Unit-1

Introduction and definitions

Histology

 It is the scientific study of biological tissues.

 Histology is the microscopic study of the structure of biological
tissues using special staining techniques combined with light
and electron microscopy.
 Histology is the study of the microscopic structures of cells and
tissues of plants and animals. It is often carried out by examining a
thin slice (called a 'section') of tissue under a light microscope
or an electron microscope. In order to distinguish different
biological structures more easily and accurately histological
stains are often used.
Histopathology
▶ Histopathology is the microscopic examination of biological
tissues to observe the appearance of diseased cells and tissues
in very fine detail.
The word 'histopathology' is derived from a combination of three
Greek words:
▶ histos meaning tissue,
▶ pathos meaning disease or suffering, and
▶ logos which refers to study in this context*.
▶ Hence histopathology is the study of microscopic changes
or abnormalities in tissues that are caused as a result of
diseases.
Biopsy

▶ A biopsy is a medical procedure that involves taking a small

sample of tissue so that it can be examined under a
microscope.
▶ A tissue sample can be taken from almost anywhere on, or in
the body, including the skin, stomach, kidneys, liver and lungs.
▶ The term biopsy is often used to refer to both the act of taking
the sample and the tissue sample itself.
▶ An examination of tissue removed from a living body to
discover the presence, cause, or extent of a disease.
Autopsy
▶ An autopsy (post-mortem examination, necropsy) is a surgical
procedure that consists of a thorough examination of a corpse by
dissection to determine the cause, mode, and manner of death or
to evaluate any disease or injury that may be present for
research or educational purposes.
▶ Autopsies are performed by pathologists, medical doctors who
have received specialty training in the diagnosis of diseases
by the examination of body fluids and tissues.
▶ In academic institutions, autopsies sometimes are also
requested for teaching and research purposes.
▶ Forensic autopsies have legal implications and are performed to
determine if death was an accident, homicide, suicide or a
natural event.
Autolysis
▶ When a body dies, there is an organized process of decomposition
that begins almost immediately. One part of this process
is autolysis (auto = self and lysis = breakdown), which is cellular
self-digestion. This self-destruction of cells occurs
as endogenous or internal cellular enzymes (endo = inside and
genous = originating from) are released and work to break
down cellular material.
▶ Autolysis is the breakdown of cell component or organism by
its own enzyme.
Putrefaction
▶ Putrefaction is the decay of the organic matter by the action of
microorganisms resulting in the production of a foul smell. It occurs
between 10 to 20 days of the death of an organism. It is the fifth
stage of death.
▶ Putrefaction involves the decomposition of proteins, breakdown of
cohesiveness between the tissues, and liquefaction of most organs.
The body is decomposed by the action of putrefying bacteria and fungi
which releases certain gases that infiltrate and deteriorates the body
tissues and organs. Putrefying bacteria play a major role in recycling
nitrogen from the dead organism.
▶ Cytology is a study of structure, composition and function of
cells.
▶ Cytopathology is a study of abnormal or diseased cells
▶ Autolysis is a cell death due self enzymes digestion
▶ Fixation is a process of preserving tissue or cells using
chemical agent(s) in as life-like manner as possible.
▶ Fixative is a chemical agent that is used to preserve tissue or
cells.
▶ Decalcification is a process of removing calcium from bone and
calcified tissue.
▶ Dehydration is a process of removing free water (not molecularly
bound water) from the tissue.
▶ Clearing is a process of replacing the dehydrating agent with a reagent
that is miscible with paraffin wax.
▶ Infiltration/impregnation is a processing of filling in tissue,
intracellular and extracellular, spaces with a medium which supports
it during sectioning.
▶ Tissue blocking/ embedding/ casting is a process of enclosing
