Histopathology Made Easy

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DEDICATED TO MY FAMILY
AND MY TEACHERS
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TABLE OF CONTENTS
Type Of Samples Obtained In Laboratory .................................................................. 12
Types Of Histological Preparation .............................................................................. 12
Responsibility Of A Technician.................................................................................... 12
Histopathology Laboratory.......................................................................................... 14
Design Of Histopathology Lab .................................................................................... 14
Component Histopathology Lab ................................................................................. 14
Laboratory Hazards And Safety Measures ................................................................. 16
Types Of Hazards Include The Following: .................................................................. 16
Factors Contributing To Laboratory Accidents ............................................................ 16
Safety Precautions ..................................................................................................... 16
Light Microscopy......................................................................................................... 17
Histopathological Techniques ..................................................................................... 20
Fixation ....................................................................................................................... 21
Aims And Effects Of Fixation ...................................................................................... 21
Amount Of Fixative ..................................................................................................... 22
Properties Of Fixatives ............................................................................................... 22
Preparation Of The Specimen For Fixation ................................................................ 25
Microanatomical Fixatives .......................................................................................... 25
Cytological Fixatives ................................................................................................... 28
Cytoplasmic Fixatives ................................................................................................. 29
Histochemical Fixatives .............................................................................................. 29
Specimen Preparation ................................................................................................ 30
Specimen Reception .................................................................................................. 30
Assessment Questions ............................................................................................... 32
Gross Examination Of Tissues ................................................................................... 33
Decalcification ............................................................................................................ 34
The Criteria Of A Good Decalcifying Agents Area. ..................................................... 34
Formic Acid Sodium Citrate Method Procedure ......................................................... 36
Decalcification Of Bone Marrow Biopsy...................................................................... 36
Use Of Ion Exchange Resins ..................................................................................... 36
Chelating Agents ........................................................................................................ 37
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Electrolytic Method ..................................................................................................... 37
Determination Of End Point Of Decalcification ........................................................... 38
Tissue Processing ...................................................................................................... 41
Dehydrating Agents .................................................................................................... 42
Clearing ...................................................................................................................... 43
Impregnation............................................................................................................... 44
Impregnation With Wax .............................................................................................. 44
Properties Of Paraffin Wax ......................................................................................... 44
Points To Be Remembered During Use Of Paraffin Wax ........................................... 44
Time Of Impregnation ................................................................................................. 44
Microtomes ................................................................................................................. 49
Types Of Microtomes:................................................................................................. 49
Trouble Shooting For Poor Sections .......................................................................... 53
Staining....................................................................................................................... 57
Theories Of Staining ................................................................................................... 60
Special Stains ............................................................................................................. 69
Classification .............................................................................................................. 69
Stains For The Detection Of Connective Tissue ........................................................ 69
Connective Tissue ...................................................................................................... 72
Cells............................................................................................................................ 72
Fibers.......................................................................................................................... 72
Carbohydrates ............................................................................................................ 81
Classification Of Carbohydrates ................................................................................. 81
Mucins ........................................................................................................................ 83
Techniques For The Demonstration Of Carbohydrates .............................................. 84
Pigments And Minerals ............................................................................................... 86
Microorganisms .......................................................................................................... 93
Detection And Identification Techniques Of Microorganisms In Tissue ...................... 93
Bacteria ...................................................................................................................... 93
Fungi........................................................................................................................... 94
Viruses........................................................................................................................ 94
Parasites / Protozoa ................................................................................................... 95
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Cytological Techniques ............................................................................................... 99
Type Of Samples Obtained In Laboratory .................................................................. 99
Sampling Techniques In Cytology .............................................................................. 99
Cytological Staining Techniques ................................................................................. 102
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PREFACE
Most people do not know what a Histotechnologist is or does. Histotechnologist fight
against disease, also he is a vital and critical link in the chain of medical research.
Basically, the responsibility of the Histotechnologist is to prepare sections of human
tissues for microscopical examination by a pathologist in the interest of diagnosing
disease and conducting medical research. In many cases, Histotechnologist are
compelled to work under extreme pressure such as when a body tissue from a patient in
surgery needs to be diagnosed immediately.
The purpose of this study guide and workbook is to introduce theory and histopathological
techniques as well as to provide with an organized tool to understand laboratory aspects
of Histopathology using an interactive format of lab quizzes.
The lack of sufficient books in the histopathology has always been a problem in laboratory
technologists. Hence, the authors hope that this book would be immensely useful in
solving this existing problem at significant level.
I hope it will prove to be a valuable to the many types of reader in the future.
Despite the many advances in the subject and the profession, the title under this book be
published is Practical Histopathology Made easy for laboratory professionals.
Shafie Abdulkadir Hassan
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INTRODUCTON
Histopathology- 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 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.
Figure 1 - Cells, Tissue, Organs, Organ Systems and Organism
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Type Of Samples 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.
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.
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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.
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HISTOPATHOLOGY LABORATORY
Histopathology laboratory is a unit performing clinical activity associated with diagnosis
during and post-surgery. The lab examines tissue materials, based on which an
emergency diagnosis is needed during surgery (i.e. Gefrier or frozen section procedure),
and then, based on the result, clinicians determine the range of the surgery.
Most often this occurs when there is suspicion of a malignant tumor. If the tumor is
confirmed, a radical surgery is carried out, avoiding making two interventions which
would significantly traumatize the patient.
Design of Histopathology Laboratory

• It should be consist of three rooms, each one prepared with proper ventilation,
good light, proper distribution of equipment and proper storage of chemicals
and reagents.
• It should be prepared with suitable furniture.
Component of Histopathology Laboratory

I. Equipment and apparatus: include the following:-
• Microtome: Use to cut section.
• Hot air oven: Use for impregnation of section
• Tissue processing machine: Use for tissue processing.
• Water bath: A laboratory water bath is used to heat samples in the lab
• Wax dispenser: Use for embedding the section
• Cryostat: Use to cut the frozen section
• Centrifuge: a machine with a rapidly rotating container that applies centrifugal
force to its contents, typically to separate fluids of different densities
• Microscope: A microscope is an instrument used to see objects that are too
small to be seen by the naked eye.
• Safety cabinet: Is an enclosed, ventilated laboratory workspace for safely
working with materials contaminated with pathogens requiring a defined
biosafety level.
• Electronic balance: is a balance use for weighting powders
II. Ware: general term use for glass and plastic materials which includes tissue
box, mold, bottles, specimen containers, volumetric glass and cylinder...etc.
III. Chemicals and reagents: It include xylene, alcohol, etc.
IV. Dyes: such as haematoxylin, eosin, alcian blue. etc.
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Cryostat Wax dispenser
Tissue processing machine Floating bath
Microtome 11
Oven
LABORATORY HAZARDS AND SAFETY MEASURES
The Occupational Safety and Health Administration (OSHA) is the governmental agency
responsible for workplace safety
Types of hazards include the following:

A. Biological Hazard: Infectious agents, including airborne or blood borne
organisms such as bacteria and viruses.
B. Physical Hazard: Wet floors, heavy lifting (e.g., boxes and patient transfers).
C. Electrical Hazard: Dangerous high-voltage equipment
D. Chemical Hazard. Toxic, flammable, eg. Alcohol, HCL.
Factors contributing to laboratory accidents

A poorly designed laboratory and overcrowding can increase the risk of accident
occurrence. Most lab, accidents are the result of bad lab. Practices like:
• Poor training;
• Lack of concentration;
• Noisy environment;
• Untidy working and not using racks to hold sample
Safety precautions
• Most of the equipments present in this laboratory are functioning 24x7 days.
Electrical connections should be checked before leaving the laboratory every
day.
• Many chemicals are inflammable, hence care should be taken to avoid any
fire hazard.
• Fire extinguisher should always be available.
• Minimum inflammable substances should be kept in the laboratory.
Substances like wax, xylene alcohol, and acetone should be stored at a
separate place.
• Some chemicals are carcinogenic or harmful to the skin. Therefore staining
and other work should be performed with the gloves on.
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LIGHT MICROSCOPY
A microscope is an instrument that uses visible light and magnifying lenses to examine
small objects not visible to the naked eye, the word of microscope is combination of two
words; "micro" meaning small and "scope" meaning view.
Function of microscope:
• Magnification: to magnify the object being examined.
• Resolution: to resolve the image seen.
Resolution: The ability to distinguish between two points at short distances from each
other.
Microscope parts:
1. Eye piece (ocular):
» To provide further magnification of image (X10).
» To look at the image of object through it.
2. Eye piece distance scale: To adjust the distance between the two eye pieces.
3. Revolving Nose-piece: It holds the objective lenses.
4. Objective lenses: To produce a magnified image with different magnification.
(X4, x10, x20, x40, and x100).
» X100 objective lens called: Oil immersion objective.
• To calculate the total magnification of the microscope: Total mag. = objective
lens mag. X ocular lens mag.
» The lowest mag. = 4X10 = X40
» The highest mag. = 100X10 = X1000
5. Arm: To hold the microscope and connect its parts together.
Figure. 2.light microscopy
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6. Mechanical stage: To put the slide on it.
7. Stage control: To move the stage right and left, forward and backward.
8. Slide holder: To hold the slide and prevent it from moving.
9. Condenser: To collect the light in a cone shape from the light source to the
object.
10. Condenser knob: Move the condenser up and down.
11. Iris diaphragm: Control the intensity of light that goes to the condenser.
12. Coarse adjustment knob: Move the stage up and down rapidly to get
approximate focusing.
13. Fine adjustment knob: move the stage slowly to get fine focusing.
14. Light source.
15. Power switch.
16. Microscope base: Hold all parts of the microscope.
Bright field microscope:
» Used for stained slide.
» Magnification= X1000
» Resolution= 0.2 Mm
• Working distance: The distance between the objective and the object when the
object is in focus.
• Par focal: When move from one objective lens to another you are still in
approximate focusing.
Other types of light microscope:

