Histopathology techniques

Histopathology definition:

It is a branch of pathology which deals with the study of disease in a tissue

section. The tissue undergoes a series of steps before it reaches the diagnosis. To
achieve this it is important that the tissue must be prepared in such a manner
that it is sufficiently thick or thin to be examined microscopically and all the
structures in a tissue may be differentiated.
The term histochemistry means study of chemical nature of the tissue
components by histological methods. The cell is the single structural unit of all
tissues, the study of cell is called cytology .A tissue is a group of cells specialized
and differentiated to perform a specialized function, collection of different type of
cells forms an organ.

Type of material obtained in laboratory

The human tissue comes from the surgery (Biopsy) and/or from the dissection
room (Autopsy).
From surgery two types of biopsy could be obtained:
1. Incisional Biopsy: A small piece of lesions or tumor which sent for diagnosis
before final removal of the lesion or the tumor .
2. Excisional Biopsy: If the whole of the tumor or lesion is removed for
examination and diagnosis it is called excisional biopsy.

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 FIXATION
It is a complex series of chemical events which brings about changes in the
various chemical constituents of cell like hardening, however the cell morphology
and structural detail is preserved. Unless a tissue is fixed soon after the removal
from the body it will undergo degenerative changes due to autolysis and
putrefaction so that the morphology of the individual cell will be lost.

Principle of fixation

The fixative brings about crosslinking of proteins which produces denaturation or

coagulation of proteins so that the semifluid state is converted into semisolid
state; so that it maintains everything in vivo in relation to each other. Thus
semisolid state facilitate easy manipulation of tissue.

Aims and Effects of fixation

If a fresh tissue is kept at room temperature it will become liquefied with a foul
odour mainly due to action of bacteria i.e. putrefaction and autolysis so the first
and fore most aim of fixation is
1. To preserve the tissue in as life like manner as possible.
2. To prevent postmortem changes like autolysis and putrefaction.
Autolysis :is the lysis or dissolution of cells by enzymatic action probably as a
result of rupture of lysosomes.
Putrefaction: The breakdown of tissue by bacterial action often with formation of
gas.
3. Preservation of chemical compounds and microanatomic constituents so
that further histochemistry is possible.

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 4. Hardening : the hardening effect of fixatives allows easy manipulation of
soft tissue like brain, intestines etc.
5. Solidification: Converts the normal semifluid consistency of cells (gel)to an
irreversible semisolid consistency (solid).
6. Optical differentiation it alters to varying degrees the refractive indices of
the various components of cells and tissues so that unstained components
are more easily visualized than when unfixed.
7. Effects of staining certain fixatives like formaldehyde intensifies the staining
character of tissue especially with haematoxylin.

Properties of fixatives

1. Coagulation and precipitation.

2. Penetration Fixation is done by immersing the tissue in fluid containing the
fixative. Faster fixative can penetrate the tissue better it is penetration
power depends upon the molecular weight e.g. formalin fixes faster than
osmic acid.
3. Solubility of fixatives - All fixatives should be soluble in a suitable solvent,
preferably in water so that adequate concentrations can beprepared.
4. Concentration - It is important that the concentration of fixative is isotonic
or hypotonic.
5. Reaction - Most fixatives are acidic. It may help in fixation but can affect
staining so has to be neutralized e.g. formalin is neutralized by adding of
calcium carbonate.

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 6. Amount of fixative: The fixative should be at least 15-20 times the bulk of
tissue. For museum specimens the volume of fixative is > 50 times. Note : If
the specimen is large then see that the sections are made to make slices
which have a thickness of 3 – 5 cm so that fixative can penetrate the tissue
easily.
Simple fixatives
(I) Formaldehyde:

Formaldehyde is a gas but is soluble in water to the extent of (37 - 40%) w/v. This
solution of formaldehyde in water is called formalin or full strength formalin.
Formalin is one of the commonly used fixative in all laboratories since it is cheap
penetrates rapidly and does not over harden the tissues.

• It preserves the proteins by forming cross linkage with them and the tissue
component.

Autolysis It denatures the proteins.

• Glycogen is partially preserved hence formalin is not a fixative of choice for

carbohydrates.

• Some enzymes can be demonstrated in formalin fixed tissues.

• It neither preserves nor destroys fat.

• Complex lipids are fixed but has no effect on neutral fat.

• After formalin fixation fat may be demonstrated in frozen section.

• Pure formalin is not a satisfactory fixative as it over hardens the tissue. A 10%
dilution in water (tap or distilled) is satisfactory. Since it oxidizes to formic acid if
kept standing for long period so it should be neutralized by phosphates or calcium
carbonate otherwise it tends to form artifact; a brown pigment in tissues. To
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remove this pigment picric alcohol or saturated alcoholic sodium hydroxide may
be used.
• Concentrated formalin should never be neutralized as there is a great danger of
explosion.
• The commercial formalin becomes cloudy on standing especially when stored in
a cool place due to formation of precipitate of paraformaldehyde which can be
filtered.
• Formalin on prolonged exposure can cause dermatitis its vapour may damage
the nasal mucosa and cause sinusitis.
• Time required for fixation at room temperature (12 hours) for small biopsies, (4-
6 hours) at (65°C) fixation occurs in (2 hours).

(II) Alcohol (Ethyl Alcohol)

• Absolute alcohol alone has very little place in routine fixation for
histopathology. It acts as a reducing agents, become oxidized to acetaldehyde and
then to acetic acid.

• It is slow to penetrate, hardens and shrinks the tissue.

• Alcohol penetrates rapidly in presence of other fixative hence in-combination

e.g. Carnoy's fixative is used to increase the speed of tissue processing.

• Ethanol preserves some proteins in relatively un-denatured state so that it can

be used for immunofluorescence or some histochemical methods to detect
certain enzymes.

• It is a fat solvent hence it dissolve fats and lipids

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• Methyl alcohol is used for fixing blood and bone marrow smears.

(III) Acetone

• Cold acetone is sometimes used as a fixative for the histochemical

demonstration of some tissue enzymes like phosphatases and lipases.

• Its mode of action as fixative is similar to that of alcohol

(IV) Mercuric Chloride (HgCl2)

• Mercuric chloride is a very good salt employed in fixing but is rarely used alone
because it causes shrinkage of the tissue.
• It brings about precipitation of the proteins which are required to be removed
before staining by using potassium iodide in which they are soluble.
•The size (thickness) of the tissue to be fixed in mercuric chloride is important,
since if the tissue is more than 4 mm, then it hardens the tissue at the periphery
whereas the Centre remains soft & under fixed.
• It penetrates rapidly without destroying lipids.
• It neither fixes nor destroys carbohydrates.
• Treatment of the tissue with mercuric chloride brings out more brilliant staining
with most of the dyes.
• Tissues fixed with mercuric chloride containing fixatives contain black
precipitates of mercury which are removed by treating with 0.5% iodide solution
in 70% ethanol for 5-10 minutes, sections are rinsed in water, decolourized for 5
minutes in 5% sodium thiosulphate and washed in running water.

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 (V) Picric acid

• It produces marked cells shrinkage hence it is not used alone.

• It has to be stored in a damp place because of its explosive nature it is
preferably stored under a layer of water.
• It penetrates well and fixes rapidly.
• It precipitates proteins and combines with them to form picrates some of the
picrates are water-soluble so must be treated with alcohol before further
processing where the tissue comes into contact with water.
• Note : All the tissues fixed in picric acid containing fixatives should be
thoroughly washed to remove the yellow discoloration to ensure proper staining
of tissue sections. If the fixative is not removed by washing thoroughly with time
even the embedded tissue loses its staining quality.

(VI) Potassium dichromate

• It fixes the cytoplasm without precipitation.

• Valuable in mixtures for the fixation of lipids especially phospholipids.
Used for fixing phosphatides and mitochondria.
• Note: Thorough washing of the tissue fixed in dichromate is required to avoid
forming an oxide in alcohol which cannot be removed later.
(VII) Osmium tetroxide

• It is a strong oxidizing agent and brings about fixation by forming cross links with
proteins.
• It gives excellent preservation of details of a cell, therefore exclusively used for
electron microscopy.
• It fixes fat e.g. myelin.
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• It also demonstrates fat when (0.5 - 2%) aqueous solution is used it gives a black
color to fat.

(VIII) Acetic acid

•It causes the cells to swell hence can never be used alone but should be used
with fixatives causing cell shrinkage.

(IX) Glutaradehyde

• It is used alone or in combination with osmium tetroxide for electron

microscopy.

Compound fixatives:
• Some fixatives are made by combining one or more fixative so that the
disadvantage of one are reduced by use of another fixative.
• All these compound fixative have their own advantages and disadvantages.
• Choice of fixative - The choice of fixative depends on the tissue is going to be
receive e.g. what is the chemical structure that needs to be stained ? If fat is to
be demonstrated the formalin fixed tissue is better. For demonstration of
glycogen formalin should never be used.

Preparation of the specimen for fixation

1. For achieving good fixation it is important that the fixative penetrates the
tissue well hence the tissue section should be (3 – 5 mm) thick, so that
fixation fluid penetrates from the periphery to the Centre of the tissue.
2. For fixation of large organs perfusion method is used i.e. fixative is injected
through the blood vessels into the organ.