the tissue in the infiltration medium used for processing and then
allowing the medium to solidify.
▶ Microtomy/section cutting is a cutting of thin sections of tissue for
microscopic examination using a microtome.
▶ Adhesive materials are materials which are thinly smeared on the
microscope glass slided before mounting for the purpose of
increasing adherence of tissue section to slide. E.g. Mayer’s egg-
albumin.
▶ Stain is a substance used to impart colour to tissue or cells,
facilitate microscopic study and identification.
▶ Staining is a process of imparting colour to tissue or cells so as
to facilitate microscopic study and identification.
▶ Mounting/coverslipping is a process of covering the stained slide
with coverslip using special media in between them.
▶ Mountants are special media with gluing property used to
facilitate adhering of coverslip to stained slide.
▶ Cytological aspirates is a specimen obtained by sucking fluid from
the body for the purpose of harvesting cells for investigation.
 Unit-2
Preparation of Tissue
 Unfixed Tissue preparations
▶ Imprint preparation:-
These are prepared by touching a freshly cut piece of tissue with the
surface of clean microscope slide. This way, cells are transferred and
adhere to the slide. The smear can be examined with the phase contrast
microscope or by using vital stain.
▶ Impression smears:-
Smearing a piece of fresh specimen of tissue evenly on the surface of
microscope slide is an acceptable practice in histopathology. The making of
such smears depends on the type of tissue to be examined. The smear can
be examined fresh in which case it is stained as for teased preparation or by
using supravital stain in conjunction with a warm stage. The preparation is
never permanent.
▶ Teased preparation –
The fresh specimen of tissue, immersed in saline or. Ringer's solution,
is dissected with mounted needles. Pieces of the tissue are picked onto a
microscope slide and mounted as a wet preparation under a coverslip.
The slide is then examined by the ordinary light microscope or better
still by phase contrast microscope.
▶ Frozen section:-
Fresh tissue frozen on microtome with CO2 can be cut into sections of
about 10 to 15 um in thickness. The section are transferred to a dish and
attached onto the slide before staining or from the dish carried on a
glass rod through staining solution.
▶ Squashed preparation:-
Small pieces of tissue not more than 1mm in diameter are placed
in the Center of a microscope slide. A coverslip is forcibly pressed
down on them. Vital staining can be done by placing a drop of the
stain at the junction of the slide and the coverslip. The stain 1s
drawn in by capillary action and absorbed by the tissues.
Fixed Tissue preparations
▶ Paraffin embedding
Paraffin is not miscible with water, but tissues are mostly water.
In addition, most fixatives are aqueous solutions.
This means that water must be removed from tissues before they
are infiltrated with paraffin.
It is done by dehydration of tissues with alcohol, mostly ethanol, from
a graded series of alcohols from 50º to 100º (absolute o pure alcohol).
All the water needs to be removed for a good embedding.
After dehydration, samples are transferred to an intermediary liquid,
like xylene, benzene, propylene oxide, or toluene, which are miscible
with both absolute alcohol and paraffin.
These are clearing substances and we can check their infiltration in
the sample by observing how translucent the sample is.
The immersion of the sample in the intermediary liquid, like xylene
and toluene, must not last very long because they harden the
samples and getting sections might be more difficult.
The last step of the embedding procedure is to plunge the
sample in melted paraffin.
It is done in an oven at a temperature properly set for the paraffin
type we are working with. For a complete replacement of the
intermediary liquid with paraffin, three changes in fresh paraffin are
recommended.
How long the samples are incubated in paraffin depends on the
intermediary liquid, size of the sample, and type of paraffin.
▶ Celloidin embedding
Celloidin is dissolved in equal parts of absolute alcohol and
