lubricants used in tissue sectioning.
10. Describe errors and remedies in paraffin wax sectioning.

3.1 Introduction
Staining is a biochemical technique of adding a classic specific dye to a substrate:
 DNA
 Proteins
 Lipids and
 Carbohydrates
to qualify or quantify the presence of a specific compound.

3.2 Types of stain

Negative stain is the type of stain that does not color the tissue or cells but surrounds the tissue
as a result, uncolored tissue can be seen against a background color.
Principle of staining

The stained slide must go through the reverse process that it went through from paraffin
section to water .
The stained slide is taken through a series of alcohol solutions to remove the water, then
through clearing agents to a point at which a permanent resinous substance beneath the
glass cover slip, or a plastic film, can be placed over the section.

3.3 Affinity of staining

 Affinity means the force, which binds the dye to the tissues.
 The word crisp is used in textile industry instead of affinity.
 Affinity is not a one-way procedure but a two-way procedure as it depends on the tissue
as well as on the dye.

3.3.1 Factors contributing to dye-tissue affinity

Factors that can adversely affects staining of tissues:
 Solvent-solvent interaction (Hydrophobic bonding)
 Reagent-reagent interaction
 Reagent-tissue interaction (Vander -Waal forces)
 Columbic attraction
 Hydrogen bonding
 Covalent bonding.
3.3.1.1 Solvent-solvent interaction
 There is a tendency of hydrophobic grouping or chemicals or substances to come together
even though initially dispersed in aqueous (water) solution.
 The water molecules are transiently held together in clusters by hydrogen bonding.
 These clusters are stabilized by hydrophobic group
 Any process that involves breakup of the clusters in to disorganized water molecules will
tend to occur spontaneously.
 Phenylalanine and tryptophane side chains or biphenyl naphthol are hydrophobic
grouping.

3.3.1.2 Reagent-reagent interaction

 Dye interacts with each other and form aggregates.
 Dye aggregation increases with concentration.
 Cations or basic dyes build up in tissues where there is high negative charge such as,
o polysaccharides in mast cell granules and
o cartilage matrix.

Metachromatic staining
 This is due to dye aggregation having spectral properties unlike those of monomeric dyes.
Silver impregnation technique
  There is stain-stain interaction.
Gomer’s type enzyme histochemistry
 There is stain-stain interaction.
3.3.1.3 Dye - tissue interaction or Vander- Walls reaction
Van-deer-Walls force
 acts between dye and tissues.
 is of short range.
Therefore, Van-deer-Walls force is strong where close reagent contact is possible.
Substrates and chemicals that favor Van-der-walls bonding include:
 Tyrosine and tryptophane residues of proteins;
 Heterocyclic bases of nucleoproteins;
 Halogenated dyes such as,
o rose Bengal,
o phloxine,
o enzyme substrate,
o naphthyl indoxyl system.

3.3.1.4 Columbic attraction

Columbic attraction
 termed as salt link or electrostatic bond.
 the electrostatic bonds arise from electrostatic attraction of dissimilar ions
 is the most widely acknowledged reagent tissue interaction.
Example
The colored cation of basic dyes and tissue structures rich in anion such as - phosphated
DNA and RNA
- carboxylated or
- sulphated mucosubstances.
In case of columbic attraction, the amount of dye able to enter a given tissue will depend on:
- charge of dye,
- magnitude of charges,
- amount of non-electrolyte present in dye bath
- ability of tissue to sink or swell.

3.3.1.5 Hydrogen bonding

Hydrogen bonding(H-bonding)
 is a localized bond when a hydrogen atom lies b/n two electronegative atoms such as ,H
or N .
 It will rarely be an important source of reagent-tissue affinity when aqueous solvents are
used.
 if dye is taken by other process, H- bonding may subsequently contribute to affinity.
3.4 Factors that determine selectivity of stain
Dye or stains are not taken by every part of tissue, and this is called selectivity of stain.
Factors that determine selectivity of stain include:
 Number and affinity of binding sites
 Rates of reagent uptake
 Rate of reaction
 Rate of reagent loss.