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 3. For hollow viscera fixative is injected into the cavity e.g. urinary bladder,
eyeball etc.
4. Ratio of volume of fixative to the specimen should be 1:20.
5. Time necessary for fixation is important routinely 10% aqueous formalin at
room temperature takes 12 hours to fix the tissue. At higher temperature
i.e. 60-65°C the time for fixation is reduced to 2hours.
Classification of fixatives:

Fixatives are divided into three main groups:

1. Microanatomical fixatives; such fixatives preserves the anatomy of the
tissue.
2. Cytological fixatives ; such fixation are used to preserve intracellular
structures or inclusion.
3. Histochemical fixatives : Fixative used to preserve the chemical nature of
the tissue.
Microanatomical fixatives

1. 10% (v/v) formalin (10 ml formalin in 90 ml distilled water) in 0.9% sodium

chloride (normal saline). This has been the routine fixative of choice for
many years, but this has now been replaced by buffered formal or by
formal calcium acetate
2. Buffered formation(a) Formalin 10ml(b) Acid sodium phosphate - 0.4
gm.(monohydrate)(c) Anhydrous disodium - 0.65 gm. phosphate(d) Water
to 100 ml- Best overall fixative
3. Formal calcium (a) Formalin : 10 ml(b) Calcium acetate 2.0 gm.(c) Water to
100 ml

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 • Specific features

i. They have a near neutral pH

ii. Formalin pigment (acid formaldehyde haematin) is not formed.
4. Buffered formal sucrose (a) Formalin : 10ml(b) Sucrose : 7.5 gm.(c) M/15
phosphate to 100 ml buffer (pH 7.4)
 Specific features
i. This is an excellent fixative for the preservation of fine
structure phospholipids and some enzymes.
ii. It is recommended for combined cytochemistry and electron
microscopic studies.
iii. It should be used cold (4°C) on fresh tissue.
5. Alcoholic formalin: Formalin 10 ml 70-95% alcohol 90 ml
6. Acetic alcoholic formalin: Formalin 5.0ml Glacial acetic acid 5.0 ml Alcohol
70% 90.0 ml
7. Formalin ammonium bromide: Formalin 15.0 ml Distilled water 85.0 ml
Ammonia bromide 2.0 gm.
 Specific features :
i. Preservation of neurological tissues especially when gold and
silver impregnation is employed
8. Heidenhain Susa (a) Mercuric chloride 4.5gm(b) Sodium chloride 0.5 gm.(c)
Trichloroacetic acid 2.0 gm.(d) Acetic acid 4.0 ml(e) Distilled water to 100
ml
• Specific features

i. Excellent fixative for routine biopsy work.

ii. Allows brilliant staining with good cytological detail.

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 iii. Gives rapid and even penetration with minimum shrinkage.
iv. Tissue left in its for over 24 hours becomes bleached and
excessively hardened.
v. Tissue should be treated with iodine to remove mercury
pigment.
9. Zenker's fluid (a) Mercuric chloride 5gm(b) Potassium dichromate 2.5
gm.(c) Sodium sulphate 1.0 gm(d) Distilled water to 100 ml(e) Add
immediately before use : Glacial acetic acid : 5 ml
 Specific features
i. Good routine fixative
ii. Give fairly rapid and even penetration
iii. It is not stable after the addition of acetic acid hence acetic
acid (or formalin) should be added just before use
iv. Washing of tissue in running water is necessary to remove
excess dichromate.
10. Zenker formal (Helly's fluid) (a) Mercuric chloride - 5 gm.(b) Potassium
dichromate 2.5 gm.(c) Sodium sulphate 1.0 gm.(d) Distilled water to 100
ml(e) Add formalin immediately before use 5 ml
 Specific features
i. It is excellent microanatomical fixative
ii. Excellent fixative for bone marrow spleen and blood containing
organs
iii. As with Zenker's fluid it is necessary to remove excess
dichromate and mercuric pigment

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 11.B5 stock solution: Mercuric chloride 12 gm. Sodium acetate 2.5gm,
Distilled water 200ml, B5 Working solution: B5 stock solution 20ml
Formalin (40% w/v formaldehyde) 2 ml
• Specific Features
i. B5 is widely advocated for fixation of lymph node biopsies both
to improve the cytological details and to enhance
immunoreactivity with antiimmunoglobulin antiserum used in
phenotyping of B cell neoplasm.

Procedure

Prepare working solution just before use

Fix small pieces of tissue (7x7x2.5mm) for 1-6 hours at room temperature

Process routinely to paraffin.

12. Bouin's fluid(a) Saturated aqueous picric acid 75ml(b) Formalin 25ml(c)
Glacial acetic acid 5 ml

• Specific features

i. Penetrates rapidly and evenly and causes little shrinkage

ii. Excellent fixative for testicular and intestinal biopsies because
it gives very good nuclear details, in testes is used for
oligospermia and infertility studies
iii. Good fixative for glycogen
iv. It is necessary to remove excess picric acid by alcohol
treatment

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 13. Gender's fluid - better fixative for glycogen.(a) Saturated picric acid in 95%
v/v/ alcohol 80ml(b) Formalin 15ml(c) Glacial acetic acid 5ml

Cytological fixatives

Subdivided into:
(A) Nuclear fixatives (B) Cytoplasmic fixatives.
(A) Nuclear fixatives : As the name suggests it gives good nuclear fixation. This
group includes
1. Carnoy's fluid.(a) Absolute alcohol 60ml(b) Chloroform 30ml(c) Glacial
acetic acid 10 ml
• Specific features
- It penetrates very rapidly and gives excellent nuclear fixation.
- Good fixative for carbohydrates.
- Nissil substance and glycogen are preserved.
- It causes considerable shrinkage.
- It dissolves most of the cytoplasmic elements. Fixation is usually complete in 1-2
hours. For small pieces 2-3 mm thick only 15 minutes is needed for fixation.
2. Clarke's fluid(a) Absolute alcohol 75 ml(b) Glacial acetic acid 25 ml.
• Specific features
- Rapid, good nuclear fixation and good preservation of cytoplasmicelements.
- It is excellent for smear or cover slip preparation of cell cultures or chromosomal
analysis.
3. New Comer's fluid.(a) Isopropranolol 60 ml(b) Propionic acid 40ml(c)
Petroleum ether 10 ml.(d) Acetone 10 ml.(e) Dioxane 10 ml.
• Specific features
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- Devised for fixation of chromosomes

- It fixes and preserves mucopolysacharides. Fixation in complete in12-18 hours.

(B) Cytoplasmic Fixatives

(1) Champy's fluid(a) 3g/dl Potassium dichromate 7ml.(b) 1% (V/V)
chromic acid 7 ml.(c) 2gm/dl osmium tetraoxide 4 ml.
• Specific features
- This fixative cannot be kept hence prepared fresh.
- It preserves the mitochondrial fat and lipids.
- Penetration is poor and uneven.
- Tissue must be washed overnight after fixation.
(2) Formal saline and formal CalciumFixation in formal saline followed by
postchromatization gives goodcytoplasmic fixation.

Histochemical fixatives

Vapour fixatives

1. Formaldehyde- Vapour is obtained by heating paraformaldehyde at

temperature between 50° and 80°C. Blocks of tissue require (3-5) hours
whereas section require( ½- 1) hours.
2. Acetaldehyde- Vapour at 80°C for (1-4) hours.
3. Glutaraldehyde- 50% aqueous solution at 80°C for 2 min to 4 hours.
4. Acrolein /chromyl chloride- used at 37°C for 1-2 hours.
5. Other more commonly used fixatives are (1) formal saline (2) Cold acetone
Immersing in acetone at 0-4°C is widely used for fixation of tissues intended
to study enzymes especially. phosphates.

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Secondary fixation

- Following fixation in formalin it is sometimes useful to submit the tissue to

second fixative eg. mercuric chloride for 4 hours. It provided firmer texture to the
tissues and gives brilliance to the staining.
Post chromation

- It is the treatment the tissues with 3% potassium dichromate following normal

fixation. Post chromatization is carried out either before processing, when tissue
is for left for 6-8 days in dichromate solution or after processing when the
sections are immersed in dichromate solution, In for 12-24 hours, in both the
states washing well in running water is essential. This technique is used a mordant
to tissues.
Washing out

- After the use of certain fixative it in urgent that the tissues be thoroughly
washed in running water to remove the fixative entirely. Washing should be
carried out ideally for 24 hours. Tissues treated with potassium dichromate,
osmium tetraoxide and picric acid particularly need to be washed thoroughly with
water prior to treatment with alcohol (for dehydration).
Tissue fixative of choice and time for fixation

 Routine Formalin (10-12) hours.

 GIT biopsies buffered formaldehyde (4-6) hours.
 Testicular biopsy Bouin's fixative (4-6) Hours.
 Liver Biopsy Buffered formaldehyde (4-12) hours .

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 Bone marrow biopsy Bouin's fixative in running (2½) hours followed
by washing in running water over night.
 Spleen and blood filled cavities Zenker's fluid (1-6) hours.
 Lymph node B5 (12-18) hours.
 Mitochondria, phosphatides and Nissil substance Carnoy's fluid (1-2)
hours
 Chromosome / cell culture Clarke's fluid (1-2) hours.

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 DECALCIFICATION
Definition

Decalcification is a process of complete removal of calcium salt from the tissues

like bone and teeth and other calcified tissues following fixation.
Aim of decalcification

Decalcification is done to assure that the specimen is soft enough to allow cutting
with the microtome knife. Unless the tissues is completely decalcified the sections
will be torn and ragged and may damage the cutting edge of microtome knife.
Note:

1. To ensure adequate fixation and complete removal of the calcium it is

important that the slices are 4-5 mm thick. Calcified tissue needs (2-3) hours only,
for complete decalcification to be achieved so it is necessary to check the
decalcification after (2-3) hours.
2. Fixative of choice for bone or bone marrow is Zenker formal or Bouin's fluid.
Unfixed tissue tends be damaged 4 times greater during decalcification than a
properly fixed tissue.
Methods of Decalcification.