1. Dark field microscope:
» The condenser condenses the light on the object or specimen but out of the
objective. The result is dark background and bright object.
» Used: to see the motility of bacteria.
2. Phase contrast microscope:
» Produce contrast between the cell and the background.
» The cells appear darker against a brighter background.
» Used: in examination of wet preparation.
3. Inverted microscope:
» The condenser is above the stage while the objectives below the stage.
» Used: to see the effect of virus on the cells (cell culture flasks).
4. Dissecting microscope:
» It is a simple microscope.
» Used: in mycology to see the plate of fungi + dissecting of insect.
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Non-light microscope:
1. Fluorescent microscope:
» Produce ultra violet light.
» The slides stained with fluorochrome.
» Used: in immunology.
2. Electron microscope (E.M):
» Produce electrons (electron beam).
» Magnification= X100 000- X300 000
» Resolution= 0.0003 Mm
» Used: to see viruses and the cell ingredients.
Figure 3. Parts of light microscopy
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HISTOPATHOLOGICAL TECHNIQUES
Cell structure is most commonly studied in slices of the tissue, called sections that are
thin enough to allow transmission of light or an electron beam.
There are many methods of sectioning tissues, and sometimes particular tissues require
special techniques. The method most widely employed is called the paraffin method.
Although this technique is not universally applicable, e.g. it does not work well with hard
tissues such as woody parts of plants or bones from animals, it does present many
advantages over alternative methods. The necessary reagents are inexpensive, readily
available, and much less toxic to humans than those used in most other techniques
All histological procedures can be divided into a similar series of steps.
A. Paraffin method: these steps are as follows:
1. Fixation: is the preservation of biological tissues from decay due to autolysis
or putrefaction
2. Dehydration: is the removal of water from the tissues.
3. Clearing: is the replacing dehydrating fluids
4. Impregnation: is the saturation of tissue cavities and cells by a supporting
substance
5. Embedding: is the process by which tissues are surrounded by a medium
which when solidified will provide sufficient external support during sectioning
6. Microtomy: is the process by which tissue can be sectioned and attached to a
surface so that microscopic examination can take place
7. Mounting:
8. Staining: is the concept referred to process of stain
9. Cryostat frozen section: When the tissue is frozen the water within it turn to
ice and tissue is firm. The ice acting as the embedding medium. The section
produced either: - Fixed or unfixed
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FIXATION OF TISSUES
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 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
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 intensifies the staining
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character of tissue especially with haematoxylin.
Properties of fixatives
1. Coagulation and precipitation as described above.
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 osimic 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 atleast 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 crosslinkage with them and the tissue
component.
» It denatures the proteins.
» Glycogen is partially preserved hence formalin is not a fixative 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
overhardens 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
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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%
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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.
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
looses 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. Osimium 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.
VII. Acetic acid - It causes the cells to swell hence can never be used alone but
should be used with fixatives causing cell shrinkage
VIII. 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 disadvantage 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
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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.
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 (monohydrate) -0.4 gm
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.
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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
» This is an excellent fixative for the preservation of fine structure phospholipids
and some enzymes.
» It is recommended for combined cytochemistry and electron microscopic
studies.
» It should be used cold (4°C) on fresh tissue.
5. Alcoholic formalin
A. Formalin 10 ml
B. 70-95% alcohol 90 ml
6. Acetic alcoholic formalin
A. Formalin 5.0ml
B. Glacial acetic acid 5.0 ml
C. Alcohol 70% 90.0 ml
7. Formalin ammonium bromide
A. Formalin 15.0 ml
B. Distilled water 85.0 ml
C. Ammonia bromide 2.0 gm
Specific features :
» 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
» Excellent fixative for routine biopsy work
» Allows brilliant staining with good cytological detail
» Gives rapid and even penetration with minimum shrinkage
» Tissue left in its for over 24 hours becomes bleached and excessively hardened.
» Tissue should be treated with iodine to remove mercury pigment
9. Zenker's fluid
A. Mercuric chloride 5gm
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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
» Good routine fixative
» Give fairly rapid and even penetration
» It is not stable after the addition of acetic acid hence acetic acid (or formalin)
should be added just before use
» 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
» It is excellent microanatomical fixative
» Excellent fixative for bone marrow spleen and blood containing organs
» As with Zenker's fluid it is necessary to remove excess dichromate and
mercuric pigment
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
» enhance immunoreactivity with antiimmunoglobulin antiserum.
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
C. Glacial acetic acid 25ml 5 ml
Specific features
Penetrates rapidly and evenly and causes little shrinkage
» Excellent fixative for testicular and intestinal biopsies because it gives very
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good nuclear details, in testes is used for oligospermia and infertility studies
» Good fixative for glycogen
» It is necessary to remove excess picric acid by alcohol treatment
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 in 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 cytoplasmic elements.
» It in 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.
24
Specific features
» Devised for fixation of chromosomes
» It fixes and preserves mucopolysacharides. Fixation in complete in 12-18
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 Calcium
Fixation in formal saline followed by postchromatization gives good cytoplasmic fixation.
Histochemical fixatives
For a most of the histochemical methods. It is best to use cryostat. Sections are rapidly
frozen or freeze dried. Usually such sections are used unfixed but if delay is inevitable
then vapour fixatives are used.
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
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 esp.
phosphates. (3) Absolute alcohol for 24 hours. 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 and 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
25
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, osimium tetraoxide and picric acid
particularly need to be washed thoroughly with water prior to treatment with alcohol (for
dehydration).
Tissue Fixative of choice Time for fixative

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.
Bone marrow biopsy Bouin's fixative in 2½ hours followed by
running washing in running water
overnight
Spleen and blood filled cavities Zenker's fluid 1-6 hours
Lymph node B5 12-18 hours
Mictocondria, phosphatides Carnoy's fluid 1-2 hours
and Nissil substance
Chromosome / cell culture Clarke's fluid 1-2 hours
Specimen Preparation
There are many reasons to examine human cells and tissues under the microscope.
Medical and biological research is under-pinned by knowledge of the normal structure
and function of cells and tissues and the organs and structures that they make up. In
the normal healthy state the cells and other tissue elements are arranged in regular
recognizable patterns. Changes induced by a wide range of chemical and physical
influences are reflected by alterations in structure at a microscopic level and many
diseases are characterized by typical structural and chemical abnormalities that differ
from the normal state. Identifying these changes and linking them to particular diseases
is the basis of histopathology and cytopathology.
Specimen reception
Specimens received for histological examination may come from a number of different
sources. They range from very large specimens or whole organs to tiny fragments of
tissue. For example, the following are some of the specimen-types commonly received
in a histopathology lab.
26
• Excision specimens (surgical biopsies), where whole organs or affected areas
are removed at operation
• Incisional biopsy specimens, where tissue is removed for diagnosis from
within an affected area
• Punch biopsies, where punches are used to remove a small piece of
suspicious tissue for examination (often from the skin)
• Shave biopsies, where small fragments of tissue are “shaved” from a surface
(usually skin)
• Curettings, where tissue is removed in small pieces from the lining of the
uterus or cervix
• Core biopsies, where a small tissue sample is removed using a special needle
sometimes through the skin (percutaneously).
Specimens are usually received in fixative (preservative) but sometimes arrive fresh
and must be immediately fixed. Before specimens are accepted by a laboratory the
identification (labelling) and accompanying documentation will be carefully checked,
all details recorded and “specimen tracking” commenced. It is vital that patient or
research specimens are properly identified and the risk of inaccuracies minimized.
Figure 4. A fresh, unfixed specimen after surgical removal. To prevent degeneration or drying-out
the specimen should be fixed as soon as possible.
27
Fixation
Aim
To preserve the cells and extracellular substances of tissues/organs and prevent autolytic
(degenerative) changes,
Principle:
Fixatives cross-link proteins thus maintaining life-like image of the tissue the resulting
coagulation of tissue proteins has a hardening effect on soft tissues. Fixatives also
lock into position a number of carbohydrate- and fat containing macromolecules that
otherwise would be lost during tissue processing
Materials and equipment:

• 10% formalin
• Biopsy or Autopsy
• Cassete (tissue box)
• Wide necked bottle
Procedure:
The fixation process must be started as quickly as possible after removal of the sample.
1. Label the tissue cassettes in pencil.
2. Fill the vial about 2/3 full with the fixative.
3. Remove the organs from your sample.
4. Place the section in tissue cassettes and then into the vial containing 75 ml of
fixative in overnight.
5. Store the vial on the bench top at your work area.
Assessment questions
1. Define fixation:
2. Write principle of fixation
28
GROSS EXAMINATION OF TISSUES
Grossing, often referred to as “cut-up”, involves a careful examination and description
of the specimen that will include the appearance, the number of pieces and their
dimensions. Larger specimens may require further dissection to produce representative
pieces from appropriate areas. For example multiple samples may be taken from the
excision margins of a tumour to ensure that the tumour has been completely removed. In
the case of small specimens the entire specimen may be processed. The tissues selected
for processing will be placed in cassettes (small perforated baskets) and batches will be
loaded onto a tissue processor for processing through to wax.
Figure 5. This surgical specimen of stomach has been fixed in formalin. Slices about 4mm thick
will now be taken from appropriate areas and placed in the labelled cassettes for processing.
29
DECALCIFICATION
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.
A. Dissolution of calcium by a dilute mineral acid.
B. Removal of calcium by used of dilute mineral and along with ion exchange
resin to keep the decalcifying fluid free of calcium.
C. Using Chelating agents EDTA.
D. Electrolytic removal of calcium ions from tissue by use of electric current.
The Criteria of a good decalcifying agents area.

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.
A. 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.
B. 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.
30
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.
6. Nitric acid is the solution of choice for decalcifying temporal bones.
Perenyi’s fluid
10% nitric acid 40.0ml
Absolute alcohol 30.0 ml.
0.5% chromic acid. 30.0 ml.
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 eg.
calcified arteries, coin lesions and calcified glands. Also good for human globe which
contains calcium due to pathological conditions. 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 stainability which partially inhibited by
formaldehyde. Old nitric acid also tends to develop yellow discolouration which may
be prevented by stabilization with 1% urea. Aqueous formic acid
31
90% formic acid 5- 10 ml
Gooding and Stelwart’s fluid.