ether. The tissue is dehydrated in alcohol in the same way as for
paraffin except that it is transferred from absolute alcohol to a
dilute solution of celloidin. As the alcohol and ether evaporate,
they are replaced by more concentrated celloidin. It is finally
hardened in chloroform and stored in 80 percent alcohol. It is a
much longer process than paraffin but causes much less
shrinkage and distortion. It is used especially in examination of
the eye and brain.
▶ Gelatin embedding
A method is described for embedding tissues in gelatin which makes
it possible to cut thin sections for electron microscopy. With this
method it is possible to embed the tissue without passing it through
organic solvents extracting the lipid soluble components.
A method is described for embedding tissues in gelatin which makes
it possible to cut thin sections for electron microscopy. With this
method it is possible to embed the tissue without passing it through
organic solvents extracting the lipid soluble components.
 Unit-3
Reception of Specimen
Reception
▶ Specimen Reception is the first point of contact with Laboratory
Medicine and is the department where specimens requiring analysis
are received.
▶ Specimen reception plays one of the most important roles in
the pathology department.
▶ It is here that patient samples from many different wards, clinics,
departments, other hospitals and GPs arrive so they can be sorted and
sent, with relevant information, to the appropriate laboratory
including immunology for testing.
▶ Reception staff provide support to the Biomedical Scientists
(BMS’s), Clinical Scientists and Medical Staff and also carry out
the pre-analytical preparation of samples.
▶ This includes the inputting of patient demographics and
investigations into the laboratory computer system, labelling
and sorting of pathological samples including blood, urine,
Cerebrospinal Fluid (CSF), faeces and other body fluids.
Recording
▶ The number of specimen received daily may be small or
large depending on whether the laboratory caters to a small
or large hospital.
▶ However, it is essential that a records are kept from the
outset.
This is best done by having a reception book in which all
specimens are recorded, including all the relevant
details.
▶ These consist of the name, age, and sex of the patient, the OPD
number, with hospital ward and bed number of the inpatient,
the name of the clinician and the organ biopsied or excised with
the clinical diagnosis.
▶ Give an identification number as lab record to every specimen.
All the detail from request form which is sent by doctor with
the specimen is recorded in the lab record as name of doctor and
patient, date and time of collection, size of the specimen and
name of fixative etc. are recorded in personal record of
laboratory.
▶ On arrival each specimen is given an accession number. This
is followed by the year of entry, e.g. 1/85, continuing
▶ throughout the year and starting again as 1/86. The specimen
will
carry this number until it is processe sectioned, reported and
field.
labeling
▶ It is the process in which is done by the technician after receiving the
sample in the histopathology lab. It can be done to every specimen
after grossing for correct resulting and easy working.
▶ Once tissue have been selected for processing they are accompanied
through all stage by a label bearing the number given to the
specimen. The label is retained as a permanent record during
sectioning and storage of tissue blocks.
▶ Very small biopsies like needle biopsies of kidney and liver, small
curetting, etc. may be wrapped in filter paper soaked in formalin
before being put in the capsules. Printed, graphite penciled, type
written or India ink written labels are satisfactory. Ordinary ink should
not be used as this may be dissolved in the reagents used during
processing.
▶ Remains of all specimens are preserved in formalin until the
reported on are discarded. This may be indicated by writing
SK (stock kept) at the end of grossing notes.
▶ All specimen kept on the shelves are to be identified by legibly
written number for future. All specimen of potential teaching
value may be photographed and if considered worthy of display
in the museum may be mounted.
Preservation:

▶ The act or process of preserving, or keeping safe; the state of

being preserved, or kept from injury, destruction, or decay;
security; safety; as, preservation of life, fruit, game, etc.; a
picture in good preservation.
▶ The specimen is placed in a liquid fixing agent (fixative) such
as formaldehyde solution (formalin). This will slowly penetrate
the tissue causing chemical and physical changes that will harden
and preserve the tissue and protect it against subsequent
processing steps.
 Unit-4
Fixation (Histological Specimens)
Fixation & Fixative
▶ Fixation: It is the preservation of biological tissues from decay due to
autolysis or putrefaction. It terminates any ongoing biochemical
reactions and may also increase the treated tissues' mechanical strength
or stability.
▶ Fixation is done to maintain the structure of tissues in almost lifelike
conditions before they are ready to be examined under the
microscope.
Fixation also serves the following important functions.
 It prevents the autolysis and bacterial decomposition/
Putrefaction. Autolysis is most rapid in brain and
Kidney.
 It coagulates the tissue to prevent the loss of diffusible
substances.
CLASSIFICATION OF FIXATIVES
Fixatives can be functionally classified into two major groups:

▶ Simple Fixatives – These fixatives are made up of simple chemical

compounds and take more time for the fixation of tissues. For
example, Formalin, Picric acid, Mercuric oxide, osmic acid,
Osmium tetroxide etc.
▶ Compound Fixatives – These are the mixtures of a number of
fixatives in definite proportion and require a lesser amount of time
for fixation. For example, Susa fluid, Carnoy’s fluid, Bouin’s Fluid,
Formal saline, buffered formalin etc.
The compound fixatives can further be classified into three types
as follows:

1. Micro anatomical fixatives: These fixatives are used for routine work
of normal and histopathological study. For example, buffered formalin,
Zenker’s fluid, Bouin’s fluid etc.
2. Cytological fixatives: These are intended to preserve the constituents
elements of the cells themselves.
3. Histochemical fixatives: These are used for the Histochemical studies
of the tissues where the minimum or no changes in the components to be
demonstrated are required. for example, Buffered formalin or vapor
fixatives include Formaldehyde, Glutaraldehyde, Acrolein etc.
SIMPLE FIXATIVES
▶ FORMALDEHYDE:
 Commercial formaldehyde is saturated solution of formaldehyde
(H.CHO) gas in water, approximately 40% gas by weight.
 10% of formalin used for fixation is prepared by adding 10ml
of formalin to 90ml of saline.
ADVANTAGES:
i) It fixes the proteins without precipitation.
ii) Has no effect on Carbohydrates.
iii) Preserves Glycogen and Lipids.
DISADVANTAGES
iv)It causes little Shrinkage.
v) Over hardens the tissue if left for a long time in formaldehyde
solution.
▶ GLUTARALDEHYDE
 Stable at 0 to 4oc and at PH 3.0 to 5.0
 To remove the impurities in Glutaraldehyde which are polymers of
glutaraldehyde (eg Acrolein, Ethanol, Glutaric acid etc)
Charcoal is added.
 For fixation 2.5 % to 4% conc. is required.
▶ Advantages
1. Formation of more cross linkages with better preservation of
cellular
& fluid proteins
2. Resists acid hydrolysis
3. Causes less shrinkage than formalin
4. More pleasant & less irritant
5. Does not cause dermatitis
▶ Disadvantages:
1. Expensive
2. Less stable
3. Penetrates tissue more slowly from formalin
4. Inferior formalin for PAS satin.
▶ Formal Mercuric chloride
 Mercuric chloride -30g
 Distilled water -900ml
 Formalin -100ml
ADVANTAGES:
i) It precipitates the proteins and hardens the tissue.
ii) Has beneficial effect on staining.
iii) Causes neither Shrinkage nor Swelling.
DISADVANTAGES
iv)It damages the tissue lipids.
v) It is difficult to make frozen sections after fixing with
Mercuric
Chloride.
▶ OSMIUM TETROXIDE
 Used in electron microscopy
 Used in fixing material for ultrathin sections for
electron microscopy
ADVANTAGES:
i) It fixes fats, conjugated lipids and mitochondria.
ii) It preserves all the details of tissues.
iii) Excellent fixative for the Electron microscopy.
DISADVANTAGES:
iv)May produce Black coloration on the tissue.
v) It is very expensive.
vi) Its vapors are irritating and can cause Conjunctivitis.
▶ PICRIC ACID
 It gives better preservation of alcohol
 Picricacid forms protein picrates, some of which are
water soluble until treated with alcohol
ADVANTAGES:
i) It precipitates & combines with proteins to form picrates.
ii) Preferred fixative for connective tissues.
iii) Prevents over hardening of tissue during dehydration.
iv) Preserves glycogen well & does not shrink the tissues.
DISADVANTAGES:
v) It does not fix the carbohydrates.
vi) Picric acid is Highly explosive.
▶ Chromic acid:
 Chromic acid is a strong oxidizer hence used with
other fixatives, but not alcohols and formalin.
 Coagulate proteins and fixes carbohydrates.
 If tissue is not washed well after fixation in chromic
acid, an insoluble precipitates will be formed.
▶ Potassium dichromate
 Potassium dichromate is an orange crystalline substance
used at 2% solution with water, fixes tissue by oxidizing
proteins.
 If mixed with ethanol it forms insoluble lower oxide that
can not be removed from issue.
 Tissue fixed in potassium dichromate must be washed
thoroughly in water before commencing
dehydration in alcohols.
COMPOUND FIXATIVES