1) Dissolution of calcium by a dilute mineral acid.

2) Removal of calcium by used of dilute mineral and along with ion
exchange resin to keep the decalcifying fluid free of calcium.
3) Using Chelating agents EDTA.
4) Electrolytic removal of calcium ions from tissue by use of electric
current.

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 The Criteria of a good decalcifying agents area :
1. Complete removal of calcium.
2. Absence of damage to tissue cells or fibers.
3. Subsequent staining not altered.
4. Short time required for decalcification.

Removal of calcium by mineral acids

 Acid decalcifies subdivided into:

I. Strong acid and
II. Weak acid.

Strong acid - e.g. Nitric acid and hydrochloric acid.

Nitric acid

- 5-10% aqueous solution is used.

- They decalcify vary rapidly but if used for longer than 24-48 hrs. will cause
deterioration of stain ability specially of the nucleus
Hydrochloric acid

- 5-10% aqueous solution decalcification slower than nitric acid but still
rapid. Fairly good nuclear staining.

Weak acid e.g. formic acid, acetic acid and picric acid of these formic acids is
extensively used as acid decalcifier. 5-10% aqueous solution or with additives like
formalin or buffer are used.
Formic acid

- Brings out fairly rapid decalcification

- Nuclear staining in better.
- Requires neutralization and thorough washing prior to dehydration.

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 Aqueous nitric acid

Nitric acid 5-10 ml , Distilled water to 100 ml.

Procedure:

1. Place calcified specimen in large quantities of nitric acid solution until

decalcification is complete (change solution daily for best results).

2. Wash in running water for 30 minutes.

3. Neutralize for a period of at least 5 hours in 10% formalin to which excess of

calcium or magnesium carbonate has been added.

4. Wash in running water over night.

5. Dehydrate, clear and impregnate in paraffin or process as desired.

Note: Overexposure to nitric acid impairs nuclear staining.

Nitric acid is the solution of choice for decalcifying temporal bones.

Perenyi's fluid

10% nitric acid 40ml , Absolute alcohol 30 ml. 0.5% chromic acid. 30ml.
Note:
- all these ingredients may be kept in stock and should be mixed immediately
before use.
- This solution may acquire of blue violet tinge after a short while but this will
have no effect in the decalcifying property.
- It is slow for decalcifying hard bone but excellent fluid for small deposits of
calcium e.g. calcified arteries, coin lesions and calcified glands. Also good
for human globe which contains calcium due to pathological conditions.

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 There is little hardening of tissue but excellent morphologic detail is
preserved.
Formalin nitric acid

Formalin 10 ml, Distilled water 80 ml, Nitric acid 10ml.

Nitric acid causes serious deterioration of nuclear stain ability which partially
inhibited by formaldehyde. Old nitric acid also tends to develop yellow
discoloration which may be prevented by stabilization with 1% urea.

Aqueous formic acid

90% formic acid 5-10 ml, Distilled water to 100 ml.

Surface decalcification

The surface of the block to be decalcified is trimmed with scalpel. The block is
then placed in acid solution at 1%hydrochloric acid face downwards so that acid
bathes the cut surface for 15-60 min.
As penetration and decalcification is only sufficient for a few sections be cut the
block shall be carefully oriented in microtome to avoid wastage of decalcified
tissue.
Decalcification of Bone marrow biopsy.

Tissue after fixation in Bouin's or Zenker's fixative is decalcified for (2½) hours
followed by an hour of washing. The tissue is then dehydrated beginning with
alcohol.

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 Use of Ion exchange resins

Ion exchange resins in decalcifying fluids are used to remove calcium ion from the
fluid. Therefore ensuring a rapid rate of solubility of calcium from tissue and
reduction in time of decalcification. The resins an ammoniated salt of sulfonated
resin along with various concentrations of formic acid are used. The resin is
layered on the bottom of a container to a depth of = ½ inch, the specimen is
allowed to rest in it. After use, the resin may be regenerated by washing twice
with dilute N/10HCL followed by three washes in distilled water. Use of Ion
exchange resin has advantage of (i) faster decalcification (ii) tissue preservation
and(iii) cellular details better preserved.

Chelating agents

Chelating agents are organic compounds which have the power of binding certain
metals. Ethylene-diamene-tetra-aceticacid (EDTA), disodium salt called Versenate
has the power of capturing metallic ions. This is a slow process but has little or no
effect on other tissue elements. Some enzymes are still active after EDTA
decalcification. Versenate 10 gm.Distilled water 100 ml(pH 5.5 to 6.5)Time 7-21
days.

Electrolytic method

This is based on the principle of attracting calcium ions to a negative electrode

into a decalcifying solution.

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 Washing after decalcification:

Through washing of the tissue before processing is essential to remove acid (or
alkali if neutralized has been carried out) which would otherwise interfere with
staining)

Determination of end point of decalcification

(1) Flexibility method

Bending, needling or by use of scalpel if it bends easily that means decalcification

is complete .

This method is Unreliable, causes damage and distortion of tissue.

(2) X-ray method

Best method for determining complete decalcification but very costly.

Tissue fixed in mercuric chloride containing fixatives cannot be tested as they will
be radio opaque.
(3) Chemical Method

It is done to detect calcium in the decalcifying fluid, when no further calcium is

detected, decalcification is considered complete.
 Procedure:
 Take 5 ml of decalcifying fluid from the bottom of container which has been
in contact with the tissue for 6-12 hrs.
 Add 5 ml each of 5% ammonium oxalate and 5% ammonium hydroxide.
 Mix and let it stand for 15-30 min.
 A cloudy solution caused by calcium oxalate indicates that specimen is not
thoroughly decalcified.
 Absence of turbidity indicates completeness of decalcification.

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Treatment of hard tissues

Keratin and chitin are softened by use of concentrated sulphuric and with that aid
of heat keratin is completely dissolved from the tissue sections. But much tissue
distortion will also occur.
Prenyi's fluid

Immersing hard tissues in this solutions for 12-24 hours will make sectioning
easier and excellent preparation of calcified arteries, thyroid and calcified glands
is possible.
Lendrum's technique

It is very useful for tissues which became hard at the time of fixation. Following
washing out of the fixative, tissue is immersed in a 4% aqueous solution of phenol
for 1-3 days.

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 TISSUE PROCESSING
Specific objective;

The tissue processing is the heart of any tissue section which will be cut
adequately only if the tissue is properly preserved and processed. The study of
this topic is to understand the coarse and fine details of tissue processing so that
excellent sections are obtained.
Definition

 The term tissue processing refers to treatment of the tissue necessary to

impregnate it into a solid medium so that the tissue is rendered sufficiently
firm yet elastic for the tissue sections of desirable thickness to be cut on
microtome.
This is not the only technique employed for tissue sections. Sections can also be
produced by means of cryostat or freezing microtome on frozen tissues. The fixed
impregnated tissues have an advantage that they can be more easily stored and
reproducibility of sections at a later date is easier. Before proceeding on tissue
processing as soon as the tissue is received it in very important that the tissue be
properly labeled so as to avoid any confusion regarding duplication of same name
or giving a wrong diagnosis to the patient.

The labeling

 The label should remain throughout the entire processing and later as
permanent record keeping. To ensure this most laboratories have a
numbering system for each specimen. As soon as the specimen is received
it is given a specific individual number, which is also recorded in the register

24
 with the details like patient's name, name of the doctor referring it, nature
of tissue is noted.
 Labeling should not be done using ordinary ink as it gets dissolved in the
reagent used during processing.

 Thin white card with a soft lead pencil, typed or printed labels are
satisfactory. To ensure that the label remains with their correct specimens
tissues processing baskets can be used. These are small perforated metal
containers in which the tissue and labels are placed. these containers can
be transferred as such from reagent to reagent. Alternatively use of tissue
tek system in which the tissue identity is written on the cassette and
retained as permanent record during sectioning and storage of tissue
blocks.
Principle of tissue processing

The tissue is embedded in a solid medium by the help of first removing the tissue
water which is then replaced by any solid medium such as paraffin wax so that
the tissue is rendered firm enough to enable thin sections to be cut, at the same
time, the tissue is soft (not so hard) to enable microtome knife to cut the sections.
The embedding medium has to thoroughly permeate the tissue in fluid form so
that it solidifies without any damage to the tissue. The most satisfactory
embedding medium used in routine histology is paraffin wax. Most of the tissue
fixatives are aqueous fixatives so before the tissue can be embedded in paraffin
wax it is necessary that the water and some of the lipid tissue fluids be removed
completely by a variety of compounds through a process called dehydration. Prior

25
to paraffin wax embedding and impregnation the tissue must be subjected to the
following steps:
1. Fixation
2. Dehydration.
3. Clearing - with a substance which is totally miscible with both the dehydrating
agent which precedes it, and embedding agent which follows it.
4. Embedding.
All these 4 processes depend upon complete impregnation of the tissue by the
agent like paraffin wax being used. Before going into the details of these 4 stages
it is important to understand the factors which influence the rate and efficiency of
tissue impregnation
Factors influencing the rate of impregnation

A tissue immersed in fluid interchange occurs between tissue fluid and

surrounding fluid. The process continues through all stages of processing from
fixation to final impregnation.
Agitation: Tissue placed in liquid is agitated so that the fluid immediately in
contact with the surface of tissue which is mixed by tissue fluid is replaced by the
fresh immersing liquid. This can be achieved by a pumping system which removes
and replaces fluid at selected intervals or by rotation and vertical oscillation
method. Efficient agitation reduces the processing time by 25-30% with improved
impregnation of the tissue.