90% formic acid 5-10ml.
Formalin 5ml
Evans and Krajian fluid
20% aqueous trisodium citrate 65 ml
90% formic acid 35 ml
This solution has a pH of - 2-3
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).
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. This method of choice for all orbital decalcification including
the globe.
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.
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.
32
The resin in 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/10 HCL followed
by three washes in distilled water. Use of Ion exchange resin has advantage of (ii) 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, 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.
(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 in to
addition to the solution.
Decalcifying solution
HCL (Conc.) 80ml
Formic acid 90% 100 ml
Distilled water 1000 ml.
Decalcify with electrolyte apparatus with the above mentioned decalcifying fluid. This
method has no added advantage over any other method.
Neutralization : It has been said that following immersion in mineral acids, tissues
should be deacidified or neutralized, before washing by treatment with alkali. This may
be effected by treatment over night in 5% lithium or sodium sulphate.
Washing : 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)
33
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
3. Washing running water for 3 hours
4. Dehydrate, clear and impregnate with paraffin.
34
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.
Wax blocks - The treatment of wax embedded block of hard tissue may be done by
soaking in soap water overnight.
Decalcification
Aim - The aim of the study is to ensure staining of hard bony lesions so that the study
of pathological lesions is possible.
Principle:
Since calcium is soluble at a pH of 4.5, acids quickly and easily dissolve the calcium
salts.
Materials:
• Take tissue with 4-5 mm thickness
• Forceps
• Decalcifying solution
Procedure
1. The fixative used is 10% neutral formal saline
2. Specimens should be decalcified in hydrochloric acid/formic acid working
solution 20 times their volume.
3. Change to fresh solution each day until decalcification is complete. It may take
24 hours up to days or months depending on size of the specimens.
4. After decalcification , the tissue is transferred to 70% alcohol for dehydration
Result:
• A cloudy solution caused by calcium oxalate indicates that specimen is not
thoroughly decalcified.
• Absence of turbidity indicates completeness of decalcification
35
Determination of end Point of Decalcification
Chemical Test:
Aim: To ensure the complete removal of the calcium.
Principle:
When ammonium oxalate are added to the decalcifying solution. A cloudy solution is
formed when specimen is not thoroughly decalcified this is caused by calcium oxalate
and absence of turbidity indicates completeness of decalcification. Materials
The following solutions are needed to chemically test for residual calcium: 5% Ammonium
Oxalate Stock:
• Ammonium oxalate 5 ml
• Distilled water 95 ml
• Mix well
Procedure:
1. Insert a pipette into the decalcifying solution containing the specimen.
2. Withdraw approximately 5 ml of the hydrochloric acid/formic acid decalcification
solution from under the specimen and place it in a test tube.
3. Add approximately 10 ml of the ammonium hydroxice/ammonium oxalate
working solution, mix well and let stand overnight.
Result
• Decalcification is complete when no precipitate is observed on two consecutive
days of testing.
• Repeat this test every two or three days.
36
TISSUE PROCESSING
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.
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 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.
37
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. 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.
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.
38
Disadvantage: It is more expensive than alcohol.
» It is toxic to man
3. Isopropyl alcohol
» It is miscible with water and other organic solvents
» It does not harden the tissue like alcohol
» It is expensive
Clearing
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 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.
Time 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.
39
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.
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.
Plastic embedding for light microscopy 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.
Paraffin wax is a suitable embedding medium for most tissues, combining adequacy of
tissue support with ease of sectioning on a standard microtome.
40
Dehydration
Aim
To remove water from the tissue block using alcohol so that the water is fully leached out
and replaced with alcohol.
Principle:
The collected tissues, once fixed, are then dehydrated in graded solutions of alcohol or
other dehydrating agents, because a large fraction of the tissue is composed of water, a
graded series of alcohol baths, beginning with 70% alcohol and progressing in graded
steps to 100% alcohol, are used to remove water (dehydration).
• (70% ethanol
• 80% ethanol
• 95% ethanol
• 100% ethanol
• Distilled water
Procedure:
1. Pass your tissue capsules through the following series of solutions.
• (70% ethanol 1hr
• 80% ethanol 1hr
• 95% ethanol 1hr
• 100% ethanol 1hr
1. Define dehydration
2. Write principle of dehydration
41
Clearing
Aim:
To removal of dehydrated agent
Principle:
Dehydrated agent by replacing it with a fluid that is miscible both with the dehydrating
agent used and with the type of embedding medium chosed to make the tissue sample
firm throughout.
Materials:
• Xylene-1
• Xylene-2
Procedure
1. Pass your tissue block through the following solutions
Step Inculpation period
1. Xylene-1 1hr
2. Xylene-2 1hr
1. Define clearing?
2. Distinguish between a dehydration and a clearing
42
Impregnation
Aim
Prior to sectioning, the tissue block must be infiltrated with a material that acts as a
support during the sectioning process.
Principle
During infiltration, the paraffin will equilibrate within the tissue block, eventually
occupying all
Of the space in the tissue.
Materials
• Melted paraffin in metal pitchers
• Oven
• Tissue block
Procedure
1. Fill the vial about full with melted paraffin
2. Pass the tissue block with melted paraffin
3. Allow tissue to equilibrate for 1 hour in an incubator set at 58OC
4. Pour the paraffin into the container labeled for paraffin disposal
5. Repeat step 1 using fresh melted paraffin
6. Take the tissue block from the melted paraffin
7. Go to the next step
1. Define impregnation?
2. What is the purpose of infiltration?
43
Embedding
Aim
Is the saturation of tissue cavities and cells by a supporting substance, embedding tissue
into paraffin blocks supports the tissue structure and enables very thin sections to be cut
and mounted onto microscope slides for analysis.
Principle
When tissues are surrounded by a medium which when solidified will provide sufficient
external support during sectioning
Materials
• Paraffin wax
• Mold
• Tissue block
• Heater
• Metal pitchers
• Petri plate
Procedure
1. Place base-pieces for two embedding molds in a plastic Petri plate – label the
plate along the edge with your name.
2. Decant the paraffin from the second infiltration step into the waste container.
3. Working quickly but carefully, use forceps to transfer the tissue blocks to the
well of separate base mold, snap the base of tissue cassette into the base
mold and then fill the mold with paraffin.
4. Allow the paraffin to solidify at room temperature. If the paraffin begins to
solidify homogeneously around the tissue block, allow the paraffin in the base
mold to melt in the incubator, and then allow it to solidify.
1. Define imbedding?
2. What is the purpose of imbedding of the tissue?
44
MICROTOMES
Microtome: (from the Greek mikros, meaning "small", and temnein, meaning "to cut")
is a tool used to cut extremely thin slices of material, known as sections. Important in
science, microtomes are used in microscopy, allowing for the preparation of samples for
observation under transmitted light or electron radiation.
Microtomy: is a process by which tissue can be sectioned and attached to a surface so
that microscopic examination can take place.
Types of microtomes:
1. Rocking microtome.
2. Rotary Microtomy.
3. Base sledge microtome
4. Ultra-microtome.
5. Hand microtomes – limited for use in botanical sections
6. Freezing microtome
7. Vibrating knife microtome
Microtome knives: The knife is probably the greatest single factor in producing good
sections.
Classified according to:
1. Materials.
A. Steel
B. diamond
C. Glass.
2. Shape.
A. Planoconcave: hallow ground on one side
B. Wedge shaped: plane on both sides.
C. Biconcave: hallow ground on both sides
D. Tool-edge: plane on both sides with a steep cutting edge.
Size of Knives
110 mm Knife - Frozen sections
120 & 185 mm - Routine paraffin block
45
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
require section adhesive.
46
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 – This is the most popular adhesive for routine use :
• Fresh egg white 50 ml
• Glycerol 50 ml
• 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.
Sectioning
Sectioning is accomplished by using a cutting apparatus called a microtome. Aim
The objective is to produce a continuous “ribbon” of sections adhering to one another
by their leading and trailing edges. The thickness of the sections can be preset, and a
thickness between 5 - 10 μm is optimal for viewing with a light microscope.
Principle
The microtome will drive a knife across the surface of the paraffin cube and produce a
series of thin sections of very precise thickness.
Materials
• Water bath
• Container with ice
Glass microscope slides Microtome and blade Oven
Procedure
1. Chill paraffin-embedded tissue blocks on ice before sectioning. Cold wax
allows thinner sections to be obtained by providing support for harder elements
within the tissue specimen. The small amount of moisture that penetrates the
block from the melting ice will also make the tissue easier to cut.
2. Fill a water bath with ultrapure water and heat to 40-45oC.
3. Place the blade in the holder, ensure it is secure and set the clearance angle.
47
The clearance angle prevents contact between the knife facet and the face of the block.
Follow the microtome manufacturer’s instructions for guidance on setting the clearance
angle. For Leica blades this is normally between 1o and 5o (Figure 1).
4. Insert the paraffin block and orientate so the blade will cut straight across the
block.
5. Carefully, approach the block with the blade and cut a few thin sections to
ensure the positioning is correct. Adjust if necessary.
6. Trim the block to expose the tissue surface to a level where a representative
section can be cut. Trimming is normally done at a thickness of 10-30 µm.
7. Cut sections at a thickness of about 4-5 µm (you will probably need to discard
the first few sections as they are likely to contain holes caused by trimming).
8. Using tweezers, pick up the ribbons of sections and float them on the surface
of the water in the water bath so they flatten out. Use the tweezers to separate
the sections.
9. Use microscope slides to pick the sections out of the water bath and store
upright in a slide rack.
10. Place the slide rack into an oven and allow sections to dry overnight at 37oC.
Figure 6. A microtomist creating a “ribbon” of very thin sections for staining
1. Define sectioning?
2. What is the purpose of tissue sectioning?
3. Sectioning is accomplished by using
4. Write the types of microtome?
48
Mounting of sections on microscope slides
It will be apparent during sectioning that the sections are not perfectly flat, but rather
slightly crinkled. This is normal, and the sections will become flattened by floating them
on water held at 45O C
Aim:
To preserve and support a stained section for light microscopy.
Principle:
Mounting the section give reflective index similar to glass, the specimen can be viewed
under a microscope. The solution also contains an adhesive, which causes the
Tissue section to bind to the slide.
Procedure
1. Carefully transfer the sections to a solution held in a 45O C water bath. Within
a few seconds you should see the sections flatten and the wrinkles disappear.
2. Dip a clean microscope slide into the adhesive solution, and slowly pull it
upward, out of the solution, allowing sections to adhere to the surface. Make
sure that the slide is oriented with the label facing upward.
3. Dry the bottom of the slide and carefully blot excess adhesive from around the
sections (be careful not to touch the sections themselves).
4. Allow the slides to dry overnight in the storage box.
1. What is mounting in histopathology?
2. What is mounting medium?
3. Why would you want to wet mount a specimen?
4. What is DPX mounting medium?
49
STAINING
Routine H&E staining and special stains play a critical role in tissue-based diagnosis or
research. By colouring otherwise transparent tissue sections, these stains allow highly
trained pathologists and researchers to view, under a microscope, tissue morphology
(structure) or to look for the presence or prevalence of particular cell types, structures or
even microorganisms such as bacteria.
In the histopathology laboratory, the term “routine staining” refers to the hematoxylin
and eosin stain (H&E) that is used “routinely” with all tissue specimens to reveal the
underlying tissue structures and conditions.
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 nucleus and cytoplasm differently.
Tissue components often demonstrated by metachromatic stains : Amyloid material,
Mast cell granules
Mucin Cartilage
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Direct staining
Application of simple dye to stain the tissue in varying shades of colours.
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 decolorised because there is no overstaining of tissue constituents.
Staining is controlled by frequent observation under microscope
Regressive staining
Tissue is first overstained 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.
Decolourization
Partial or complete removal of stain from tissue sections. When the colour is removed
selectively (usually with microscopic control) it is called differentiation. In case
decolourization is to restain the selection with some other stain, acid alcohol treatment
is the method of choice.
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
51
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.
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 :
1. According to source 2. Affinity to tissues 3. Chemical composition
a. Natural a. Acidophilic a. Thiazines
b. Synthetic b. Basophilic b. Azo-dyes
c. Rosailins
Synthetic dyes have greater staining capacity, much greater spectrum of colours.
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 in Aniline base and derived from coal tar. These aniline dyes offer wide
range of colour and action. These aniline dyes offer wide range of colour and action.
Chemical composition may be basic, acidic, amphoteric (neutral). According to these
characters stain different components of tissue.
Basic dyes
These are cationic dyes and stain nuclei, basophilic granules or bacteria.
52
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 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. Hydration
3. 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 3 changes in xylol are given for
suitable length of time. Sections of 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.
53
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.
3. Removal of mercury 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 tetrathionate by thiosulphate, 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 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.
54
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.
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
merthiolate to prevent the growth of fungi.
55
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: For formulation
see the appendix.
1. Apathy’s medium (R.I- 1.52)
2. A very useful medium for mounting sections for fluorescent microscopy.
3. Farrant’s medium (R.I. 1.43) Recommended for fat stains.
4. Glycerine jelly (R.I. 1.47)
5. An excellent routine mountant for fat stains.
6. Highman’s medium (R.I. 1.52)
Recommended with the metachroamtic dyes especially methyl violent.
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 (R.I. - 1.52)
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. (R.I. 1.52)
Polystyrene resin dissolved in xylene as a 20% solution. It is most commonly used.
3. There are many other synthetic resins sold under various trade names e.g.
Coverbond (R.I. 1.53), H.S.R. (Harlew synthetic Resin), Histoclad (R.I. - 1.54),
Permount (R.I. 1.54), Pro-Texx (R.I. 1.495).
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.
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 packings.
Following sizes are commonly available
22 x 22 mm 25 x 50mm
56
22 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 upto 6-8 weeks, preferably
in sunlight 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.
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
There are many formulations for preparing haematoxylin stains. Use of many is a matter
of personal preference of whether progressive or regressive staining is being used. In
situations where haemotoxylin staining is followed by acidic stains, Iron haemotoxylin is
preferred as it resists decolourisation by these counter stains. Various formulations differ
mainly in regards to mordant and the shorter oxidiser used.
Cytoplasmic stains
» EOSIN (AFIP)
Eosin 1% stock
Dissolve 1gm of eosin Y water soluble in 20ml of distilled water and 80ml of 95% alcohol.
57
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 - 100ml Stock Phloxine 1% in distilled water - 10ml
Alcohol 95% 780ml
Acetic acid 4 ml
Working solution to changed weekly
» Nuclear fast red (Kernechtrot)
Aluminium sulphate 5 gms
Heat and dissolve and cool
nuclear fast red 0.1gm
Dissolved with the aid of heat, cool and filter. Add a crystal of thymol.
Nuclear stains
» Hematoxylin Ehrlich’s
2% haematoxylin in alcohol 100 ml
3% ammonium or potassium alum in distilled water 100ml Mix the two above and add
the following in order Glycerol 100 ml
Acetic aid 10ml
Keep the bottle loosely plugged Let it ripen for 1-3 months
» Hematoxylin Harris
10% ammonium or potassium alum in distilled water 100 ml 10% 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
58
» Hematoxylin phosphotungstic acid
Mallory’s Haematoxylin1.0gm
Phosphotungstic acid 20gm
Dissolve haematoxylin and phosphotungstic acid separately in distilled water with the
aid of heat, when cool, combine the solution and make upto 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. Haematoxyln 1gm Alcohol 100 ml
Let it ripen for a week
B. 30% solution of ferric chloride 4ml Distilled 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
» Mayer’s Hematoxylin
Hematoxylin 1gm
Distilled water 1000ml
Heat distilled water to 55 to 60°C and add hematoxylin rotate till dissolved.
Ammonium or potassium alum 50 gms 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 increased 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
59
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.
Haematoxylin and Eosin staining