▶ 10% of buffered neutral formalin

 Water -900ml
 NaH2Po4 (anhydrous) -3.5gm
 Na2HPo4 (anhydrous)-6.5gm
 Formalin -100ml
 Hydrated salts –
 NaH2Po4.H2O-4.02g

 Na2HPo4 .12H2O-16.37g/l
ADVANTAGES:
i) It fixes proteins without precipitation.
ii) Fats are preserved and can be stained by suitable methods.
Formalin pigment is not formed.
DISADVANTAGES:
iii)High strength of formalin can causes the shrinkage of
tissues.
iv) Over-exposure may over-hardens the tissue.
▶ HEIDENHAIN SUSA
 HgCl2 -45gm
 Nacl – 5gm
 Formalin (40% formaldehyde solution )- 200 ml
 Glacial acetic acid – 40 ml
 Trichloroacetic acid – 20 gm
 Distilled water – 800 ml
ADVANTAGES:
i) Tissues are fixed quickly.
ii) Gives Rapid and even penetration with minimum Shrinkage.
DISADVANTAGES:
iii)Over exposure can bleaches the tissue and over hardens it.
iv) Tissue requires a treatment with iodine to remove mercury
pigments.
▶ CARNOY's FLUID
 Absolute ethylalcohol – 60 ml
 Choloform – 30ml
 Glacial acetic acid -10ml
ADVANTAGES:
i) It is one of the most penetrating fixative.
ii) It rapidly fixes the tissue.
iii) After fixation the tissues can be directly transferred to 90-
100%
Alcohol.
DISADVANTAGES:
iv)It causes lysis of Red blood cells and much shrinkage.
v) Some cytoplasmic granules may be preserved.
▶ BOUIN's FLUID
 1.2% aqueous picric acid -75ml

 Formalin – 25ml
 Glacial acetic acid – 5ml

ADVANTAGES:
i) It penetrates evenly and rapidly.
ii) Causes less shrinkage and can be used to demonstrate glycogen.
iii) Tissues may be left in it for months without any harm.
DISADVANTAGES:
iv)It is not suitable for tissues containing mucin, since it
becomes greatly swollen.
v) The cortex of Kidney is badly preserved.
vi)It is necessary to remove excess picric acid by washing or
by alcohol treatment.
▶ ZENKER's FLUID
 HgCl2 – 50gm
 Potassium dichromate – 25gm
 Sodium sulphate – 10gm
 Distilled water – 1000ml
 Add 50ml glacial acetic acid before use (5 ml/dl of
stock)
ADVANTAGES:
i) It rapidly and evenly penetrates the tissue.
ii) It is a good routine fixative.
DISADVANTAGES:
iii)It is unstable after the addition of Acetic acid, hence acetic acid
(or formalin) should be added just before use.
 Unit-5
Processing (by Paraffin
Technique)
▶ Tissue processing: A procedure which need to take place after
gross examination between tissue fixation and the embedding
and then sectioning of paraffin blocks is called tissue processing.

There are some basic steps for tissue processing:

 Dehydration
 Clearing/Dealcoholization
 Infilteration and impregnation
 Paraffin embedding
 Sectioning
 staining
DEHYDRATION
▶ The first stage in tissue processing is dehydration (the removal of
water). In tissues, water is present in both free and bound forms and
needs to be removed before processing can continue.
▶ Dehydration is usually carried out using alcohols (such as ethanol)
but these can dissolve certain cellular components such as lipids.
▶ Although dehydration can also cause tissue shrinkage, the stage
is necessary in all infiltration methods, except where tissues are
supported by an aqueous embedding medium (such as water-soluble
waxes).
▶ In paraffin wax processing, dehydration from aqueous fixatives such
as formalin is usually initiated in 70% alcohol before progressing
through 90%-95% to absolute alcohol before proceeding to the
clearing stage.
CLEARING
▶ Clearing is the transition step between dehydration and infiltration
with the embedding medium.
▶ The term clearing arises because some solvents have a high refractive
index. When dehydrated tissues are placed into these reagents, they
are rendered transparent.
▶ This property is used to determine the endpoint and duration of the
clearing step since the presence of opaque areas indicates incomplete
dehydration. Clearing agents are fat solvents and therefore remove
fat from the tissue.
▶ It must be noted that shrinkage occurs when tissues are transferred from
the dehydrating agent to the clearing agent and from the clearing agent
to wax.
▶ In the final stage shrinkage may result from the extraction of
fat by the clearing agent.
▶ Xylene is the most popular clearing agent and several changes
of it are required to completely displace the ethanol.
▶ The choice of a clearing agent depends upon the type of tissue
processor used, the processing conditions such as temperature,
safety factors and cost.
Infiltration
▶ This is the saturation of tissue cavities and cells by a supporting
substance which is generally the medium in which they are
finally embedded.
▶ The most common agent of choice is paraffin wax which is
molten when hot and solid when cold.
▶ An infiltrating and embedding medium should ideally be molten
between 30°C and 60°C and suitable for sectioning.
▶ Additionally, the properties of the medium should be similar to those
of the tissues to be sectioned with regard to density and elasticity.
▶ Various substances have been used to infiltrate and embed tissues in
readiness for eventual section cutting or microtomy.
Embedding
▶ Paraffin embedding is the standard method used in histology
laboratories to produce blocks of tissue for section cutting
(microtomy).
▶ After tissue have been dehydrated, cleared and infiltrating
with
embedding material like paraffin ,agar ,gelatin, which is then
hardened.
▶ This is achieved by placing tissue in a metalic angle or leuckharts
moulds and cooling in case of paraffin and heating in case of
epoxy resin.
▶ In case of automated tissue processor tissues are still in the casettes
and pick the tissue out of the casettes and pour molten paraffin
over them.
MICROTOMY
▶ A microtome is a tool used to cut extremely thin slices or sections
of tissue for light microscopy studies.
▶ The most commonly used microtomes in the histology laboratory
are the rotary and sledge varieties (see images below). Microtomes
use steel, glass, or diamond blades depending upon the specimen
and thickness of the section required.
▶ Nowadays, disposable steel blades are generally used to
prepare paraffin sections of tissues for light microscopy
histology.
AUTOMATIC TISSUE PROCESSOR MACHINE
(ATPM)

▶ A tissue processor is a device that prepares tissue samples

for sectioning and microscopic examination in the diagnostic
laboratory.
▶ Microscopic analysis of cells and tissues requires the preparation
of very thin, high quality sections (slices) mounted on glass
slides and appropriately stained to demonstrate normal and
abnormal structures.
▶ The ATP machine plays a big role in the preparation of the
tissue by passing them through various chemicals; a major
process called TISSUE PROCESSING.
TISSUE PROCESSING

▶ The ATPM works by following through an already established

processing steps.
▶ Tissues to be processed are cut into small pieces to ensure the
tissue fits into the tissue cassettes.
▶ Smaller tissues (2-4 um) will be processed faster than the whole
tissue or organ.
▶ These tissue cassettes are packed into the oscillating tissue basket
to tissue prior to fixation.
▶ FIXATION – this is the process of preserving or fixing tissues
by passing them through chemicals called fixatives. The fixatives will
help protect the tissue from decay and autolysis. Routine fixative of use
is 10% formalin.
▶ DEHYDRATION – this is the process of removing water molecules
from the tissue by passing the tissue through ascending grades of
alcohol. E.g methanol, acetone, 70-100% alcohol.
▶ CLEARING – this is the process of removing alcohol from the tissue
by passing it through chemicals that will remove the alcohol
molecules. These agents are called clearing agents. Xylene is mostly
used for clearing.
▶ INFILTRATION – this is the process of filling intracellular spaces left
in the tissue by paraffin wax. This will help confer a bit of rigidity to
the processed tissue.
▶ EMBEDDING- this last step is manually done. This has to do with
immersing the processed tissue into a mould containing liquid
paraffin wax. This is for external support so that the tissue won’t
crumble during microtomy
PARTS OF THE ATPM
▶ Oscillating tissue basket
▶ 10 beakers or jars
▶ 2 thermostatically controlled beakers
▶ An electric rotor at the base
▶ Lifting mechanism
▶ Time disc and alarm system
▶ Control unit - with display screen and control
buttons
WORKING PRINCIPLE OF AUTOMATIC TISSUE
PROCESSOR MACHINE