Heat: Heat increases the rate of penetration.

Viscosity: Larger the molecule the higher is the viscosity slower is the rate of
penetration.

Ultrasonic: Use of ultrasonics increases the penetration rate.

26
Vacuum: Use of reduced pressure in well known in the impregnation of tissue by
molten paraffin wax. It hastens the process. Use of vacuum during dehydration
and clearing has little advantage except removal of air bubble trapped within the
tissue.
STEPS OF PARAFFIN WAX EMBEDDING

1. Fixation

Usually tissue that is received at the laboratory is already fixed but before
proceeding further check if the fixation is complete.
2. Dehydration

After fixation in aqueous solvent the delicate tissue needs to be dehydrated

slowly starting in 50% ethyl alcohol. The other routine tissue specimen may be
put in 70% alcohol. A higher concentration of alcohol initially is inadvisable
because this may cause very rapid removal of water may produce cell shrinkage.
An exception to this is in case of Heidenhain's Susa fixed tissue where it may be
placed directly in 95% alcohol. Tissue transferred from alcoholic based fixative like
Carnoy's fixative may be placed in higher grades of alcohol or even in absolute
alcohol. For routine biopsy and postmortem tissue of 4-7 mm thickness 70%,90%
and absolute alcohol (2-3 changes for 2-4 hours each) are sufficient to give
reasonably satisfactory result.
Use of solid dehydrants

Anhydrous copper sulphate is used in higher grade of dehydrating alcohols. A

layer 1-2.5 cm thick is placed at the bottom of a dehydrating vessel or beaker and
is covered with 2 or 3 filter papers to prevent contamination of the tissues.
Anhydrous copper sulphate is white, it removes water from alcohol which in turn

27
has been diluted upon absorption of water from the tissues. The change of colour
of copper sulphate from white to blue indicates that both alcohol and water
should be changed. Use of copper sulphate enhances the process of dehydration
and also prolongs the life of alcohol.
Other dehydrating agents:

1) Acetone: It is clear, colorless volatile inflammable fluid.

• It has a rapid action in dehydrating the tissue but produces shrinkage and
distortion and subsequent brittleness to thetissue.
• Low cost is also an advantage. Acetone usually dehydrates within 20-30 minutes
but four changes of acetone should be used, it is preferable to use acetone after
low strength of alcohol so that distortion of the tissue is less.
2) Dioxane - It dehydrates and clears at the same time. It is miscible
withparaffin and with water and alcohol, tissue from dioxane can be
transferred straight to paraffin.
• There is less shrinkage of tissues
• Tissues can be left in dioxane without danger of hardening for longer period of
time.
Disadvantage: It is more expensive than alcohol.* It is toxic to man3.
3) Isopropyl alcohol
• It is miscible with water and other organic solvents
• It does not harden the tissue like alcohol
• It is expensive

28
 3. Clearing

Definition:
Clearing means appearance of tissue after it has been treated by the fluid chosen
to remove the dehydrating agent.
Most of these tissues have similar refractive index to that of protein, therefore
the tissue is left translucent. Clearing agent is required when the dehydrating
agent is not miscible with the impregnating medium. It is essential for a clearing
agent to be miscible both in dehydrating agent as well as embedding agent.
Commonly used clearing agents are as follows :

1) Xylene: It has a rapid action. Biopsy specimens of 3-4 mm thickness are

cleared in 2-4 hours .Immersion time must not be prolonged otherwise the
tissue become brittle.
2) Toluene and Benzene: are similar in properties to xylene but are less
damaging to the tissues on prolonged exposure.
3) Chloroform: It is slower in action but it causes less brittleness therefore
tissue can be left in it overnight.
• It does not affect the refractive index of the tissue is notrendered translucent.
• It is expensive.
• It is inflammable.
4) Carbon tetrachloride: It has similar properties to chloroform but
ischeaper.5.
5) Cedar wood oil: (Histological): It is good for treatment of delicate tissues as
it has the least hardening effect.
• It is very slow in action.

29
• It is very expensive .Care should be taken not to confuse it with cedar wood oil
(microscopic) used with oil immersion lens.
Techniques of clearing

If the tissue is being cleared in chloroform or carbon tetrachloride it may be left

overnight. In automatic tissue processor three changes of one hour each are
usually satisfactory.
In Xylene, benzene or toluene one change after 30-60 minutes is satisfactory to
give a clear translucent appearance to the tissue.

4. Impregnation

Definition:

It is the complete removal of clearing reagents by substitution of paraffin or any

such similar media.

Impregnation with wax

Impregnation with paraffin wax takes place in an oven heated to 56-60°C
depending upon the melting point of the wax in use. Frequent check of the
temperature of paraffin baths is required since temperature 5°C above the
melting point of the paraffin will cause tissue shrinkage and hardening.
Properties of paraffin wax
1. Easy to prepare large number of tissue blocks in comparatively short time.

2. Minimum supervision is required

3. It is cheaper than other impregnating media

4. During staining there is very little difficulty than other media.

30
Points to be remembered during use of paraffin wax
1. It should be free from dust, grit and other foreign matter.

2. It should not contain water, which causes it to crystallize and turn it white.

3. The wax has to be filtered before use by use of ordinary filter paper.

4. Higher melting point waxes are hard to ribbon . For impregnation the wax oven
has to be kept at high temperature, making the tissue hard, too low melting point
wax may not be hard enough to support the tissue during cutting. If the wax is
overheated and remains in that state for a long time, it tends to crystallize and
become useless.
Impregnation with Paraplast
 This is mixture of highly purified paraffin and several plastic polymers.
 It has greater elasticity than normal paraffin wax, therefore, the results are
superior.
 It ribbons well allowing almost wrinkle free serial sections to be cut with
ease at 4 micron thickness.
 It should not be used for thin walled structures as it prevents complete
expansion of the specimen.

Impregnation with Bioloid

Good embedding medium in which thin walled structures can be sectioned

satisfactorily.

31
Technique of impregnation

The tissue is transferred from clearing agent to molten paraffin wax. The amount
of wax should be 25-50 times the volume of tissue. The tissue must be submitted
to 3 changes in wax. The temperature of the wax bath should be 2-3°C above the
melting point of wax.
Time of impregnation

Depends on the following factors:

1. The size and type of tissue.

2. The clearing agent employed.

3. The use of vacuum embedding oven.

1. Size and type of tissue: The thicker the tissue the longer will be the time
required for wax to penetrate to the center in addition a thick tissue has more of
clearing agent so more changes of wax are necessary to remove it. If even small
amounts of clearing agents remains with the wax this will cause crystallization and
produce crumbling of the sections during cutting. The type of tissue is also
important since bone, skin, CNS needs twice as long as soft tissue like liver or
kindly. Tissue like muscle and fibrous tissue tends to over harden and become
brittle in wax bath so the time for impregnation must be kept to a minimum. The
reduction of time can be achieved by using vacuum embedding medium.

2. Clearing agent employed, Some clearing agents are more rapidly and easily
cleared than other e.g. Xylene, benzene and toluene are easiest to remove, and
one change of wax is normally sufficient; whereas for chloroform and carbon
tetrachloride 2-3changes are needed.

32
3. Use of vacuum embedding oven With the use of normal paraffin oven, 2
changes of paraffin wax for a period of 4 hours are needed but by using vacuum
embedding oven this time may be halved

5. Embedding

It is the orientation of tissue in melted paraffin which when solidified provides a

firm medium for keeping intact all parts of the tissue when sections are cut.

Types of moulds:

a) Leuckhart's L pieces - These are two 'L' which are resting metal usually brass,
which are resting on a flat metal or glass plate.

b) Compound embedding units - consists of square shaped brass or metal plates

in a series of interlocking plates.

c) Others like plastic embedding blocks (tissue Tek system).

Techniques of casting

1. Molten paraffin wax which is heated at a temperature 2-3° above the melting
point is poured into the mould to an adequate depth so as to cover the thickest
tissue block.

2. The wax touching the mould will quickly form a thin semi solid layers, Now
introduce the tissue with a pre warmed forceps to prevent the wax to stick to it.
The tissue is pressed in this semisolid wax to orient it at the bottom of mould in a
correct plane.

3. Fix the label in position by pressing one edge against solidifying wax usually
sides of the mould are preferred.

33
4. As soon as a film of solid wax is formed on the surface, the whole block with
mould are submerged in cold water at 20°C. If this is not done there will be
crystallization of wax, using ice water to do initial cooling will also cause the block
to crack.

5. When blocks are set hard they are removed from mould. The tissue surface
towards the mould base is from where the sections are to be cut this surface
should be trimmed lightly with a scalpel so as to expose the tissue.

Following points must be taken care off during casting :

1. Paraffin should not be allowed to cool around the tissue to be blocked for this
before introducing the tissue in the mould it should be kept in heated wax or in
cassette placed over thermostatic hot plate.

2. To prevent excess of wax solidifying on the bottom of the block during winter
pre warmed moulds may be used.

3. The cutting surface of the tissue should be facing at the bottom of the mould.

4. If 2 or more tissues have to be casted remember to keep them both at the

same depth.

5. If small biopsy fragments have to be casted, the largest piece should be first
blocked and other pieces should be as near it as possible.