Aim
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.
Principle:
Acid base reaction
Procedure
» Deparaffinize in hot air oven.
» Hydrate the section.
I. 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 2 changes
(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.
60
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).
7. Insufficient differentiation of eosin during washing or dehydration.
8. Insufficient dehydration and clearing of sections.
9. Contamination of stains.
Review questions
1. Define the following terms?
A. Dye
B. Stain
C. Auxochrome
D. Chromogen
E. Chromophore
2. Write the different mechanisms of staining?
Microscopy
Aim:
To view the coloured section under microscope

• Mounting section Slide
• light microscope
Procedure:
1. Place the slide under the microscope, and locate the focal plane.
2. Use first an objective lens of 10x, and then 40x magnification.
3. Observe the tissue cytoplasm and nucleus
Result
• Cytoplasm: Pink
• Nucleus: Blue
Review questions
1. Define the following terms?
A. Regressive stain
B. Progressive stain
2. Write the different types of hematoxylin?
3. Write the types of eosin?
61
SPECIAL STAINS
The term “special stains” has long been used to refer to a large number of alternative
staining techniques that are used when the H&E does not provide all the information the
pathologist or researcher needs.
Used in addition to H & E staining to selectively stain cells and cellular components
Used when needed Gives information on:
» Presence of certain class of molecules
» Their localization
» Number of molecules present
Classification
Can be grouped into:
• Stains for detection of microorganisms
• Connective tissues and lipids
• Carbohydrates
• Amyloid
• Minerals, pigments and miscellaneous
Stains for the detection of Connective Tissue

Toluidine Blue
• Used to stain mast cells
• These cells are widely distributed in connective tissue
• Mast cells stain Red-purple (Metachromatic staining) and the background stain
blue (orthochromatic staining)
Van Gieson Stain

• Used to differentiate collagen and smooth muscle
• Can be used to demonstrate the presence of collagen in pathological
conditions
• Stains nuclei blue, Collagen bright red, Cytoplasm, muscle, fibrin and red
blood cells yellow
Elastic Stain
• Used to stain elastic fibers
• Based on the affinity of elastin for hematoxylin complex
• Retains dye longer than other tissues elements
62
• Elastin stains dark brown/ black whereas nucleus stains black.
Gomori’s method
• Used for the identification of Reticular fibers
• Used for the diagnosis of carcinomas, Sarcomas, lymphosarcomas
• Reticulin stains black without any counter stain
Stains for the detection of Carbohydrates

Alcian Blue
• Stains acid mucins and mucopolysaccharides
• Copper in the stain is responsible for the blue stain
• Strongly acidic muco substances stain blue, nuclei pink to red and cytoplasm
pale pink.
Acid-Schiff
• Used to detect glycogen, glycoproteins, mucopolysaccharides, basement
membrane and mucin
• Based on the reaction of the free aldehyde group of monosaccharrides with
Schiff’s reagent
• PAS stains glycogen, mucin, mucoprotein, and glycoproteins magenta. The
nuclei will stain blue. Collagen will stain pink.
Alcian Blue PAS

• Combination of Alcian Blue and PAS technique
• Demonstrates both acidic- neutral and mixtures of acidic and neutral mucins
• Stains acid mucopolysaccharides blue and Neutral polysaccharides magenta
Stains for the detection of Minerals

Von Kossa Stain
• Used for demonstrating calcium or its Salts and is not specific for calcium
• Tissue sections are treated with silver nitrate solution, the calcium is reduced
by the strong light and replaced with silver deposits, visualized as metallic
silver
• Stains Calcium salts black, Nuclei red, Cytoplasm pink.
Stains for the detection of Microorganisms

Gram Staining
• Used to stain both bacilli and cocci
• Basic classification of bacteria are based on this staining
63
• Bacteria with large deposits of peptidoglycan in their cell walls retain methyl
violet and are termed Gram positive Bacteria with large deposits of lipids and
lipopolysacharrides are termed Gram negative.
Acid Fast stain

• Acid fast refers to cell walls containing high lipid content (mycolic acid and long
chain fatty acids)
• These bind to Carbol-fuchsin dye after decolorization
• Used to stain Mycobacteria, oocysts of Cryptosporidium parvum, Cyclospora,
Isospora; also hooklets of cysticerci
• Acid fast cells stain Red and non acid fast cells stain Blue.
Giemsa Staining
• Used for the detection of fungi in tissue sections
• Argentaffin reaction forms the basis for the identification of fungi
• Stains fungi, Pneumocystis carnii, histoplasma spp Black, inner parts of
mycelia and hyphae old rose, leishmania spp, toxoplasma spp negative, mucin
dark grey, background pale green
64
CONNECTIVE TISSUE
It is the group of tissue predominantly composed of intercellular matrix. It supports and
connects the other tissues in the body.
It differs from epithelium in having few cells and large amount of intercellular substance.
Main components of connective tissue are cells, fibers and ground substance.
Cells
• Fibroblasts
• Histiocytes
• Plasma cells
• Mast cells
• Fat cells
• Pigment cells
• Migratory cells
Fibers
• Collagen fibers
• Reticular fibers
• Elastic fibers
Ground substance
It contains proteoglycan and glycoprotein. Proteoglycan are:
• Hyaluronic acid
• Chondriotin sulphate
• Keratin sulphate
• Heparin sulphate.
Connective Tissue Cells:

1. Fibroblasts
» They are large cells with branching processes.
» Nucleus is large with prominent nucleolus.
» Cytoplasm is basophilic in active cells.
» Organelles are clearly visible.
» Their function is the production of fibers and ground substance.
2. Histiocytes
» They are as numerous as fibroblasts.
» They are irregularly shaped cells, with short and blunt processes.
» Nucleus is small and round.
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» Cytoplasm is filled with granules and vacuoles of ingested material.
» Inactive cell is difficult to differentiate from fibroblasts.
» Active cell is large with more prominent nucleus
» Ingested material is in the form of phagosome.
» If the ingested material is large, many macrophages surround it to form foreign
body giant cell.
» Their function is phagocytosis
3. Plasma Cells
» They are ovoid or irregularly shaped cells.
» Nucleus is small and eccentric.
» Chromatin forms clumps along the nuclear membrane, so the nucleus shows
typical cartwheel appearance.
» Plasma cell in blood smear
» Cytoplasm is basophilic due to large amount of rough endoplasmic reticulum.
» Function of plasma cells is the production of antibodies.
4. Mast Cells
» They are large cells with pale nucleus.
» Cytoplasm is filled with basophilic granules, which are not visible in routine
sections as the granules are dissolved in water during tissue processing.
» hese granules contain histamine and a vasoactive substances heparin.
» When stained by toluidine blue (dye) they are stained red, this property is
known as ‘Metachromasia’.
» They are found in skin, wall of GIT, and around blood vessels.
» Their function is to produce heparin, which is an anticoagulant and histamine,
which causes hypersensitivity reaction.
5. Fat Cells
» They are also known as ‘adipocytes’.
» They are usually found in groups.
» Fat cells are large, and filled with fat droplet.
» The nucleus and cytoplasm is pushed to one side of cell.
» In routine histological sections cells appear empty because the fat is dissolved
out.
6. Pigment Cells
» They are satellite cells with long processes.
» They contain numerous dark black pigment granules in their cytoplasm.
» The pigment is synthesized by melanocytes.
» These cells are found in the dermis of skin, especially in dark races, in iris and
choroids of eye.
» Their function is absorption of light.
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7. Migratory Cells
» Migratory cells from blood and lymph are also seen in connective tissues.
» hey include lymphocytes, neutrophil and eosinophils.
Fibers of connective tissue

» Collagen fibers
» Reticular fibers
» Elastic fibers
1. Collagen Fibers
» Collagen fibers are made up of a protein collagen.
» Molecules of collagen are aligned side by side in a staggered fashion with
three quarter of length of each molecule.
» Length of each molecule is 300nm.
» Collagens are of 14 types.
» Type-I is found in dermis, fascia, bone, tendon, ligament, blood vessels.
» Type-II is found in cartilage and viterous body.
» Type-III is found in dermis of skin.
» Type-IV is found in basal lamina.
» Collagen I, II, III, V, and XI form fibrils, others are in dissolved form.
» Type-III collagen when glycosylated, forms ‘reticular fiber’.
2. Reticular fibers
» They are very thin, 0.5-2 µm in diameter usually not visible in H&E stained
sections.
» They are found in loose connective tissue all over the body.
» Reticular fibers form the framework of certain organs, like liver, bone marrow.
3. Elastic Fibers
» They are made up of a protein elastin.
» Elastic fibers are yellow in color.
» The fibers branch and rejoin freely.
» They are thinner than the collagen.
» No banding pattern.
» The fibers are stained by special stain.
» They are stretched easily and then recoil perfectly.
» Elastic fibers are present in lungs, skin and large blood vessels
Classification of Connective Tissue