▶ The tissue basket oscillates up and down in each station at

three- second intervals to ensure thorough and even mixing of
the reagents and optimum tissue infiltration.
▶ Infiltration time is separately programmable for each station.
Up to nine programs may be run with immediate or delayed
starting times.
▶ When it’s time for tissue to be transferred to the next beaker
or jar, the cover of the machine is raised up, and the lifting
mechanism carefully removes the tissue basket and gently
transfers it to the next beaker.
▶ When the infiltration time for any particular station is exceeded,
a warning message will display, indicating the station number
and excess time.
▶ Controls are arranged by functionality with an LCD to indicate
operational parameters. Reagent container lids have seals to
minimize operator exposure to hazardous fumes.
▶ Tissue basket immediately immerses in a station in the event
of
power loss to protect samples from drying out.
▶ When power is restored, program will resume. In the
event of long-term power failure, wax is liquified. Capacity
of tissue basket is 80 cassettes.
▶ Vacuum configurations hasten infiltration, allowing pressure to be
applied to any station in either manual or automatic operation.
▶ Fume control configurations extract fumes with a fan and pass
them through an internal carbon filter.
▶ For added efficiency, these models feature a two-part containment
shield surrounding the reagent container platform.
ADVANTAGES OF ATPM
▶ It’s very efficient
▶ Saves time and energy to operate
▶ Cost effective and user friendly
▶ Can process different tissues same time
▶ The machine does the transfer of tissue from one bath to
another.
 Unit-7
Theory of staining
(Routin
e)
H&E Staining
▶ For routine diagnosis, the use of Hematoxylin and Eosin (H&E)
is by far preferred for viewing cellular and tissue structure
detail by pathologists.
▶ The variation of stain intensity is often driven by the
pathologist’s learning experience and personal preference.
▶ Because this stain demonstrates such a broad range of
cytoplasmic, nuclear, and extracellular matrix features, nearly
all teaching texts use H&E images.
▶ In a high quality H&E there are subtle differences in the
shades of color produced by the stains, particularly eosin, and
this aids in the detection and interpretation of morphological
changes associated with disease.
The staining procedure for H&E follows a
basic protocol
 Dewaxing

 Dehydration

 Hematoxylin

 Differentiation

 Bluing

 Eosin

 Dehydration

 Clearing

 Cover-slipping
Remove the Wax

▶ Following the preparation of a paraffin section, all the

elements are infiltrated with and surrounded by paraffin wax
which is hydrophobic and impervious to aqueous reagents.
▶ The majority of cell and tissue components have no natural
color and are not visible.
▶ The first step in performing an H&E stain is to dissolve all
the wax away with xylene (a hydrocarbon solvent).
Hydrate the Section
▶ After thorough de-waxing, the slide is passed through several
changes of alcohol to remove the xylene, then thoroughly
rinsed in water. The section is now hydrated so that aqueous
reagents will readily penetrate the cells and tissue elements.