6. All four corners of the block should be in one horizontal plane.

7. The tissue should have at least 2 mm wax around its edges.

34
8. Smear mineral or machine oil on the inner surface of the mould for facilitating
easy removal of block.

9. Whitish areas around tissue in block denotes crystallization which may be due
to moisture or due to incomplete removal of clearing agent. Most tissue sections
are cut from the largest area but some tissue needs special mention such as:.

(1. Tissue of tubular nature are cut transversely so should be embedded vertically.

2. Skin is cut in a plane at right angles to the surface so should be embedded at

right angles to the bottom.3. Muscle biopsy should be sectioned in both
transverse and longitudinal planes).

Automatic tissue processor

It has 2 advantages

1. Transferring the tissue mechanically from one reagent to another can be done
both by day and night.

2. Reduces processing time by the action of continuous agitation. This eliminates

the possibility of human errors of leaving the tissue for long time in one solution
due to forgetfulness.

VARIOUS PARTS OF THE MACHINE :

(a) Tissue containers - These are also the cassettes. The tissue to be processed is
placed in an appropriate container, together with a label and the lid snapped on.
These containers are placed in the tissue basket in which they remain throughout
the whole process.

(b) Beakers and wax baths - Most machines are equipped with ten beakers and 2
wax baths thermostatically controlled at 56°C + 4°C.The beakers are filled with

35
appropriate fluids and wax is placed in the wax baths after ensuring that main
switch is on, so as to keep the wax in molten state.

(c) Stirring mechanism - The basket is attached to the arms of the machine on
which one arm is designed in such a manner so as to bring about the rotation of
the basket nearly at the rate of one revolution per minute.

(d) Timing mechanism - Timer is meant to keep the tissue in different reagents
and wax for an optimum time. If kept for longer or shorter period than necessary,
tissue will not be adequately processed.

Points to noted

1. Fluid and wax beakers must be filled up to appropriate mark and located in
their correct position in the machine.

2. Any spillage of the fluid should be wiped away.

3. Accumulations of wax must be removed from beaker, covers, lids and

surrounding areas.

4. Wax bath thermostats should be set at satisfactory levels usually 2-3°C above
the melting point of wax.

5. Particular attention should be paid to fastening the processing baskets on the

crousel type of machines, if the baskets are shed they will remain in one particular
regent for a long period till it gets noticed.

6. Timing should be set with utmost care when loading the machine.

7. Paraffin wax baths should be checked to ensure that the wax is molten.

36
Automated processing schedule

1. 80% alcohol (holding point) 1 hours,

2. 95% alcohol 2 hours.
3. 95% alcohol 1 hour.
4. 100% alcohol 1 hour.
5. 100% alcohol 1 hour.
6. 100% alcohol 1 hour.
7. Chloroform 1 hour.
8. Chloroform 1 hour.
9. Chloroform 1 hour.
10. Paraffin wax 2 hours.
11. Paraffin wax 2 hours.
12. Paraffin wax 2 hours.
Note :
• Keep watch on paraffin temperature
• Tissue should not be left in any solution for a longtime
• Frequent filtration and changes of solution are needed

Schedule for hard & delicate tissues

1. 80% alcohol two changes 1 hour each.

2. 95% alcohol two changes 1 hour each.
3. Absolute alcohol three changes 1 hour each.
4. Absolute alcohol and xylene equal parts 1 hour.
5. Cedar wood oil 2 changes 2 hours each.

37
6. Cedar wood oil 1 change 1 hour.
7. Paraffin wax one change 2 hours.
8. Paraffin wax four changes 1 hour each, Vacuum the last paraffin change.
Embed and cool quickly. Cut as desired.
Processing schedule for skin

1. 70% alcohol 1 hour.

2. 95% alcohol 2 hours.

3. Absolute alcohol 2 hours.

4. Absolute alcohol 3 hours.

5. Cedar wood oil over night.

6. Xylene 20 minutes.

7. Wax I 3 hours.

8. Wax II overnight.

38
 SECTION CUTTING

Introduction:
To master in the art of good section cutting it is required
1. To have a thorough knowledge of the equipment used.
2. Quality of equipment.
3. Quality of processing the tissue.
Microtome Knives :
The knife is probably the greatest single factor in producing good sections.
Types of microtome knives :
Microtome knives are classified by the manner in which they are ground and seen
in their cross section.
1. Plane wedge.
2. Plano concave.
3. Biconcave.
4. Tool edge
Plane wedge : It is used for paraffin and frozen sections.
Planoconcave : used for celloidin section since the blade is thin it will vibrate
when used for other harder materials.
Biconcave : It is recommended for paraffin section cutting on rocking and sledge
type of microtome.
Tool edge :This is used with a heavy microtome for cutting very hard tissues like
un-decalcified bone.

39
General description:
In the description of knives the expressions “Heel” and “Toe” are used to indicate
which end of the cutting edge is referred to. The heel of the knife is the angle
formed by the cutting edge and the end of the knife nearest to handle. The “toe”
of knife is the angle formed by the cutting edge and the end of the knife farthest
from the handle.
Sharpening of microtome knives

The cutting edge of an ideal microtome knife is a straight line formed by

intersection of 2 planes, the cutting facets. The angle between the planes is called
the bevel angle and is greater than the wedge angle between the sides of knife.
The standard microtome knife has a wedge angle of approximately 15º and bevel
angel varying between 27 and 32º.
Honing : Grinding of knife on a hone to restore straight cutting edge and correct
bevel. There are various types of hones:
1. Belgian black vein or Belgian yellow It is a yellow stone ½ inch thick and is
backed with a black stone of same thickness. Only yellow side should be used for
honing. It is the best hone. It is quite a fast hone and may be used for coarse
grinding and finishing.

2. Arkansas – Not very fast.

3. Aloxide – Fairly fast but coarse and not good for finishing a knife.

4. Carborundum – These hones can be obtained in a variety of grade sonly the

finest of which should be used that too for coarse work.

5. Plate glass – May be used as a hone by applying an abrasive such as aluminium

oxide to the surface and then using in the same way as ordinary hone.
40
52The advantage of such a hone is that it can be used for all types of honing by
changing the abrasive powder.

Lubricants for hone

1. Soap water. 2. Liquid paraffin. 3. Castor oil. 4. Clove oil

Method of honing

1. The hone is placed on a bench on a nonskid surface.

2. A small quantity of light lubricant oil is poured on the center of the hone and
lightly smeared over the surface.
3. The knife complete with handle and backing sheath is laid on the hone with the
cutting edge facing away from the operator, and the heel in the center of the
nearest end of hone. Correct positioning of the fingers is achieved by holding the
handle of the knife between the thumb and forefinger with the cutting edge
facing away from the operator (so that the thumb in on the back). When the knife
is on the hone the tips of finger and thumb of other hand rest on the other end of
knife ensuring even pressure along the whole edge of knife during honing.
4. The knife is pushed forward diagonally from heel to toe, turned over on its back
and moved across the hone until the heel is in the center with the cutting edge
leading, and then brought back diagonally. It is turned to its original position, thus
completing figure of 8 movement.
5. The process is continued until all jagged edges have been removed. The knife is
ready for stropping.
Stropping: It is the process of polishing an already fairly sharp edge. It removes
burrs formed during honing .Fine quality leather is used leather strops may be

41
either flexible /hanging or rigid. In stropping usually firm surface is preferred.
Action is reverse of honing toe to heel direction of stropping is also opposite.
Assessment of the sharpened knife edge:
Examine the edge of the knife by reflected light and under microscope to assess
the honing and stropping.
Care of the knife
1. Keep the knife covered in the box when not in use.
2. Oil the knife to prevent corrosion.
3. Always clean knife with xylol before and after use.

4. It should always be stropped before use.

5. Knife should be sharpened as and when required.

Automatic microtome knife sharpeners

There are many automatic knife sharpeners available Shandon type Is most
commonly used which consists of a glass plate on which fairly coarse abrasive
powder like alumina powder is applied. First matting is done followed by lapping
to remove all finer scratches. In all stages of use of abrasive powder care must be
taken to remove by thorough washing any traces of abrasive powder from both
knife and plate.
Microtomes
These are mechanical devices for cutting uniform sections of tissue of appropriate
thickness. All microtomes other than those used for producing ultra-thin sections
for election microscopy depend upon the motion of a screw thread in order to
advance the tissue block on knife at a regulated number of microns. Motion of
screws can be direct or through system of gears or levers to magnify the
movement.

42
Types of microtome
1. sliding microtomes.

2. Rocking microtome.

3. Rotary microtome.

4. Freezing microtome (Cryostat).

5. Base sledge microtome.

6. Vibrating knife microtome

7. Ultra microtome.

Paraffin section cutting

Equipment required:

1. Microtome.
2. Water bath preferably thermostatically controlled.
3. Hot plate or drying oven thermostatically controlled.
4. Fine pointed forceps.
5. Small hair brush.
6. Seeker.
7. Scalpel.
8. Clear cloth or paper towel.
9. Slide rack.
10. Clean glass slides.
11. Section adhesive.
12. Fluff less blotting paper.
13. Ice cubes.
14. Diamond marker pencil

43
Water bath: Thermostatically controlled for paraffin wax of melting point56ºC, a
water temperature of 45ºC is sufficient ordinary distilled water is satisfactory;
addition of a trace of detergent to water is beneficial in flattening of sections.
Hot plate or drying oven : Drying of sections at around the melting point of wax is
satisfactory
Brush, seeker, forceps: needed to remove folds and creases in sections after
floating out.
Slides: Majority of sections fit comfortably on a 76 x 25 x 1.2 mm slide.
Diamond pencil: needed to write the identification details like name or specific
number.
Section adhesives: An adhesive is a substance which can be smeared on to the
slides so that the sections stick well to the slides. Most of the tissue sections
which are adequately thin and thoroughly dried without any air bubble trapped
under them do not require an adhesive, as in case of routine H and E staining, but
for histochemical methods requiring alkaline solutions e.g. ammonia tend to
remove sections from slide for such cases adhesive is required. Also adhesive is
required for tissues like brain, spinal cord, blood clot, decalcified tissues which
have a tendency to detach themselves from the slide. Tissue impregnated with
ester wax also require section adhesive.
Types of adhesive

Albumin, Gelatin, Starch, Cellulose, Sodium silicate Resin, Poly L Lysine Adhesive
are either added to water bath or smeared thinly on slide.

