HISTOTECHNOLOGY

INTRODUCTON

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 examiners desk to be thoroughly
examined microscopically to arrive at a particular 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 objective of the subsequent discussions will be to acquaint the staff with their responsibility; the
basic details of tissue handling, processing and staining. 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 and the autopsy room from surgery two types of tissue are
obtained.
1. As biopsy- A small piece of lesions or tumor which in sent for diagnosis before final removal of the
lesion or the tumor (Incisional biopsy).
2. If the whole of the tumor or lesion is sent for examination and diagnosis by the pathologist, it is called
excisional biopsy.
3. Tissues from the autopsy are sent for the study of disease and its course, for the advancement of
medicine.

Types of Histological preparation

The histological specimen can be prepared as
1. Whole mount 2. Sections 3. Smears.

1. Whole mounts- These are preparation entire animal eg. Fungus, parasite. These preparations
should be no more than 0.2-0.5 mm in thickness.

2. Sections- The majority of the preparations in histology are sections. The tissue is cut in about 3-5
mm thick pieces processed and 5 microns thick sections are cut on a microtome. These are then
stained and permanently mounted.

Microtomes are special instruments which have automatic mechanism for cutting very thin sections. To
cut the sections on the microtome; the tissue must be made hard enough to not get crushed. There are
2 methods of hardening the tissues. One is by freezing them and the other is by embedding them in a
hard material such at paraffin wax or gelatin.

3. Smears- Smears are made from blood, bone marrow or any fluid such as pleural or ascitic fluid.
These are immediately fixed in alcohol to presence the cellular structures are then stained. Smears are
also made by crushing soft tissue between two slides or an impression smear in made by pressing a
clean slide in contact with the moist surface of a tissue. By doing this, the cells are imprinted on the
slide and these may be stained for cytological examination.

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Responsibility of a technician

The technician is responsible for

1. Specimen preservation.
2. Specimen labeling, logging and identification.
3. Preparation of the specimen to facilitate their gross and microscopy.
4. Record keeping.

To obtain these aims the following point need consideration.

1. As soon as the specimen is received in the laboratory, check if the specimen is properly labeled with
the name, age, Hospital Registration No. and the nature of tissue to be examined and the requisition
form is also duly filled.

2. Also check if the specimen is in proper fixative. Fixative should be fifteen to twenty times the volume
of the specimen add fixative if not present in sufficient amount.

3. Check if the financial matters have been taken care off.

4. Make the entries in biopsy register and give the specimen a pathology number called the accession
number. Note this number carefully on the requisition form as well as the container. This number will
accompany the specimen everywhere.

5. If the specimen is large inform the pathologist who will make cut in the specimen so that proper
fixation is done. Container should be appropriate to hold the specimen without distorting it.

6. Blocks of tissues taken for processing should be left in 10% formalin at 60°C till processing. These
would be fixed in 2 hours.

7. Slides should be released for recording after consultation with the pathologist.
8. Specimens should be kept in their marked container and discarded after checking with pathologist.
9. Block must be stored at their proper number the same day. Note the blocks have to be kept
preserved for life long. Slides should be stored in their proper number after 3 days. It gives time for the
slides to be properly dried.

FIXATION

Definition 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 facilitates easy manipulation of tissue.

Aims and Effects of fixation

If a fresh tissue in kept as such 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:

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(1)To preserve the tissue in as lf 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.

(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 intensify 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 a 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 be prepared.

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.

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 1.5 cm so that fixative can penetrate the tissue easily

Reagents employed as fixatives (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.
 It denatures the proteins.
 Glycogen is partially preserved hence formalin is not a fixative choice for carbohydrates.

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  Some enzymes can be demonstrated in formalin fixed tissues.
 It neither preserves nor destroys fat. Complex lipids are fixed but have 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 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 either 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 undenatured 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
 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.

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.
Advantage It penetrates well and fixes rapidly.

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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 discolouration 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 tetraoxide - 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.
 It also demonstrates fat when 0.5-2% aqueous solution is used it gives a black colour 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 osimium tetroxide for electron microscopy.

Compound fixatives - Some fixatives are made by combining one or more fixative so that the
disadvantages of one are reduced by use of another fixative.

All these compound fixative have their own advantages and disadvantages. They should be used
judiciously.

Choice of fixative - The choice of fixative depends on the treatment a tissue is going to receive after
fixation 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 instead
alcohol should be the choice of fixative

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 > 4mm thick, so that fixation fluid penetrates from the periphery to the centre of the
tissue. For fixation of large organs perfusion method is used i.e. fixative is injected through the blood
vessels into the organ. For hollow viscera fixative is injected into the cavity e.g. urinary bladder, eyeball
etc.

2. Ratio of volume of fixative to the specimen should be 1:20.

3. 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 2 hours.

Fixatives are divided into three main groups

A. Microanatomical fixatives - such fixatives preserves the anatomy of the tissue.
B. Cytological fixatives - such fixation are used to preserve intracellular structures or inclusion.

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C. Histochemical fixatives: Fixative used to preserve he chemical nature of the tissue for it to be
demonstrated further. Freeze drying technique is best suited for this purpose.

Microanatomical fixatives
1. 10% (v/v) formalin 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 (Lillie : 1965)

(a) Formalin : 10 ml
(b) Calcium acetate 2.0 gm
(c) Water to 100 ml

Specific features
- They have a near neutral pH
- Formalin pigment (acid formaldehyde haematin) is not formed.

Cytological fixatives
Subdivided into
(A) Nuclear fixatives
(B) Cytoplasmic fixatives

DECALCIFICATION
Specific Objective - The aim of the study is to ensure staining of hard bony lesions so that the study of
pathological lesions is possible.