Connective tissue is classified into two types
1- Embryonic
2- Adult type.
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1. Embryonic connective tissue It is further divided into:
A. Mesenchyme
B. Mucous
» The mesenchyme is the most primitive connective tissue. Here the fibroblasts
are embedded in a jelly like matrix.
» Mucoid tissue is found primarily where the connective tissue is developing
from mesenchyme. It consists of mucoid substance, fine meshwork of type II
collagen fibers and few fibroblasts which are highly branched.
2. Adult connective tissue
It varies from place to place in their appearance and composition according to their
functional requirements and can be subdivided into the following groups:
• Connective tissue proper: – includes loose or areolar, dense, regular and
adipose irregular, reticular.
• Bone
• Cartilage
• Blood
1. Loose connective tissue.
» Loose connective tissue is characterized as being composed of meshwork of
collagen bundles, intermingled with elastic fibers.
» Gel like ground substance and variety of cell types are present.
» This type of connective tissue forms superficial and deep fascia, and part of
framework of most of the organs.
2. Dense connective tissue
» It is either irregularly arranged or it may be regular.
» Dense irregularly arranged connective tissue is composed mainly of collagen
fibers, fibroblasts and macrophages.
» Fibers interlace and form coarse network.
» Examples are dermis of the skin, periosteum and capsule of the testis
» 3- Reticular connective tissue
» It is special tissue, forming the supporting framework of bone marrow and most
lymphoid tissue.
» In contrast to other sites, reticular tissue of thymus is derived from endoderm.
3. Adipose tissue
» It is composed of adipocytes contained in loose areolar tissue.
» All the other cell types are also present.
» Adipose tissue is divided into loculi by fibrous septa, which contain blood
vessels.
» Adipocytes are round or polygonal in shape with signet ring appearance.
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Functions of adipose tissue
» Adipose tissue is the energy storehouse of nutrition.
» It serves as insulation against cold external environment.
» It serves as shock absorber.
» Adipose tissue fills empty spaces in the body.
» In time of nutritional deficits, it releases stored fat.
» In some races it has cosmetic function.
» In new born infants there are areas of specialized adipose tissue called brown
fat, which is related to the temperature regulation.
» Brown fat is also present in adults at few sites in the body.
» In pigmented connective tissue the cytoplasm is filled by a brown or black
pigment, usually melanin.
» This type of connective tissue is found in choroids and iris of eye.
Connective tissue stains: Trichrome stains

» Is selective demonstration of muscle, collagen fibers, fibrin, and erythrocytes.
Factors affecting trichrome staining

1. Tissue permeability and dye molecular size
2. Heat
3. pH
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TECHNIQUES FOR THE DEMONSTRATION OF
CONNECTIVE TISSUES
VAN GIESON METHOD

Aim: Staining of the connective tissue
Principle:
In the routine staining method collagen, elastic fibers and smooth muscle appear pink
or reddish in colour.
In the Van Gieson stain, collagen and most reticulin stain selectively with acid aniline
dyes (acid fuchsin). Picric acid acts as counter stain for muscle and cytoplasm and form
complex with the dyes. This complex has special affinity for collagen.
Reagents
1. Solution A
A. Haematoxylin 1.0 gm
B. Alcohol 95% 100 ml
2. Solution B
A. 29% (w/v) ferric chloride 4 ml
B. Conc. Hydrochloric acid 1.0 ml
C. Distilled water 95.0 ml
3. Weight's iron heamatoxylin solution :mix equal quantities of solution A and
solution B. Colour of this reagent should appear violet black.
4. Van Gieson’s solution
A. Saturated aqueous picric acid – 10 ml
B. 1% (m/v) acid fuchsin – 1.5ml
It should be freshly prepared
Procedure
1. Deparaffinize with xylene
2. Hydration take sections to water
3. Stain with Weigert’s haematoxylin for 20-40 minutes
4. Wash in distilled water
5. Van Gieson stain 1-3 min
6. Rinse well in distilled water
7. Dehydrate in absolute alcohol (2 changes)
8. Clear in xylene (2 changes)
9. Mount in DPX
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Results
1. Collagen - Red
2. Muscle and Cornified epithelium - Yellow
3. Nuclei - Blue to Black
Clinical significance
Several pathological changes are associated with cellular changes in connective
tissues. Histological diagnosis of collagen diseases is based on the study of the section
of various connective tissues.
Verhöeff’s method
Verhöeff’s method is the classical method for elastic fibers and works well after all routine
fixatives.
Aim:
Elastic fiber techniques are used for the demonstration of pathologic changes in elastic
fibers. This include atrophy of the elastic tissue, thinning or loss that may result from
arteriosclerotic changes, and reduplication, breaks, or splitting that may result from
other vascular diseases. The techniques also may be used to demonstrate normal
elastic tissue, as in the identification of veins and arteries, and to determine whether or
not the blood vessels have been invaded by tumor.
Principle
The tissue is overstained with a soluble lake of hematoxylin-ferric chloride-iodine. Both
ferric chloride and iodine serve as mordants, but they also have an oxidizing function
that assists in converting hematoxylin to hemtein. The mechanism of dye binding is
probably by formation of hydrogen bonds, but the exact chemical groups reacting with
the hematoxylin have not been identified. The dye will be attracted to the larger amount
of mordant in the differentiating solution and will be removed from the tissue. The elastic
tissue has the strongest affinity for the iron hematoxylin complex and will retain the dye
longer than the other tissue elements, this allows other elements to be decolorized and
the elastic fibers to remain stained.
Materials
• Any well fixed tissue
• Coplan jars
• Verhoeff’s elastic tissue stain
• Van Gieson's stain
71
Procedure:
1. Deparaffinize and hydrate to distilled water.
2. Verhoeff's hematoxylin for 30 minutes (save solution until stain is completed).
3. Wash in tap water.
4. Differentiate in 2% ferric chloride solution, check microscopically for black
fibers on a gray background.
5. Rinse in water.
6. Hypo for 1 minute to remove iodine.
7. Wash in water.
8. Counterstain in Van Gieson's for 5 minutes.
9. Dehydrate, clear in xylene, and coverslip.
Results
Elastic fibers and nuclei………. black
Collagen………………………. red
Other tissue elements………… yellow
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Johnson’s method for metachromasia (toluidine blue)
Aim:
To demonstrate metachromatic tissue, the toluidine blue stain has a good affinity for
molecules negatively charged like nucleic acids etc. toluidine blue is quick and easy
to apply and is alternative to hematoxylin and Eosin staining. It’s particularly used
by pathologists t observe the abnormal presence of mastocytes due to cancer, an
inflammatory or a gastrointestinal disease like irritable bowel syndrome. It’s also used to
highlight cartilage and mucins composed of Mucopolysachrides. Moreover toluidine blue
staining is an interesting method for the early diagnostic of cancer because DNA labeling
in cancer cells is more intense than in healthy cells.
Principle:
Toluidine blue is a basic (or acidophilic) staining based on the principle of metachromasia
due to molecular properties, the stain intensity varies in different tissue components
depending on their pH, temperature and concentration variations. The components
stained in purple or red are called metachromatic while the other components are
orthochromatic because they keep the blue staining.
Materials
• 0.1% toluidine solution
• Cut paraffin sections at 6 microns
Procedure
1. Deparaffinize and hydrate to distilled water
2. Toluidine blue solution for 2 minutes.
3. Rinse in distilled water.
4. Cover slip section with distilled water.
5. Blot around edge of cover glass and seal with fingernail polish.
6. Examine the sections as soon as possible after preparation.
Results
Metachromatic tissue- pink
Review questions
1. Define metachromatic?
2. Mention uses of toluidine blue?
73
CARBOHYDRATES
Introduction
The word ‘carbohydrate’ is used to encompass a large group of compounds with the
general formula Cn(H2O)n.
Classification of carbohydrates
Carbohydrates are broken down into two broad categories: simple carbohydrates or
those molecules composed purely of carbohydrates, and glycoconjugates, those
molecules composed of carbohydrates and other molecules such as protein or lipid.
The simple carbohydrates are further categorized as monosaccharides, oligosaccharides,
or polysaccharides.
Glycoconjugates may further be broken down into proteoglycans, mucins, and ‘other’
glycoproteins.
Monosaccharide
The most basic or simple form of a carbohydrate is the monosaccharide. Typical
monosaccharides are of the empirical formula (CH2O)n, where n is a value between 3
and 9. The basic monosaccharide is the six-carbon.
Glucose is not charged or ionized and for this reason is referred to as a neutral sugar.
Other neutral sugars include mannose, galactose, and fructose. Monosaccharides
contain asymmetric carbons referred to as chiral centers.
The letters D or L at the beginning of a name refer to the conformation of one of the
chiral carbons within the molecule. The high number of hydroxyl (OH) groups present on
the monosaccharide renders most of them extremely water soluble. Monosaccharides
within a tissue specimen are lost during fixation and tissue processing due to the small
size and the water solubility of these molecules. As a result, the monosaccharides are
not easily demonstrated by most histochemical techniques.
Polysaccharides
A polysaccharide is a large macromolecule composed of multiple monosaccharides
joined by covalent bonds referred to as glycosidic linkages.
Both glycogen and starch consist of glucose units with α1–4 as well as α1–6 glycosidic
linkages, differing only in size and branching structure.
Glycogen is the only polysaccharide found in animals that frequently is evaluated by
histochemical techniques. Glycogen serves as a major form of stored energy reserves
in humans.
74
Carbohydrates absorbed following a meal are converted to glycogen by the hepatocytes
of the liver.
There are a number of disease processes or pathological conditions in which histochemical
assessment of glycogen content or accumulation may be of value diagnostically.
There are several well-characterized glycogen storage diseases which are the result of
inherited defects of one or more of the enzymes involved in the synthesis or breakdown
of glycogen.
In most of these disorders, the liver shows massive accumulation of glycogen. In some
diseases, glycogen accumulation is also observed in skeletal and cardiac muscle.
Connective tissue glycoconjugates
Proteoglycans also are commonly referred to as connective tissue mucins or
mucopolysaccharides. These molecules are large glycoconjugate complexes that are
found in high concentrations within the extracellular matrix of connective tissues.
The carbohydrate components of proteoglycans are known as glycosaminoglycans.
Glycosaminoglycans are large polysaccharide polymers that are covalently bound to
the protein core of proteoglycans. The typical glycosaminoglycan is a long unbranched
polysaccharide composed of repeating disaccharide units each made up of two different
monosaccharides.
The negatively charged sulfate and/or carboxyl groups together with numerous hydroxyl
groupings render most proteoglycans extremely hydrophilic.
This property accounts for the gel-like consistency of the extracellular matrix and
connective tissues such as cartilage.
The proteoglycans of cartilage in particular contain many glycosaminoglycan chains and
thus are capable of binding a large volume of water. Proteoglycans act in stabilizing
and supporting fibrous elements of connective tissue. Tissues that contain high
concentrations of proteoglycans include cartilage, tendons, ligaments, blood vessels,
heart valves, and skin.
Hyaluronic acid is found in high concentrations in synovial fluid and in the ground
substance and connective tissue matrices where other proteoglycans are found.
Several pathological conditions involve accumulation of glycosaminoglycans or
proteoglycans. The mucopolysaccharidoses are a group of genetic disorders that result
from a deficiency of one or more of the enzymes that are involved in the degradation
of heparin sulfate and dermatan sulfate. This results in the abnormal accumulation
of glycosaminoglycans in connective tissues as well as cell types such as neurons,
histiocytes, and macrophages.
75
Mucins
Mucins, like the proteoglycans, consist of polysaccharide chains covalently linked to
a protein core. Typically, the carbohydrate component is attached via an O- glycosidic
linkage to serine or threonine.
Mucins are categorized into functionally distinct families (muc1, muc2, muc3, etc.) based
in part upon differences in the amino acid sequences within the tandem repeats and the
structure of their protein core.
The carbohydrate content of a mucin may account for up to 90% of its molecular
weight. In contrast to the glycosaminoglycans, which are strongly acidic polyanions, the
polysaccharide chains of the mucins vary from neutral or weakly acidic to strongly acidic
sulfomucins.
Function of Mucin
The function of the mucins depends in part upon the tissue location of the mucin-
producing cell as well as the mucin type. In most cases, the secreted mucins provide
lubrication and protection for the secreting cells and/or tissues in the immediate area.
Detection of mucin in a tumor may be a valuable clue in the identification of a malignancy.
Malignancies derived from simple epithelial tissues (carcinomas) frequently contain
detectable mucin. In contrast, melanomas, lymphomas, and sarcomas rarely exhibit
significant levels of mucins. In addition, determining the type of mucin (i.e. neutral or
acidic) may be helpful in evaluating neoplastic changes within a tissue.
The detection of acid or sulfomucins within the gastric mucosa may aid in the detection
and characterization of intestinal metaplasia, a lesion associated with gastric carcinoma.
76
Techniques for the demonstration of carbohydrates
Mcmanus For Glycogen (PAS)
Aim
Staining and identification of the various types of carbohydrates (polysaccharides
& mucopolysaccharides).
Principle : Tissue structures like liver & heart, striated muscles are studied by Periodic
acid Shiff stain. Periodic acid reacts with aldehyde group of the carbohydrates and
afterwards reaction with the schiff’s reagent produces a red or purple red colour.
Reagents
1. 0.5% w/v periodic acid solution
2. Schiff’s reagent
A. Dissolve 1.0 gm of basic fuchsin in 100 ml of boiling distilled water cool to
about 60 degrees and filter.
B. Add 20 ml of 0.1 N hydrochloric acid, cool further and add 1.0 gm of sodium
metabisulphite and mix well.
C. Keep in the dark for 24-48 hours. When the solution becomes straw coloured,
add 300 mg of activated charcoal, shake vigorously, filter and store.
4. 1 N Hydrochloric acid
5. 0.1 gm of light green in 100 ml of 0.1% (v/v) acetic acid.
6. Harris haematoxylin stain
Procedure
2. Oxidize in periodic acid solution for 5 minutes
3. Rinse in distilled water
4. Schiff’s regent solution for 15 minutes.
5. Wash in running water for 10 minutes for pink colour to develop.
6. Harris haematoxylin for 6 minutes or light green counter stain for a few seconds.
7. Wash in running water.
8. Differentiate in 1% acid alcohol solution 3-10 quick dips.
9. Wash in running water.
10. Dip in ammonia water to blue the sections
11. Wash in running water for 10 minutes
12. Dehydrate in 95% alcohol, absolute alcohol, clear in xylene two changes each.
13. Mount in DPX.
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Results
With hematoxylin counterstain
1. Nuclei – blue
2. Glycogen, mucin, hyaluronic acid, reticulin, colloid droplets, amyloid infiltration,
thrombi. – purple red
3. Fungi – Red
4. Background – pale green (with light green counter staining).
Test for schiff reagent solution