1. Agar – add 50 ml of melted agar to water bath.

2. Gelatin – Add 30 ml of melted gelatin to water bath.

3. Mayer’s glycerol albumin.

44
This is the most popular adhesive for routine use :

1. Fresh egg white 50 ml.

2. Glycerol 50 ml

3. Sodium salicylate 1 ml Mix and agitate the ingredients filter through coarse
filter paper smear fluid over the slide. This fluid may be diluted 1:20 with distilled
water and section floated on the fluid, while manipulating the albuminized slide
under water in the floatation bath to pick up the section, avoid dipping the entire
slide as the albumin may wash off.
Section cutting of paraffin embedded tissue

Fixing of block

1. Fix the block in the block holder and the microtome knife in such position that
it will be clear of the knife when it is in position, block may be fixed directly or it
may be fixed to a metal carrier which in turn is fixed to the microtome.

2. Insert the appropriate knife in the knife holder and screw it tightly in position.
Adjust if required. The clearance angle should be set at 3-4degree and angle of
slope should be set permanently at 90 degree. It is important to tighten the knife
clamp screw securely and block clamp screws most also be firm. The exposed
ends of the knife must all the times be protected by magnetic or clip on knife
guards to avoid any accidents.

3.Trimming of tissue block :

Move the block forward so that the wax block is almost touching the knife. To
trim away any surplus wax and to expose a suitable area of tissue for sectioning,
the section thickness adjusters are set at 15 microns.

45
4. On exposing a suitable area of tissue the section thickness is set to the
appropriate level for routine purposes to 4-6 microns.

5. Apply ice to the surface of the block for a few seconds and wipe the surface of
block free of water. This step is optional but makes sections cut easily.

6. Note that the whole surface of the block will move parallel to the edge of the
knife in order to ensure a straight ribbon of sections.

7. The microtome is now moved in an easy rhythm with right hand operating the
microtome and left hand holding the sections away from the knife. The ribbon is
formed due to the slight heat generated during cutting, which causes the edges of
the sections to adhere. If difficulty is experienced in forming the ribbon it is
sometimes overcome by rubbing one of the edges of the block with finger.

8. During cutting the paraffin wax embedded sections become slightly

compressed and creased. Before being attached to slides the creases must be
removed and the section flattened. This is achieved by floating them on warm
water. Thermostatically controlled water baths are now available with the inside
coated black. These baths are controlled at a temperature 4-6ºC below the
melting point of paraffin wax. It is easy to see creases if the inside of water bath is
black.

9. The action in floating out must be smooth with the trailing end of ribbon
making contact with water first to obtain flat sections with correct orientation,
floating out with the shiny surface towards the water is essential. When the
ribbon has come to rest on water the remaining wrinkles and folds are removed
by teasing apart by using forceps or seeker.

46
10. Picking up sections

The ribbon of sections floating on water is split into individual or groups of

sections by use of forceps or seekers. Picking up a section on slide is achieved by
immersing the slide lightly smeared with adhesive vertically to three fourths of its
length bringing the section in contact with the slide. On lifting the slide vertically
from the water, the section will flatten on to the slide. The sections are then
blotted lightly with moistened blotting paper to remove excess water and to
increase contact between section and slide. For delicate tissues or when several
ribbons of sections are placed on the slide, omit the blotting instead keep the
slide in upright position for several minutes to drain.

11.Drying of section :

Sections are then kept in incubator with a temperature 5-6ºC above the melting
point of wax i.e. at 60ºC for 20-60 minutes. It is better to overheat than under
heat. If the sections are not well dried they may come off during staining. The
sections should not be allowed to dry without a good contact with the slide ,such
sections will come off during staining.

SOME USEFUL HINTS IN SECTION CUTTING

Methods of removing bubbles trapped beneath the sections:

Bubbles may get trapped under a section while in the tissue flotation bath. These
need to be removed before the section are picked on the slides this may be done
by:

1. With the edge of slide.

2. Can be teased out with bent dissecting needle.

47
3. Place the sections on slide and run 2% alcohol under them. Any folder bubbles
will be removed.

To cut a tissue which has a tendency to crumble or fragment while cutting. With
the mouth open and sounding a soft long drawn 'H' thus 'h h h h h ' exhale gently
on to the section as it leaves the knife and cut very slowly. This also helps to
reduce the effect of static electricity. If sections fragment due to large amount of
blood in tissue, the block should be coated with celloidin between sections. The
surface of the block should be wiped dry, and painted with a camel hairbrush
which has been dipped in 1% Celloidin. After allowing few seconds for the
Celloidin to dry a section is cut in usual way. It must be remembered that when
floating the sections to remove the creases, the celloidin layer must be
uppermost, and the water should be a little hotter than usual to counteract the
effect of celloidin. Following drying in usual way, the cellodin is removed with
equal parts of ether and alcohol before removing wax with xylol.

Serial sectioning:

Serial sectioning may be needed to study the track of some structures or to find
the extent of a lesions. Sections are collected from the very first cut that includes
any tissue. Ribbons of ten - 1-10, 11-20, 11-20, 21-30 so on are picked up and
mounted on the slides.

Step sections:

This is an alternative for serial sections and for the same reason. Sections are
taken at periodic level through the block.

48
Cooling block and knife:

In general keep ice cubes ready at hand and cool the surface of block and knife
before cutting.

Trouble shooting for poor sections:

Main reasons are either:

 Faults occurring during section cutting or

 Faults due to poor processing.

Below are given the various defects, reasons for the defect and the remedy for
the same.

Faults in cutting

1. Fault - Tear or scratch across part of section, Cause - Calcium, Carbon, or Suture
etc., in the tissue or wax Remedy- Examine block under magnifying glass. If
calcium is present, decalcify block. Remove suture from the tissue with scalpel
point. If dust is in wax - Re-embed

2. Fault - Holes in the section. Cause - Air bubbles in the tissue or wax A piece of
hard material in tissue A soft piece of tissue in block Remedy- Re-embed Remove
hard material if possible Reprocess specimen

3. Fault - Cracks across the section parallel to knife Cause - A blunt knife Knife tilt
too small. Block too hard for thickness of specimen Remedy- change knife Adjust
tilt Warm block slightly or re-embed in soft wax.

4. Fault - Section shows thin and thick horizontal lines (chatters) Cause - A loose
knife A loose block A blunt knife Extremely hard tissue Remedy- Tighten knife

49
and/or block change the knife Soften the tissue if possible or embed in harden
wax.

5. Fault - section cut thick and thin alternative Cause - Knife tilt is too great and is
compressing the block Remedy Adjust tilt.

6. Fault - Section compress at one end. Cause - Blunt spot on the knife A soft spot
in the wax, due to presence of clearing agent Remedy- Move block along the knife
or change knife. Re infiltrate tissue and re-embed

7. Fault - Section curves to one end. Cause - Edge of block is not parallel to knife.
A dull spot on knife. Remedy- Trim edges Move block along knife or change knife.

8. Fault - Section curl as they are cut Cause - Blunt knife Sections too thick Too
much tilt to knife Remedy- change knife Adjust microtome Correct the tilt

9. Fault - Sections lift from knife on upward travel of block Cause - Blunt knife Too
much tilt to knife A buildup of wax debris behind knife A greasy knife. Remedy-
change knife Correct the tilt Clean the knife

10. Fault - Knife bites deeply into block Cause - A loose knife A loose block
Remedy- Tighten the knife and block.

11. Fault - The block no longer feeds towards knife Cause - Forward feed
mechanism had expired Remedy- Release the safety locking catch, man back off
feed mechanism and readjust knife holder

12. Fault - Sections crumble on cutting Cause - Knife is blunt Wax is too soft; has
crystallized due to slow cooling or contamination with water or clearing agent.
Defective processing e.g. incomplete fixation, dehydration, clearing or

50
embedding. Remedy- change knife .Re-embed and block with fresh wax
Reprocess

13. Fault - Failure of block to ribbon Cause - Block not parallel to knife Paraffin too
hard. Knife tilted too much Sections too thick Remedy- Correct the alignment Re-
embed Correct the tilt Adjust the section thickness.

Fault due to poor processing

1. Fault - The tissue is shrunken away from wax Cause - Insufficient dehydration
Remedy- Reprocess

2. Fault - The tissue is too soft when block is trimmed Cause - Insufficient fixation
Remedy- Reprocess

3. Fault - Specimen crumbles and drops out of the wax leaving a rim of wax as a
section Cause - Insufficient infiltration Overheated paraffin bath causing tissue to
become hard and brittle Remedy- Re infiltrate and re-embed Service the paraffin
bath

4. Fault - Tissue is dried out or mummified Cause - Mechanical failure of tissue

processing machine or a basket was out of balance and hung up. Remedy- Place
the specimen in the following rehydration solution for 18-24 hrs. Sodium
Carbonate - 1.0 gm. Dist. Water - 70.0 ml Absolute ethyl alcohol - 30.0 ml Re
hydrate the reprocess.