3.4.1 Number and affinity of binding sites

The staining affinity of dye depends on:
 the number of binding sites of the dye to tissue.
Reagent-tissue affinities and the number of binding site vary.

Example

non-ionic Sudan dye will have affinity for fat droplets, but none for the surrounding hydrated
proteins. Therefore Sudan stains intensely fat tissues and not proteins or carbohydrates.


5.4.2 Rate of reagent uptake by the tissue
Selectivity of the stain by the tissue depends on the rate of reagent uptake by the tissue.
Therefore, selectivity can be controlled by modifying the time of staining.
 Rate of reagent uptake by the tissue
Example
short period of staining
 mucin-staining method using Alcian blue or colloidal iron.
prolonged period of staining
 nucleic acid and RNA-rich cytoplasm are also stained using Alcian blue or colloidal iron.
There are methods where three or more dyes diffuse at varying rates, as a result different
structures can be stained by different dyes.
Example collagen fibers stain rapidly while the muscle fibers stain at intermediate rate

3.4.3 Rate of reaction

Selectivity of the stain also depends on the rate of reaction. This is because,
- reactive stain yield colored derivatives, &
- amount of color
depend on the selective rates of reaction.
Example
The periodic acid oxidation step of the periodic acid-Schiff procedure, if sufficiently
prolonged, oxidize various chemical structures present in tissue.

Rate of reaction
Enzyme histochemistry provides many other examples of reaction rates that affect selectivity.
Example
at low pH the hydrolysis of an organic phosphate applied to a tissue section in a suitable
incubation media will be rapid in those parts of the tissue containing acid phosphates.
However, in structures containing alkaline phosphatase , the hydrolysis rate will be very slow.

3.4.4 Rate of reagent loss

Some tissues or structures are stained and decolorized readily while other tissues or structures are
not decolorized readily.
Differentiation Is when a decolorized tissue takes up the counter stain.
Example
 staining of muscle striation with iron Haematoxyline and myelin sheath with
Luxol Fast Blue.
 In regressive staining all structures are first stained quite non-selectively some
times with the assistance of penetration aid such as heat or phenol.
 Subsequently, the tissues are extracted in a solvent. The solvent extracts the dye
from the tissue. And this is known as destaining.
The staining conditions chosen to maximize selective affinity.
 Basic dyes must be applied from neutral or acidic solution.
 the concentration of inorganic salt present in a dye bath ,called critical electrolyte
concentration.
 rate of reagent uptake, or subsequent reaction or loss of reagent or product.

3.5 Types of commonly used stains in histopathologic techniques

3.5.1 Haematoxyline (H)
 is the most popular & widely used histologic stain.
 extracted from heartwood of the tree called Haematoxyline campechium with
hot water.
 can demonstrate clearly enormous number of d/t tissue structures.

Natural oxidation of Haematoxyline

natural oxidation(ripening)
 is exposing haematoxyline to light & air and converting it to hematin.
 is a slow process, it may takes 3-4 months.
 the hematine solution retains its staining ability for a long time.
Examples
-Ehrlichs haematoxyline
-Delafield’s haematoxyline.
 Haematoxyline also stains tissues that are processed by various methods.
 In the routine H & E stain, nuclei stain blue-black with good intra nuclear
details.
 While Eosin stains cytoplasm and connective tissue in varying intensity of pink,
orange & red.
 Haematoxyline has many more uses than in E & H combination.
 Haematoxyline by itself is not stain but haematin, an oxidant product of
Haematoxyline is a stain & a natural dye.