Pour a few drops of schiff reagent solution into 10 ml of formaldehyde (37- 40%) in
a watch glass. It the solution turns reddish purple rapidly it is good. If the reaction is
delayed and the resulting colour deep blue purple the solution in breaking down.
78
PIGMENTS AND MINERALS
Pigments are defined as substances occurring in living matter that absorb visible light.
The various pigments may greatly differ in origin, chemical constitution and biological
significance. They can be either organic or inorganic compounds that remain insoluble
in most solvents.
Minerals are naturally occurring homogeneous inorganic substances having a definite
chemical composition and characteristic crystalline structure, color and hardness.
In biology they are necessary for growth and development. Pigments can be classified
under the following headings:
1. Endogenous pigments:
hese substances are produced either within tissues and serve a physiological function,
or are by-products of normal metabolic processes. They can be further subdivided into:
• hematogenous (blood-derived) pigments
• non-hematogenous pigments
• Endogenous minerals.
2. Artifact pigments:
These are deposits of artifactually produced material caused by the interactions between
certain tissue components and some chemical substances, such as the fixative formalin.
Formalin and malaria pigments are sometimes classified as a subdivision of endogenous
pigments.
3. Exogenous pigments and minerals:

These substances gain access to the body accidentally through a variety of methods (e.g.
carbon anthracotic pigment, which is seen in lung and lymph nodes). These pigments
and minerals serve no physiological function. Entry is gained either by inhalation into
the lungs or by implantation (e.g. tattoos) into the skin. Most exogenous pigments are
minerals, few of which are pigmented.
Endogenous pigments
Hematogenous: This group contains the following blood-derived pigments:
1. Hemosiderins: These pigments are seen as yellow to brown granules and
normally appear intracellularly.
2. Hemoglobin: Hemoglobin is a basic conjugated protein that is responsible for
the transportation of oxygen and carbon dioxide within the bloodstream. It is
composed of a colorless protein, globin, and a red pigmented component,
heme. Four molecules of heme are attached to each molecule of globin.
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3. bile pigments: in liver sections is a histopathological indication that the patient
has obstructive jaundice due to a blockage in the normal flow of bile from the
liver into the gallbladder and subsequently into the bowel, probably because of
gallstones or a carcinoma of the head of pancreas.
4. Porphyrins: These substances normally occur in tissues in only small amounts.
They are considered to be precursors of the heme portion of hemoglobin.
The porphyrias are rare pathological conditions that are disorders of the
biosynthesis of porphyrins and heme.
Non-hematogenous endogenous pigments

This group contains the following:
• Melanins: Melanins are a group of pigments whose color varies from light
brown to black. The pigment is normally found in the skin, eye, substantia nigra
of the brain, and hair follicles.
The most common sites where melanin can be found are:
1. Skin, where it is produced by cells called melanocytes that are usually scattered
within the basal layer of the epidermis.
2. Eye, where it is found normally in the choroid, ciliary body, and iris.
3. Brain, where it is found particularly in the substantia nigra, in such quantities
that this structure is macroscopically visible as a black streak on both sides of
the mesencephalon.
• Lipofuscins: These yellow to red-brown pigments occur widely throughout the
body and are thought to be produced by an oxidation process of lipids and
lipoproteins. This type of pigment is found in the following sites:
A. Hepatocytes.
B. Cardiac muscle cells.
C. Inner reticular layer of the normal adrenal cortex.
D. Testis
E. Ovar
F. Cytoplasmic inclusions in the neurons of the brain, spinal cord, and ganglia.
• Chromaffin: This pigment is normally found in the cells of the adrenal medulla
as dark brown, granular material. It may occur in tumors of the adrenal medulla
(pheochromocytomas).
• Pseudomelanosis (melanosis coli): This pigment is sometimes seen in
macrophages in the lamina propria of the large intestine and appendix.
• Dubin-Johnson pigment: This pigment is found in the liver of patients with
Dubin-Johnson syndrome and is due to defective canalicular transport of
bilirubin.
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• ceroid-type lipofuscins: different from lipofuscin because it failed to stain with
the ferric-ferricyanide reaction.
• Hamazaki-Weisenberg bodies: These small, yellow-brown, spindle-shaped
structures are found mainly in the sinuses of lymph nodes, either lying free or
as cytoplasmic inclusions, and their significance is unknown.
Endogenous minerals
Calcium: Insoluble inorganic calcium salts are a normal constituent of bones and teeth.
Abnormal depositions of calcium can be found in necrotic areas of tissue associated
with tuberculosis, infarction (Gandy-Gamna bodies), atheroma in blood vessels, and
malakoplakia of the bladder (Michaelis-Gutman bodies).The most common forms of
calcium salts occurring in these conditions are phosphates and carbonates.
Copper
Many enzymes in the body would fail to function without the presence of copper, although
copper deficiency is extremely rare. Copper accumulation is associated with Wilson’s
disease, the most important disorder of copper metabolism.
Uric acid and urates
Uric acid is a breakdown product of the body’s purine (nucleic acid) metabolism, although
a small proportion is obtained from the diet. Most, but not all, uric acid is excreted by the
kidneys. The uric acid circulating in the blood is in the form of monosodium urate, which
in patients with gout may be high, forming a supersaturated solution. These high levels
may result in urate depositions, which are Water soluble in tissues, causing:
• subcutaneous nodular deposits of urate crystals
• (known as ‘tophi’)
• synovitis and arthritis
• renal disease and calculi.
Artifact pigments
This group of pigments comprises:
• Formalin: This pigment is seen as a brown or brown-black deposit in tissues
that have been fixed in acidic formalin. The deposit is usually present in blood-
rich tissues such as spleen, hemorrhagic lesions, and large blood vessels filled
with blood.
• Malaria: This pigment is morphologically similar to formalin pigment and
occasionally may be identical, even though it is produced in a slightly different
manner.
• Mercury: This pigment is seen in tissues that have been fixed in mercury-
containing fixatives, although it is rarely seen in tissue fixed in Heidenhain’s
Susa.
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• Chromic oxide: This pigment is rarely seen in tissue sections and is extremely
difficult to produce intentionally.
• Starch: This pigment is introduced by powder from the gloves of surgeons,
nurses, or pathologists.
Exogenous pigments and minerals