51
 STAINING

The sections, as they are prepared, are colorless and different components
cannot be appreciated. Staining them by different clouded dyes, having affinities
of specific components of tissues, makes identification and study of their
morphology possible. Certain terminologies used in the following account are
given below.

Basophilic: Substances stained with basic dyes

Acidophilic: Substances stained by acid dyes

Vital staining: Staining of structures in living cells, either in the body (in vivo) or in
a laboratory preparation (in vitro). e.g. Janus green is taken up by living cells and
stains the mitochondria.

Metachromatic staining: There are certain basic dyes belonging to aniline group
that will differentiate particular tissue components by staining them a different
color to that of original dye. The phenomenon is known as metachromasia. The
tissue elements reacting in this manner are said to be exhibiting metachromasia.
The generally accepted explanation of this phenomenon is that change in color is
due to polymerization. Sulfated substances are highly metachromatic e.g. Mast
cell granules. These contain Heparin which is highly sulfated. Some of the
common metachromatic dyes are :Methylene blue Methyl violent Thionin Crystal
violent Toluidine blue Thionin and toluidine blue dyes are commonly used for
quick staining of frozen selection using their metachromatic property to stain

52
nucleus and cytoplasm differently. Tissue components often demonstrated by
metachromatic stains :Amyloid material, Mast cell granules Mucin Cartilage

Direct staining: Application of simple dye to stain the tissue in varying shades of
colors.

Indirect staining: It means use of mordant of facilitate a particular staining

method or the use of accentuator to improve either the selectivity or the intensity
of stain.

Progressive staining:

Stain applied to the tissue in strict sequence and for specific times. The stain is
not washed out or de-colorized because there is no over staining of tissue
constituents. Staining is controlled by frequent observation under microscope

Regressive staining:

Tissue is first over stained and then the excess stain is removed from all but the
structures to be demonstrated. This process is called differentiation and should
always be controlled under microscope.

De-colourization:

Partial or complete removal of stain from tissue sections. When the color is
removed selectively (usually with microscopic control) it is called differentiation.
In case decolourization is to re stain the selection with some other stain, acid
alcohol treatment is the method of choice.

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Differentiation:

In regressive staining differentiation is the removal of washing out of the excess

stain until the colour is retained only in the tissue components to be studies.

Impregnation:

It is the deposition of salts of heavy metals on or around cells, tissue constituents

etc. It has followed characteristics
1. Structures demonstrated are opaque and black
2. The colouring matter is particulate
3. The deposit is on or around but not in the element so demonstrated.
Histochemical staining:

Staining which is used to indicate the chemical composition of the tissue or

cellular elements.

Counter stains:

A counter stain is the application to the original stain, usually nuclear, of one or
more dyes that by contrast will bring out difference between the various cells and
tissues. A heavy counterstain is to be avoided lest it mask the nuclear stain. It can
be done either by using dilute stain or cutting down the staining time. Some
counterstains which are acidic may lighten or remove the nuclear stains.

Mordants:

Substance that causes certain staining reactions to take place by forming a link
between the tissue and the stain. The link is referred as lake. Without it, dye is not
capable of binding to and staining the tissue. e.g. Ammonium and Potassium alum
for haematoxylin.

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Accentuators:

These are substances that causes an increase in the selectively or in the staining
power of dye. Thus they lead to more intense staining. e.g. Phenol in Carbol
fuchsin, KOH in Mehtylene blue.

Leuco compounds:

Conversion of a dye into a colourless compound by the destruction of its

chromophore. Prefix leuco is applied to it, e.g. leucofuchsin used in PAS stain.

Dyes used in staining:

Dyes are classified in various ways :

 According to source.
 According to affinity to tissues.
 According to chemical composition.

Natural dyes

These are very few in numbers. They are mainly two in common use.

1. Haematoxylin

This is the most popular dye used as a nuclear stain. It is derived from the log tree
mainly found in Mexico. It develops staining property after oxidation. It is a weak
dye and to make it give sharp stain a mordant is needed.

2. Carmine

It is a scarlet dye made from the ground bodies of cochineal beetles.

Synthetic dyes

Most of these are Aniline base and derived from coal tar. These aniline dyes offer
wide range of colour and action. Chemical composition may be basic, acidic,

55
amphoteric (neutral). According to these characters stain different components of
tissue.

Basic dyes

These are cationic dyes and stain nuclei, basophilic granules or bacteria.

Acidic dyes

These are anionic dyes and stain mainly cytoplasm, eosinophilic granules.

Theories of staining

Physical theories :

1. Simple solubility e.g. Fat stains are effective because the stain is more soluble
in fat than in 70% alcohol.

2. Adsorption: This is a property by which a large body attracts to itself minute

particles from a surrounding medium.

Chemical theories:

It is generally true that acid dyes stain basic elements (Cytoplasm) and basic dyes
stain acidophilic material (nucleus) however this far from being complete truth,
Indeed hematoxylin, which is an acid dye, does not stain the cytoplasm, but (in
the presence of mordant) is one of the most widely used nuclear stains.

Staining of paraffin section:

The most common method of histological study is to prepare thin sections(3-5

micron) from paraffin embedded tissues. These are then suitably stained and
mounted in a medium of proper refractive index for study and storage.
Commonest mountants used are resinous substances of refractive index close to
that of glass. These are soluble in xylol. Hence sections are dehydrated and
cleared in xylol and mounted. Mounting in aqueous mounting media is done

56
directly after staining for sections which cannot be subjected to dehydrating and
clearing agents. The basic steps in staining and mounting paraffin sections are as
follows:

1. Deparaffinization 2. Hydration3. Removal of mercury pigments wherever

needed 4. Staining 5. Dehydration and clearing 6. Mounting

1. Deparaffinization

Removal of wax is done with xylol. It is essential to remove the wax completely,
otherwise subsequent stages will not be possible. At least 2 to 3changes in xylol
are given for suitable length of time. Sections at this stage should appear clear
and transparent. Presence of any patches indicates the presence of wax and
sections should be kept longer in the xylol.

2. Hydration

Most of the stains used are aqueous or dilute alcoholic solutions. Hence it is
essential to bring the section to water before the stains are applied. The
hydration is done with graded alcohols from higher concentration to lower
concentration. Alcohol and acetone are miscible with xylol. First change is made
to absolute alcohol or acetone followed by 90%, 70% alcohol and finally distilled
water. Sections now should appear opaque. Presence of any clear areas are
indicative of the presence of xylol. To remove this xylol, sections should be
returned to absolute alcohol and rehydrated.

57
3. Removal of (mercury formalin) pigments wherever needed

In case mercury containing fixatives e.g. Zenker, Susa etc are used, mercury
pigments are precipitated on the sections. It has to be removed before staining is
done. This is brought about by treatment with iodine solutions which changes
mercury to an iodine compound. This in turn is converted to tetra-thionate by
thio-sulphate, which is readily soluble in water. The slides are placed in running
water to wash out all extraneous chemicals.

4. Staining

Various staining procedures are applied from this hydrated stage. The most
common stain applied for histological study is Haemotoxylin and Eosin. Various
types of haemotoxylin formulations are used. Certain of the stains use strong
chemicals e.g. ammonia. Sections tend to float off the slides in such stains. This
can be prevented by coating the sections by a thin layers of celloidin. For this
sections are returned to absolute alcohol and then dipped in a dilute solution of
celloidin and finally hardened in 70% alcohol. Washing and rinsing of tissue
sections is a necessary part of most staining techniques. It eliminates carrying
over of one dye solution to the next. Excess dye, mordants, or other reagents
might react unfavourably or precipitate when placed in the fluid employed in the
next step.

5. Dehydration and clearing

Dehydration is done is graded alcohols or acetones from 70% to absolute alcohol

or acetone. Dehydrating alcohol and acetones can remove some of the stains.
Time has to be suitably modified to minimize fading of stains. Since alcohol and

58
acetone are miscible in xylol, it is used for clearing the sections. Any sections from
which water has not been completely removed would give a milky appearance
after the first xylol. Such sections should be returned to absolute alcohol and the
process repeated. Mounting is done after 2nd or 3rd xylol.

6. Cover slipping and mounting

Make quite sure that the sections are quite clear. Do not let the section go dry
before mounting.

1. Hold the slide between the thumb and the forefinger of one hand and wipe
with a clean cloth both ends of the slides. Look for the engraved number to make
sure the side the sections is present.

2. Clean carefully around the section and lay on a clean blotting paper with
section uppermost along with appropriate coverslip which has already been
polished.

3. Place a drop of mountant on the slide over coverslip. Amount of mountant

should be just enough. Invert the slide over the coverslip and lower it so that it
just adheres to the cover slip quickly turn the slide over, then lay it on a flat
surface to allow the mountant to spread. Do not press or push the slide at all. It
can damage the section.

4. After the mountant has spread to the edge of the coverslip wipe around it for
neatness. If proper care has been taken there should be no air bubbles. If many
are present, slide should be returned to the xylol to remove the coverslip. It will
slip off and remounting is done. No attempt should be made to pull the coverslip.

59
Slight warming of the slide from below will make the small air bubbles to escape
from the slide of the coverslip.

5. Coverslip should be in the center of the slide with neatly written label on one
slide. A good knowledge of various mountants and the coverslips is necessary for
proper selection of the procedure.