Definition Decalcification is a process of complete removal of calcium salt from the tissues like bone
and teeth and other calcified tissues following fixation. Decalcification is done to assure that the
specimen is soft enough to allow cutting with the microtome knife. Unless the tissues in completely
decalcified the sections will be torn and ragged and may damage the cutting edge of microtome knife.

The steps of decalcification

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 in
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.

Decalcification

Decalcification is effected by one of the following methods.

 Dissolution of calcium by a dilute mineral acid.
 Removal of calcium by used of dilute mineral and along with ion exchange resin to keep the
decalcifying fluid free of calcium.
 Using Chelating agents EDTA.

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  Electrolytic removal of calcium ions from tissue by use of electric current.

The Criteria of a good decalcifying agent are:

1. Complete removal of calcium.
2. Absence of damage to tissue cells or fibres.
3. Subsequent staining not altered.
4. Short time required for decalcification.

Removal of calcium by mineral acids - Acid decalcifies subdivided into-

Strong acid, weak acid.

Strong acid - eg. Nitric and hydrochloric acid.

Nitric acid- 5-10% aqueous solution used.
They decalcify vary rapidly but if used for longer than 24-48 hrs. cause deterioration of stainability
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, acetic 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
1. Brings out fairly rapid decalcification.
2. Nuclear staining in better.
3. But requires neutralization and thorough washing prior to dehydration.

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. Washing 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.

Formalin Nitric acid

Formalin 10 ml
Distilled water 80 ml
Nitric acid 10ml
Nitric acid causes serious deterioration of nuclear stainability which partially inhibited by formaldehyde.
Old nitric acid also tends to develop yellow discolouration which may be prevented by stabilization with
1% urea.

Formic acid sodium citrate method

Procedure
1. Place calcified specimen in large quantities of formic acid-sodium citrate solution until decalcification
is complete (change solution daily for best results).

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2. Wash in running water for 4-8 hours
3. Dehydrate, clear and impregnate with paraffin or process as desired.
This technique gives better staining results then nitric acid method, since formic acid and sodium citrate
are less harsh on the cellular properties. Therefore even with over exposure of tissue in this solution
after decalcification has been complete, causes little loss of staining qualities.

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 in then dehydrated beginning with alcohol.

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.
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
in 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.

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.

For softening of chitin foll procedure gives a satisfactory result.

1. Fix the specimen in fixative of choice.
2. Place the specimen in following solution until complete dechitinized.
Change the solution every two days for best results.

Mercuric chloride - 4 gm
Chromic acid - 0.5gm
Nitric acid (Conc.) - 10.0ml
Ethyl alcohol 95% - 50.0 ml
Distilled water - 200.0ml

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3. Washing running water for 3 hours

4. Dehydrate, clear and impregnate with paraffin.

Wax blocks - The treatment of wax embedded block of hard tissue may be done by soaking in soap
water overnight.

TISSUE PROCESSING
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 crytostat 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
has to be a full proof system.

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 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 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 that precedes it,
and embedding agent which follows it.
4. Embedding

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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.
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

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

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 in inadvisable because this may cause very rapid removal of water
may produce cell shrinkage. 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.

Other dehydrating agents

1. Acetone - It is clear, colourless volatile inflammable fluid.
 It has a rapid action in dehydrating the tissue but produces shrinkage and distortion and
subsequent brittleness to the tissue.
 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 with paraffin 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

3. Isopropyl alcohol
 It is miscible with water and other organic solvents
 It does not harden the tissue like alcohol
 It is expensive
Clearing
Definition - Clearing means appearance of tissue after it has been treated

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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 not rendered translucent.
 It is expensive.
 It is inflammable.
4. Carbon tetrachloride - It has similar properties to chloroform but is cheaper.
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.
 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.

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.

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.
Paraplast - This is mixture of highly purified paraffin and several

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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.

Time of impregnation
Depends on the following 3 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 centre in addition a
thick tissue has more of clearing agent so more changes of wax are necessary to remove it. If an even
small amount 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
overharden 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 2-3 changes are needed.

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
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.
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 layer; 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 is
preferred.
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.

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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.
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 crystalization 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.
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.
3. 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 ARE AS FOLLOWS

(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 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.

 Tissue should not be left in any solution for a long time

 Frequent filtration and changes of solution are needed

SECTION CUTTING

Microtome Knives: The knife is probably the greatest single factor in producing good sections.
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 ragg before and after use.
4. It should always be stropped before use.
5. Knife should be sharpened as and when required.
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

NZITAKERA Page 13 of 16
of microns. Motion of screws can be direct or through system of gears or levers to magnify the
movement.

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

Water bath. Thermostatically controlled for paraffin wax of melting point 56ºC, 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 eg 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 requires section adhesive.

Section cutting of paraffin embedded tissue

Fixing of block
1. Fix the block in the block holder on the microtome knife in such as 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 3-4 degree 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.

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3. Trimming of tissue block: Move the block forward so that he 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.

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 tearing apart by using forceps or seeker.

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 underheat. 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 needs 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.

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3. Place the sections on slide and run 2% alcohol under them. Any fold or bubbles are removed.

STAINING
The sections, as they are prepared, are colourless and different components cannot be appreciated.
Staining them by different coloured dyes, having affinities of specific components of tissues, makes
identification and study of their morphology possible.

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. 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.

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 and Co.
5. H.J. Conn's Biological Stains (1969) Lille, R.D. 8th edn, Baltimore; Williams and Wilkins

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