Certain types of mineral gain access to the body by inhalation, ingestion, or skin
implantation, commonly as a result of industrial exposure.
The most common minerals seen in tissue sections are carbon, silica, and asbestos.
Tattoo pigment
This is associated with skin and any adjacent lymphoid areas. If viewed using
reflected light.
Amalgam tattoo
Brown-black areas of pigmentation in the mouth may result from traumatic
introduction of mercury and silver from dental amalgam during dental procedures.
Carbon
This exogenous substance is the most commonly seen mineral in tissues and is easily
recognized in stained tissue sections. Commonly found in the lung and adjacent lymph
nodes of urban dwellers, the main sources of this material are car exhausts and smoke
from domestic and industrial pollution.
Silica
Silica in the form of silicates is associated with the majority of all mined ores, because
they are found in, or near, rocks that contain silica. Mine workers can inhale large
quantities of silica, which can give rise to the disease silicosis.
Asbestos
Asbestos has been used for many years as a fire-resistant and insulating material. There
are two groups of asbestos that cause pulmonary disease in humans:
1. serpentine (curly fibers)
2. Amphibole (straight/chain-like fibers).
There is one type of serpentine asbestos known as white asbestos (chrysotile). There
are many types of amphibole asbestos; the most common are blue asbestos (crocidolite)
and brown asbestos (amosite). Perhaps the most dangerous type is crocidolite (blue
asbestos). The fibers are 5–100 µm long and only 0.25–0.5 µm in diameter, and can
collect in the alveoli at the periphery of the lung.
82
AMYLOID
Amyloidosis is a disorder of protein folding, in which normally soluble proteins accumulate
in the
tissues as abnormal insoluble fibrils (or filaments), thus disrupting their function.
The term amyloid means ‘starch-like’ and was first used in botany to describe the starchy
cellulose material found in plants.
Composition and ultrastructure
- 90-95% non branched fibrils diam. 10-12nm.
- 5-10% p-component - glycoprotein + fibronectin, laminin, collagen 4.
Classification
Pathogenesis
Amyloidosis is considered to belong to the category of conformational diseases,
because the pathological protein aggregation is due to the reduced stability and a strong
propensity to acquire more than one conformation.
Conformational diseases arises when a constituent protein undergoes a change in size
or fluctuation in shape with resultant self - association and tissue deposition“
Several amyloid proteins are rich in β-pleated sheet conformation in their native form;
prion protein contains no β-pleated sheet and it is the formation of β-pleated sheets de
novo that is the pathological event of the prion diseases.
Demonstration
Dyes used for the demonstration of amyloid include Congo red, which was developed as
the first direct cotton dye in 1884 and has been ‘re-invented’ many times in the search
for a differential method for the detection of amyloid.
83
Congo Red
Aim:
To demonstrate amyloid deposits in tissue sections.
Principle: Amyloid is homogeneous and eosinophilic, the deposits are extracellular and
may become sufficiently large enough to cause damage to surrounding tissues. when
stained with the Congo Red Stain the amyloid, with the aide of polarizing lenses, will
birefringe an apple green color. under the microscope.
Reagents:
Mayer's Hematoxylin:
• Hematoxylin 1.0 gm
• Distilled water 1000.0 ml
• Sodium iodate 0.2 gm
Ammonium or Potassium
• aluminum sulfate 50.0 gm
• Citric acid 1.0 gm
• Chloral hydrate 50.0 gm
1% Sodium Hydroxide
• Sodium hydroxide 1.0 gm
• Distilled water 100.0 m
Sodium Chloride, Stock Solution, Saturated:
• Sodium chloride 30.0 gm
Congo Red Stock Solution, Saturated:
• Congo red stain 1.0 gm
• Stock sodium chloride 500.0 ml
Procedure
1. Deparaffinize and hydrate to water.
2. Mayer's Hematoxylin for 10 minutes.
3. Wash in tap water until blue.
4. Working sodium chloride solution, microwave, 20 power, 45 seconds. Allow
slides to sit in solution for 2-5 minutes.
5. Place directly into Working Congo red solution, microwave, 20 power, for 45
seconds. Allow slides to sit in solution for 2-5 minutes.
6. Dehydrate rapidly in absolute alcohol, 10 dips, 3 changes.
7. Clear in xylene, coverslip with Permount.
84
Results:
• Amyloid red to pink
• Nuclei blue
NOTES:
1. Sections must be cut at 8 to 10 microns for birefringence.
2. Solution must be filtered through glass wool, not paper filters for birefringence
to occur.
3. Tissue fixed in solutions other than formalin may display false positive
birefringence.
85
MICROORGANISMS
Microorganisms are living organism that can only be observed with the aid of microscopes
and they are capable of causing disease.
They are broadly classified into bacteria, viruses, parasites and fungi. Demonstration of
microorganisms in tissue sections, without the application of microbiological processes,
is important in histopathology because it is relatively accurate in making a diagnosis.
This can be achieved most especially by using the special staining techniques, examples
of which include; modified Giemsa, Ziehl-Neelsen, Grocott/Gomori methenamine silver,
Periodic acid Schiff.
The general staining technique in histopathology, Haematoxylin and eosin (H&E) stain,
can demonstrate vast majority of microorganisms. It can also demonstrate specific
inflammatory cell response as a result of specific microorganisms present in the tissue.
The specific inflammatory cells response of diagnostic importance in tissue samples
include; increase number of eosinophils in parasitic/helminthic infections, neutrophils
in acute bacterial infections, lymphocytes in viral infections, multinucleated giant cells
in chronic granulomatous inflammation (Langhans cell in tuberculosis, Touton cells in
xanthogranulomas).
The special staining technique is employed, in most cases, to take the advantage of the
properties of the microorganisms.These include poor demonstration of the microorganism
by the general H&E stain, relatively small organism size (seen with Treponema spp),
few number of organisms present in the tissue that can be visualized, high lipid content
of cell wall (seen with Mycobacteria spp), primarily intracellular organism (seen with
Legionella spp, Chlamydia spp) and absence of cell wall (seen with Mycoplasma spp).
Detection And Identification Techniques Of Microorganisms In

Tissue
Bacteria
Giemsa stain (modified) is the most commonly used bacteria stain in histopathology. It
demonstrates Helicobacter pylori in gastric tissue biopsies as purple rod-like structures
within the gastric glands. Helicobacter pylori is the causative agent for peptic ulcer
disease and the most common type of gastric cancer. Histopathological demonstration
of bacteria in tissue using the Gram stain divides bacteria into 2 major categories; Gram
Positive and Gram-negative bacteria. The Gram staining method (mostly Brown-Brenn
method) is characterised by the affinity of the organism’s cell wall for the dye used
(crystal violet-iodine complex). Gram- positive organisms stain blue-black as opposed
to Gram-negative organisms that stain pink to red. Atypical bacteria like Legionella spp,
86
because of their intracellular nature, are best demonstrated using the Dieterle silver stain.
Bartonella henselae can also be demonstrated using Dieterle sliver stain. Mycobacteria
spp, with a high lipid cell wall (hydrophobic mycolic acid) requires a acid and alcohol-fast
stain which is provided by the Ziehl Neelsen procedure (which has carbolfuchsin as the
principal dye) and demonstrate the bacilli as red rod-shaped organism in a background
of blue or green counter stain.
Fungi
These are found in nature in three forms; spores (e.g. Histoplama spp), yeast (e.g.
Candida spp, Cryptococcus spp) and mould (e.g. Aspergillus spp). Most fungi
infections are cutaneous; hence the use of potassium hydroxide (KOH) to demonstrate
dermatophytes on skin scrapings smear preparation.The global increase in incidence
and prevalence of human immunodeficiency virus (HIV) infection has led to increase
in the occurrence of systemic and tissue mycotic infections. Main examples include
Pneumocystis carinii, histoplasma spp and Crytococcus spp. Demonstration of fungi in
tissue is can be done using Periodic acid Schiff (because of the high polysaccharides
level in the fungi cell wall) and most importantly, Gomori/Grocott methenamine silver
stains (polysaccharide cell wall forms aldehyde following oxidation and upon reduction,
forms black coloured silver precipitates). Giemsa stain can also be used for Aspergillus
spp identification in tissue.
The Haematoxylin & Eosin stain can also demonstrate fungi organisms in tissue because
of their relatively large size.
Viruses
The viruses are the smallest of the entire microorganisms that are demonstrated in
histopathology laboratory; hence, the molecular methods are desirable.2 However,
due to high cost in resource limited centre, the viral effects on host cells and tissue,
the cytopathic and cytoproliferative effects, can be demonstrated in tissue sections.16
In some cases like measles, rabies, cytomegalovirus and herpes, there is formation
of intracellular inclusion bodies.These inclusion bodies can either be intranuclear or
intracytoplamic, or both, and they stain readily with haematoxylin and eosin stain most
of the times.
In addition, various staining techniques have been developed and modified over the
years to distinguish these inclusion bodies.These methods include; Mann's methyl blue-
eosin method stains intracytoplasmic Negri bodies of Rabies as red structures.
Lendrum's phloxine-tartrazine and Macchiavello techniques stain viral inclusion bodies
(intranuclear or intracytoplasmic) as red structures within the affected cell.
Modified orcein stain is used for the detection of hepatitis B surface antigen in affected
87
hepatocytes which stain brown-black (due to prominent staining of the rough endoplasmic
reticulum).
The cytopathic effects of some viruses can be visualized on cells or tissues affected with
H&E stain; example include koliocytic atypia of squamous cell in Human papilloma virus
infection.
Parasites / Protozoa
They can easily be visualised with the haematoxylin and eosin stain because of their
relatively large size.
Periodic acid Schiff and Giemsa stains can also be applied to the majority of the
organisms in this group to demonstrate them clearly in tissue sections.
Ascaris lumbricoides, Trichuris trichiura, Taenia spp (larva), Schistosoma spp (egg),
Onchocerca volvulos and Strongyloides stercoralis all stain well with either the
combination of haematoxylin and eosin and PAS or using either stain individually.
Dieterle silver stain can be used to stain spirochetes and Donovan bodies
(Leishmaniasis). Macchiavello techniques can also be used for detection of
Rickettsia in tissue.
88
Gram Bacteria - Modified Brown And Brenn
Aim:
For demonstrating gram-negative and gram-positive in tissue.
Principle: Both bacteria's, positive and negative, cell wall is composed of peptidoglycan,
(the gram-positve has a thicker wall) and both will take up the crystal violet. The gram-
negative has a layer of lipopolysaccharide external to the peptidoglycan wall, which is
disrupted in the acetone rinse, allowing the crystal violet to be differentiated out.
This allows the gram-negative bacteria to take up the basic fuchsin stain.
Reagents:
1% Crystal Violet:
• Crystal violet 1.0 gm
• Distilled water 100.0 ml.
• Lugol's Iodine:
Acetone:
Basic Fuchsin:
• Basic fuchsin 0.25 gm
Picric Acid-Acetone:
• Picric acid, saturated 100.0 ml
• Acetone 900.0 ml
Procedure:
2. Place slides on staining rack, drop crystal violet stain onto tissue section, stain
for 1 minute.
4. Lugol's iodine, 1 minute.
6. Blot sections dry, breath on section then quickly pour acetone over section until
no color runs off.
8. Place slides on staining rack, drop Basic fuchsin on tissue sections, stain 3
minutes.
9. Wash in tap water, blot gently but not completely dry.
10. Dip quickly into acetone, 2 dips.
11. Dip directly into picric acid-acetone mixture until a 'salmon' color.
89
12. Dip quickly into two changes of acetone.
13. Air dry, dip into xylene, and coverslip.
Results:
• Gram-positive bacteria : blue
• Gram-negative bacteria: red
• Nuclei: red
• Background: yellow
90
Acid-Fast Bacteria - Ziehl-Neelsen Stain (AFB)
Aim:
Used in the demonstration of acid-fast bacteria belonging to the genus 'mycobacterium',
which include the causative agent for tuberculosis.
Principle:
The lipoid capsule of the acid-fast organism takes up carbol fuchsin and resists
decolorization with a dilute acid rinse. The lipoid capsule of the mycobacteria is of such
high molecular weight that it is waxy at room temperature and successful penetration by
the aqueous based staining solutions (such as Gram's) is prevented.
Reagents:
Ziehl-Neelsen Carbol-Fuchsin Solution:
• Basic fuchsin 2.5 gm
• 100% alcohol 25.0 ml
• Phenol crystals, melted 12.5 ml 1% Acid Alcohol:
• Hydrochloric acid 10.0 ml
• 70% Alcohol 990.0 ml Methylene Blue
• Stock Solution:
• Methylene blue 0.7 gm
• Distilled water 50.0 m
Procedure:
2. *Carbol-fuchsin solution, microwave 80 power, 45 seconds, allow slides to
stand in hot solution for 5 minutes. Filter solution once a week.
3. Wash in running tap water.
4. 1% Acid alcohol until light pink and color stops running.
5. Wash in running tap water for 5 minutes..
6. Rinse in distilled water.
7. Working methylene blue for 30 seconds.
8. Rinse in water.
9. Dehydrate, clear, and coverslip.
*Conventional Method: 60°C oven for 1 hour.
Result
• Acid-fast bacilli bright red
• Background blue
91
CYTOLOGICAL TECHNIQUES
Cytology is a discipline that involves the morphologic study of cells. It is broadly divided
into exfoliative cytology and aspiration cytology.
Exfoliative cytology involves examination of specimens that contain cells exfoliated
from body cavities and surface. It is further subdivided into gynaecological cytology
(Pap/cervical smears) and non-gynaecological cytology (pleural fluid, peritoneal fluid,
cerebrospinal fluid, urine, sputum, brushing, etc).
Aspiration cytology involves examination of cells that are actively obtained by fine needle
aspiration.
Type of samples obtained in laboratory

There are two main types of cytology specimens - gynaecological and non- gynaecological
specimens.
1. Gynaecological specimens are usually Pap (cervical) smears.
2. Non-gynaecological specimens fall into two categories – exfoliative and fine
needle aspiration samples.
Cytologists, sometimes also known as cytotechnologists, are specially trained personnel
who prepare cytology slides and examine them under the microscope to diagnose various
types of diseases, including some types of cancers. Most of the abnormalities detected
by the cytologist (including all of the cancers) are then examined by a pathologist before
a final diagnosis is made.
Sampling techniques in cytology