Mountants

Histological sections which need to be examined for any length of time or to be

stored, must be mounted under a cover-slip. There are two types of mounting
media :

1. Aqueous media - Used for material which is unstained, stained for fat, or
metachroamtically stained.

2. Resinous media - For routine staining.

1. Aqueous media

There are used for mounting sections from distilled water when the stains would
be decolorised or removed by alcohol and xylene, as would be the case with most
of fat stains (Sudan methods). Some stains, e.g. methyl violent, tend to diffuse
into medium after mounting. This can be avoided by using Highman's medium.
Aqueous mountants require addition of bacteriostatic agents such as phenol,
crystal of thymol or sodium merthiolateto prevent the growth of fungi.
Permanent seal :After mounting the cover slip can be ringed by clear nail polish
for storage.
Following are some of the commonly used aqueous mounting media:
1. Apathy's medium : A very useful medium for mounting sections for fluorescent
microscopy.

60
2. Farrant's medium: Recommended for fat stains.
3. Glycerine jelly: An excellent routine mountant for fat stains.
4. Highman’s medium: Recommended with the m61metachromatic dyes
especially methylviolent.

2. Resinous mounting media

Natural or synthetic resins dissolved in benzene, toluene or xylene. These are

purchased readymade. In case they become too viscous they may have to be
diluted with xylene. Following are some of these media.

1. Canada balsam - Natural resin: It is used as 60% resin by weight in xylene. H.&E
stained slides are fairly well preserved but basic aniline dyes tend to fade and
Prussian blue is slowly bleached. Slides take few months to dry.

2. D.P.X. Polystyrene resin dissolved in xylene as a 20% solution. It is most

commonly used.

3. There are many other synthetic resins sold under various tradenames e.g.
Coverbond , H.S.R. (Harlew synthetic Resin),Histoclad , Permount , Pro-Texx .

Criteria of acceptable mounting media:

1. Refractive index should be as close as possible to that of glass i.e.1.5.

2. It should not cause stain to diffuse or fade.

3. It should not crack or appear granular on setting.

4. It should be dry to a nonsticky consistency and harden relatively quickly.

5. It should not shrink back from edge of cover-glass.

6. It should be free flowing and free from air bubbles.

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Cover glasses used in histopathology

Care has to be exercised in selecting cover glasses for mounting, these are
available in variable sizes and thickness and are supplied usually in 10 gm.
packing. Following sizes are commonly available22 x 22 mm 25 x 50mm22 x 30
mm Circular 22 x 40 mm. Cover glass should preferably be the No. 1 thickness
(0.13 - 0.16 mm), but never more than No. 1 ½ thickness (0.16 - 0.19 mm).

Haematoxylin

Haematoxylin as supplied has no staining properties until it has been ripened by

oxidation into haematin. This ripening is achieved by two methods:

1. Exposure of prepared solutions to the air for periods up to 6-8 weeks,

preferably in sun light or

2. Addition of an oxidizing agent such as sodium iodate, potassium permaganate

or mercuric oxide. In this ripening process Haemtoxylin (C16 HI & O6) loses two
hydrogen atoms to become Haematin (C16 H12).Sufficient Haematoxylin should
be left unoxidized in solution, so that natural oxidation can continue. It prolongs
shelf life of the stain.

Blueing

Alum Haematoxylin stains nuclei and red color, which is converted to blue black
color, when the section is washed in weak alkali. Tap water is usually alkaline
enough to produce this color change .

Following may be used for rapid blueing of the sections.

1. 1% Lithium carbonate.

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2. 2% Ammonia (Ammonia Water).

3. Scott's water Sod. or Pot. Carbonate 2 to 3 gm. Magnesium sulphate 20 gm.

Dist. water 1000 ml.

Cytoplasmic stains

• EOSIN (AFIP)Eosin 1% stock Dissolve 1gm of eosin Y water soluble in 20ml of

distilled water and 80ml of 95% alcohol. Eosin working Stock Eosin - 1 Part Alcohol
80% - 3 Parts Add 0.5ml of acetic acid just before use per 100 ml

• Eosin Phloxine (AFIP)Eosin B 1% and distilled water Eosin Phloxine working Stock
eosin 1% in distilled water - 100mlStock Phloxine 1% in distilled water
10mlAlcohol 95% 780mlAcetic acid 4 mlWorking solution to changed weekly

• Nuclear fast red (Kernechtrot) Aluminium sulphate 5 gm. Distilled water 100 ml
Heat and dissolve and cool nuclear fast red 0.1gmDissolved with the aid of heat,
cool and filter. Add a crystal of thymol.

Nuclear stains

• Hematoxylin

Ehrlich's2% haematoxylin in alcohol 100 ml3% ammonium or potassium alum in

distilled water 100mlMix the two above and add the following in order Glycerol
100 mlAcetic aid 10mlKeep the bottle loosely pluggedLet it ripen for 1-3 months

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• Hematoxylin Harris

8310% ammonium or potassium alum in distilled water 100 ml10% alcoholic

hematoxylin 10 ml Bring the alum to boiling point and add the haematoxylin
solution carefully till the solution is deep red. Add 0.5 gm. of red oxide of
mercury, solution becomes become deep purple. Promptly remove the flame and
plunge into ice cold water. This is the most important part. Leave over night at
room temperature and filter. Add 2 or 4 ml of acetic acid before use per 100 ml in
the stain. Note: In place of red oxide of mercury 0.177 gm of potassium
permanganate can be used but should added after cooling the solution and never
while boiling.

• Hematoxylin phosphotungstic acid Mallory'sHaematoxylin

1.0gmPhosphotungstic acid 20gmDistilled water 1000 ml Dissolve haematoxylin
and phosphotungstic acid separately in distilled water with the aid of heat, when
cool, combine the solution and make up to 1 litre with distilled water. Let it stand
for 5-6 weeks before use. Staining time - 12-24 hours Note : Quick ripening may
be done by adding 0.177 gm. of potassium permanganate. However the results
are not so good.

• Weigert's iron Hematoxylin

A. Haematoxylin 1gmAlcohol 100 ml. Let it ripen for a week

B. 30% solution of ferric chloride 4mlDistilled water 100 ml Hydrochroloric acid
(conc.) 1 ml Immediately before use mix equal parts of A&B add B to A and not
vice versa. Staining time 20-30 minutes

64
• Mayer's HematoxylinHematoxylin

1gm Distilled water 1000ml Heat distilled water to 55 to 60°C and add
hematoxylin rotate till dissolved. Ammonium or potassium alum 50 gm. Sodium
iodate 0.20gm (to be weighed exactly)Add the above in order given Citric acid
1gm. Chloral hydrate 50.0 gm. Above must added in order given. Allow to stand
overnight before use. Solution is stable for 6-8 weeks. Staining time 6-8 minutes
to increase after a month.

Some basic rules for staining

1. Keep stains and solutions covered when not in use.

2. After the slides are removed from oven these should be cooled before being
put in xylene.

3. Filter stains before use.

4. Once the slides have been put in the xylene to remove paraffin they should not
be allowed to dry out. Particular care must be taken not to let the sections dry at
the time of mounting as the xylene easily evaporates and if the section dried
before mounting preparation would become useless.

5. Care should be taken that level of any solution used during staining is such as to
cover the slides.

6. Drain the slides well and blot the bottom on filter paper before putting into the
next solution. This is particularly necessary in transferring from 95% to absolute
alcohol and absolute alcohol in xylol.

7. Xylol used to remove paraffin should not get mixed up with the clearing xylol. It
also should be frequently changed as it tends to get saturated.

8. If for blueing an alkali e.g. ammonia has been used, it should be well washed
out. Failure to do that will lead to disagreeably hazy blue colour of nuclei.

65
Haematoxylin and Eosin staining Procedure

♦ Deparaffinize in hot air oven.

♦ Hydrate the section. 3 dips in xylene (2 Min. each)ii) 3 dips in acetone / alcohol
(2 Min. each)iii) In running tap water for 5 Minutes.

♦ Mayer's haemotoxylin for 15 minutes.

♦ Wash in running tap water for 20 minutes

♦ Counter stain with eosin for 2 minutes

♦ Dehydrate the section in 95% and absolute alcohol/ acetone 2changes

(2minutes each).

♦ Clear in xylene 3 changes (2 minutes each)

♦ Mount in DPX

Results

Nucleus – blue Cytoplasm and background - pink

Causes of poor quality of staining:

1. Poor or inadequate fixation of tissue.

2. Over or under-ripened Haematoxylin.

3. Overused or worked out Haematoxylin.

4. Over or under differentiation of haematoxylin.

5. Insufficient blueing following differentiation.

6. Failure to wash blueing agent out of section before counter staining with eosin
(especially when ammonia is used).

66
7. Insufficient differentiation of eosin during washing or dehydration.

8. Insufficient dehydration and clearing of sections.

9. Contamination of stains.

67
References

1. Bancroft, J.D. and Stevens, A.: theory and practice of histological techniques
ed.3, Churchill livingstone inc. 1990. Edinburgh. London,Melbourne and New
York.

2. Lillie, R.D.: Histopathologic technique and practice histochemistry ed.3, New

York, 1965 McGraw Hill Book co.

3. Manual of histologic and special staining techniques ed. 2, New York,1960, The
Blakiston Division McGraw Hill Book Co.

4. Pearse A.G.E.: Histochemistry, ed. 2, Boston 1960, Little Brown andCo.5. H.J.
Conn's Biological Stains (1969) Lille, R.D. 8th edn, Baltimore;Williams and Wilkins.

68