Production of haematin from Haematoxyline

 Haematoxyline is extracted from heartwood or logwood. It is precipitated by
using urea.
 Haematin is produced from Haematoxyline by two ways.
1. Natural oxidation or ripening of Haematoxyline.
2. Chemical oxidation of Haematoxyline.
 Chemical oxidation of haematoxyline
 haematoxyline is oxidized by chemicals such as - sodium iodate & - Mercuric
oxide.
 Sodium is used as an oxidant in preparation of Mayer’s haematoxyline
 Mercuric oxide for Harris haematoxyline
Advantages
- Oxidize haematoxylin almost instantaneously;
- Ready to be used after immediate preparation.
Disadvantage
- have shorter useful life when compared to naturally ripened haematoxylin.

3.5.2. Classification of haematoxyline based on mordant used

Classification of haematoxyline based on mordant:
 Alum haematoxylin;
 Iron haematoxylin;
 Tungsten haematoxylin;
 Molybdenum haematoxylin;
 Lead haematoxylin;
 Haematoxylin with out mordant.

3. 5.2.1 Alum haematoxylin

Types of Alum haematoxylin
1. Ehrlichs haematoxylin;
2. Delafield’s haematoxylin;
3. Mayer’s haematoxylin;
 4. Harris haematoxyline;
5. Coles haematoxylin;
6. Carazzi’s haematoxylin.

Blueing
After staining with haematoxylin, the section is treated with:- tap water or
- Scott’s tap water or
- lithium carbonate.

The red color of the nuclei becomes blue and this is known as blueing.
Disadvantages of alum haematoxylin
 The stain is sensitive to any subsequently applied acid solution.
Example - vangieson and
- trichrome staining.

 The picric acid fuchsine mixture in vangieson stain removes most of the haematoxylin so
that nucleus becomes faint and not easily seen.
 In such situation,
o Iron-mordant haematoxylin
o a combination of alum haematoxylin
o Celestine blue is used, since Celestine blue solution is prepared in ferric acid
solution & the ferric salt in Celestine blue solution strengthens the bond b/n the
nucleus & alum haematoxylin.
For routine H & E staining, the most commonly used haematoxylin are:
 Ehrlich’s haematoxylin
 Harris haematoxylin
 Mayer’s haematoxylin
 Colles haematoxylin
 Dalfield’s haematoxylin

Ehrlichs haematoxylin
 is a naturally ripened haematoxylin
 takes about 2-4 months to ripen.
 Alum is used as mordant.
 naturally ripened solution will last in bulk for years.
 It is an excellent nuclear stain.
 It also stains mucin and polysaccharide
 is recommended for bone and cartilage.
Uses of Ehrlich’s stain
 Ehrlich’s stain stains nuclei intensely and crisply.
 The stained sections fade much more slowly, than those stained with other haematoxylin.
 It is particularly useful for tissues that have been exposed to acid.
 is useful for bone, which has been exposed to acid for decalcification.
 is also valuable for tissues that have been fixed in formalin .
Delafield’s haematoxylin
 Delafield’s haematoxylin is a naturally ripened haematoxylin.
 Its longevity is similar to Ehrlich’s haematoxylin.
Mayer’s haematoxylin
Mayer's haematoxylin
 water is used as a solvent.
 is not naturally ripened but artificially ripened with sodium iodate.
used as:
 Regressive stain like any other haematoxylin
 Progressive stain particularly when nuclear counter stain is needed to emphasize
cytoplasmic components
 Nuclear counter stain in demonstration of glycogen
 is used in various enzyme histochemical techniques.

Harris haematoxyline
Harris haematoxylin
 is not naturally ripened but artificially ripened with mercuric oxide.
 The nuclear staining deteriorates after few months
 For best result it is wise to prepare a fresh batch of stain every month.
.

Coles haematoxylin
Cole’s haematoxylin:-is artificially ripened with alcoholic solution
Carazzi's haematoxylin
 water is used as a solvent.
 is artificially ripened with potassium iodate.
 It can be used as progressive nuclear staining using a short time followed by blueing in
tape water
 is suitable for pale and precise nuclear staining.
 does not stain any of the cytoplasmic components
 it is largely confided to its use with frozen section for-urgent surgical biopsy.
 For frozen section, it is an excellent nuclear staining when used as double
strength solution (using one gm of haematoxylin).