The successful evaluation of cytological material depends, to a great extent on the
technical quality of the preparation. The material often contains only scanty diagnostic
evidence, and this may be unrecognisable at screening unless care is taken during the
preparation of the smears.
1. Vaginal smears: material is usually obtained by aspiration of the posterior
vaginal fornix with stout-walled, slightly curved, glass Pippete fitted with a
rubber bulb.
2. Cervical smears: To assist in the collection of material for this type of smear the
clinician employs an instrument known as a speculum.
3. Sputum Smears: early morning specimens are recommended and should be
produced by a deep cough. Several consecutive daily samples are advised
ranging from 3 to 9 days.
4. Smears from urine, Pleural, Ascitic fluids and Gastric washings. It is of the
most importance that smears from fluid specimens are prepared and fixed as
soon as possible after collection, otherwise much cellular detail will be lost.
92
Cells of gastric fluids in particular undergo rapid degeneration and digestion at
room temperature. Refrigeration will arrest these destructive processes for a
short time only.
5. Membrane filters: cellulose acetate membrane filters of graded pore size are
useful for the concentration of cells from most body fluids. They are particularly
useful where only a few cells are present because the total cellular content of
a fluid may be collected into a single membrane.
Guidelines For Collection And Transportation Of Cytology

Specimen
93
94
Cytological staining techniques
Papanicolaou stain
Dr. George Papanicolaou who in 1928 presented his findings that cancer cells could be
found in the vaginal fluid of women with cancer of the cervix.
Aim:
Papanicolaou staining is applied to vaginal exudates for the detection of uterine or vaginal
cancer. Papanicolaou method is designed to give sharp nuclear staining, transparency
of cytoplasm and good differential colouring of acidophilic and basophilic cells.
Principle
The technique uses a high number of dyes in its procedure.
• Hematoxylin: is the chosen nuclear staining, basically allows to reveal the
nuclei of the cells present in the sample. Harris Hematoxylin is usually used.
• Orange G: is a synthetic acid dye that reveals basic compounds such as
prequeratine (that stains pink) or keratin (that stains bright orange).
• Yellowish Eosin: stains cytoplasm of mature squamous cells, hair cells and
erythrocytes into pink-orange.
• Green SF Yellowish light: stains squamous non-superficial cells (immature or
partially mature) into greenish-blue.
• Bismark Brown R: does not stain the cellular cytoplasm but does mucin.
• Phosphotungstic acid: has a mordant function, especially important for Green
Light SF.
Material
• Vaginal exudates.
Procedure
1. Fix the sample with spray.
2. Submerge successively in alcohol 80%, alcohol 70%, alcohol 50% and water,
1 minute in each liquid.
3. Stain with Hematoxylin Harris solution for approximately 5 minutes.
4. Immerse in water 6 times for 1 second.
5. Submerge in 0.5% Hydrochloric Acid, 8 times for 1 second.
6. Rinse with tap water for 5 minutes, and pass the sample through successive
grade alcohols, 50%, 70%, 80% and 96% for 30 seconds in each of them.
7. Stain with Pap smear or OG 6 for 1 to 1.5 minutes.
8. Wash the excess dye in two 96% Ethanol baths by immersing the preparation
2 times in each of 3 to 4 seconds.
95
9. Stain with Pap smear or EA 50 for 1.5 to 2 minutes.
10. Wash in 3 different containers of Ethanol 96% v / v by immersing the preparation
2 times of 3 to 4 seconds in each of them.
11. Wash in absolute ethanol for 30 seconds.
12. Immerse the preparation for 4 minutes in a 1: 1 bath of Xylene, mixture of
isomers and absolute ethanol.
13. Rinse with Xylene, mixture of isomers by immersing the preparation for 3
minutes in a bath.
14. Mount with Mounting medium
15. Observe under a microscope
Result
• Nuclei : Blue
• Acidophilic cells : Red
• Basophilic cells : Blue Green
• Erythrocytes : Orange-red
• Keratin : Orange-red
• Superficial cells : Pink
• Intermediate and Parabasal Cells : Blue Green
• Eosinophil : Orange Red
• Candida : Red
• Trichomonas : Grey green
Review questions
1. Describe the papanicolaou’s staining and identification of cells in a normal
vaginal smear?
2. Explain the composition and applications of PAP smear?
3. What are the stains used in cytology?
4. What is exfoliative cytology?
96
Buccal smear
Aim: In this activity, you will prepare a sample of the cells that line the inside of your
cheeks (i.e. a buccal smear) and then examine it under a light microscope.
Principle
The cells which line the inside of the cheeks form a mucous membrane and are
classified as a stratified squamous epithelium tissue. These flat, scale-like buccal cells
(pronounced "buckle") resist friction and are shed constantly as the tissue is renewed. By
gently scraping the inside of the cheek, these cells can be harvested, and when smeared
and stained, may be used to illustrate a number of important biological phenomena
including cell and tissue structure, oral bacterial flora and morphology, etc. This tissue is
non-keratinized and therefore the surface cells are living and still possess their nuclei, in
contrast with shed epidermal cells.
Materials
• Soap and water
• Microscope slides
• Dropper bottle of distilled water
• Dropper bottle of0.3% methylene blue
• Tooth picks (optional)
• Bunsen burner or alcohol lamp, striker
• Paper towel (“non-linty”) or bibulous paper
Procedure
Slide preparation
1. Clean a microscope slid e well with soap & water, dry with a “non -linty” paper
tow el.
2. Cleanse very thoroughly under the nail of your index finger.
3. Place a tiny drop of distilled water in the center of the very clean slide.
Harvest the cells, prepare the smear
4. Gently scrap the inside of your cheek to pick up some of the shed stratified
squamous cells. Do NOT scrape chunks. A toothpick may be used if you
have no fingernails. Gentle scraping is the watchword, there should be no
discomfort...
5. Express the material from under your nail by pressing with your thumb, and
press into the drop of water, mix and spread the material around to the size of
a dime.
97
Fix the smear
6. Pass the slide briefly through the flame several times to warm and fix the
smear. Do NOT Heat the slide above a temp which is comfort able. You are
"gluing" the smear to the slide.
Stain the smear
7. Place a drop of 0.3% methylene blue on the specimen. Let sit for 1 minute.
8. Rinse off the excess stain with tap water. (Do not splash on your white shirt!)
9. Blot dry with a non-linty paper tow el or bibulous paper. Do not rub.
10. Flame again briefly to dry slide by warming.
Examine under the microscope
11. Examine first with the4x objective, scanning the entire field to find a well-
distributed region with individual cells (no big clumps). Then view with the10x
and 40x objectives. Illustrate The 400x view noting 1) the nucleus,2) nucleolus,
3) cell boundary and 4) the variety of bacteria colonizing the surface of the
cells. For Microbiology only: you will be instructed on oil immersion use (see
separate protocol), then view and illustrate bacterial morphologies with the
100x oil immersion objective (1000 x view).
Clean up
12. When finished, scrub the slide w ell with soap and hot water, rinse well and
drain dry in a plastic test tube holder.
Result
Intermediate Cell - Blue
Superficial Cell - Pink
98
MGG stain ( May Grunwald Giemsa’s Stain )
AIM
This stain is done for any kind of FNAC slide or any kind of FNAC smear .
For this stain the smear should fixed in Methyl-alcohol for 15-20 minute .
Preparation of MG-stain
i) Take MG-stain 0.3gm in a conical flask .
ii) Add 100ml of methanol .z
iii) Heat the mixture up-to 50*c .
iv) Alow the solution to cool at room-temperature .
v) Then filter after 24-Hrs , before use .
Preparation of Giemsa’s stain

i) Take Giemsa stain 1-gm in a conical flask .
ii) Add 66 ml of glycerol .
iii) Heat up-to 56*c for 1 and ½ hour .
iv) Then add 66ml of methanol .
v) Then filter and use .
Procedure :
i) Keep the smear in Coplin-jar containing MG – stain which is already diluted by equally
buffer whose pH is 6.8 .
ii) Stain in 1 in 10 diluted Giemsa-stain with diluted distilled water for 10-15 minutes .
iii) Differentiate in buffer whose pH is 6.8 for 5-10 minutes .That is depending upon the
fixation of the smear .
iv) Wash in running tap-water followed by distilled-water .
v) Air dry the smear .
vi) Clear in 3-changes of Xylene 3-minutes each .
vii) Mount in DPX .
Result : -
Nucleus - Blue
Cytoplasm and other component - pale / Light blue
99
BIBLIOGRAPHY
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
6. Sheehan D,Hrapchak B, Theory and practice of Histotechnology, 2nd
Ed,1980, pp235-237,Battelle Press, Ohio
7. Carson F, Histotechnology: A Self-Instructional Text, 1990, pp189- 191,ASCP,Ill
8. Crookham,J, Dapson,R, Hazardous Chemicals in the Histopathology
Laboratory, 2nd ED, 1991, Anatech
100
Tel: +252 615206907 / 682150077
Email: shafcisom10@gmail.com
101
Most people do not know what a Histotechnologist is or does. Histotechnologist fight
against disease, also he is a vital and critical link in the chain of medical research.
Basically, the responsibility of the Histotechnologist is to prepare sections of human
tissues for microscopical examination by a pathologist in the interest of diagnosing
disease and conducting medical research. In many cases, Histotechnologist are
compelled to work under extreme 102pressure such as when a body tissue from a patient
SHAFIE ABDULKADIR HASSAN in surgery needs to be diagnosed immediately.
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1

Figure 1 - Cells, Tissue, Organs, Organ Systems and Organism

Types Of Histological Preparation

Design of Histopathology Laboratory

Component of Histopathology Laboratory

Tissue processing machine Floating bath

Types of hazards include the following:

Factors contributing to laboratory accidents

Figure. 2.light microscopy

Other types of light microscope:

Figure 3. Parts of light microscopy

Aims and Effects of fixation

II. Alcohol (Ethyl Alcohol)

Fixatives are divided into three main groups

(b) Cytoplasmic Fixatives

Tissue Fixative of choice Time for fixative

Materials and equipment:

The Criteria of a good decalcifying agents area.

Aqueous nitric acid

Gooding and Stelwart’s fluid.

Formic acid sodium citrate method

Decalcification of Bone marrow biopsy.

Use of Ion exchange resins

Impregnation with wax

Properties of paraffin wax

Points to be remembered during use of paraffin wax

Hot plate or drying oven

Figure 6. A microtomist creating a “ribbon” of very thin sections for staining

Dyes used in staining

1. Haematoxylin : This is the most popular dye used as a nuclear stain.

Staining of paraffin section

3. Removal of mercury pigments wherever needed

5. Dehydration and clearing

Resinous mounting media

Cover glasses used in histopathology

Some basic rules for staining

Haematoxylin and Eosin staining

Causes of poor quality of staining

Materials and equipment:

Stains for the detection of Connective Tissue

Van Gieson Stain

Stains for the detection of Carbohydrates

Alcian Blue PAS

Stains for the detection of Minerals

Stains for the detection of Microorganisms

Acid Fast stain

Connective Tissue Cells:

Fibers of connective tissue

Classification of Connective Tissue

Connective tissue stains: Trichrome stains

Factors affecting trichrome staining

VAN GIESON METHOD

Test for schiff reagent solution

3. Exogenous pigments and minerals:

Non-hematogenous endogenous pigments

Exogenous pigments and minerals

Detection And Identification Techniques Of Microorganisms In

Type of samples obtained in laboratory

Sampling techniques in cytology

Guidelines For Collection And Transportation Of Cytology

Preparation of Giemsa’s stain

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