Apply the Hematoxylin Nuclear Stain

▶ The slide is now stained with a nuclear stain such as Harris

hematoxylin, which consists of a dye (oxidized hematoxylin or
hematein) and a mordant or binding agent (an aluminium salt) in
solution. Initially this stains the nuclei and some other elements
a reddish-purple color.
Complete the Nuclear Stain by “Blueing”

▶ After rinsing in tap water, the section is “blued” by treatment

with a weakly alkaline solution.
▶ This step converts the hematoxylin to a dark blue color.
▶ The section can now be rinsed and checked to see if the
nuclei are properly stained, showing adequate contrast and to
assess the level of background stain.
Remove Excess Background Stain
(Differentiate)

▶ On most occasions when Harris hematoxylin is employed, a

differentiation (de-staining) step is required to remove non-
specific background staining and to improve contrast.
▶ A weak acid alcohol is used.
▶ After this treatment, blueing and thorough rinsing is
again required.
▶ Staining methods that include a de-staining or differentiation
step are referred to as “regressive” stains.
Apply the Eosin Counterstain
▶ The section is now stained with an aqueous or alcoholic solution of
eosin (depending on personal preference).
▶ This colors many non-nuclear elements in different shades of pink.
Rinse, Dehydrate, Clear and Mount (Apply Cover Glass)
▶ Following the eosin stain, the slide is passed through several
changes of alcohol to remove all traces of water, then rinsed
in several baths of xylene which “clears” the tissue and
renders it completely transparent.
▶ A thin layer of polystyrene mountant is applied, followed by
a glass cover slip. If the stain and all the subsequent steps have been
properly performed, the slide will reveal all the important
microscopic components and be stable for many years.
 Unit-8
Mountants
▶ Mountant any substance in which a specimen is
suspended between a slide and a cover glass for microscopic
examination.
▶ The mounting medium is the solution in which the specimen is
embedded, generally under a cover glass. It may be liquid, gum
or resinous, soluble in water, alcohol or other solvents and be
sealed from the external atmosphere by non-soluble ringing
media.
▶ The main purpose of mounting media is to physically protect
the specimen; the mounting medium bonds specimen, slide and
coverslip together with a clear durable film. The medium is
important for the image formation as it affects the specimen's
rendition.
Properties of an Ideal Mounting
Media (Mountant)
▶ It should be colorless and transparent.
▶ It should not cause stain to diffuse or fade.
▶ It should be dry to a non-stick consistency and harden relatively quickly.
▶ It should not shrink back from the edge of cover-glass.
▶ It should be able to completely permeate and fill tissue interstices.
▶ It should have no adverse effect on tissue components.
▶ It should be resistant to contamination (particularly
microorganism growth).
▶ It should protect the section from physical damage and chemical
activity.
▶ It should be completely miscible with dehydrant or clearing agent.
Classification of Mounting Media

1. Resinous media
2. Aqueous media
Resinous media
▶ These are natural resins such as Canada balsam and gum
dammar.
For many years these were used for mounting sections.
▶ These natural resins usually dissolve in xylene.
▶ They are inherently acidic and caused fadingof some stains after
the sections were stored for several years.
▶ They also set very slowly. Sometimes taking months to harden
to non-stickiness. They also tend to yellow with age.
▶ Resinous media consists of solid resins which are dissolved in
an appropriate solvent.
▶ The viscosity of the medium should be such that the solution
will enter the tissue spaces and flow readily between the slide
and the cover glass.
▶ Air bubbles should be removed quickly. Most resinous
media are dissolved in toluene. Because slides are usually
mounted from xylene, xylene should be the solvent for the
mounting media.
▶ Toluene is more volatile than xylene so bubbles are more likely
to appear.
AQUEOUS MOUNTING MEDIA
▶ Aqueous mounting media are used when dehydrating and clearing
will adversely affect the stain.
▶ They can be classified for use in histology as simple syrups, gum
arabic media, and glycerol gelatins.
▶ Both gum arabic and glycerol gelatins media cause, or allow diffusion
of basic aniline dyes into the surrounding medium. This can be
prevented by adding large amounts of sugar (sucrose), fructose, or D-
sorbitol, to the gum Arabic or glycerol gelatin media.
▶ The syrups remain wet and sticky in most climates and will only serve
as temporary mounting media. Aqueous mounting media have an
index of the fraction that differs greatly from that of the tissue.
 Unit-9
Various Terms
associated with staining
▶ Solvent: A solvent is a substance that becomes a solution by
dissolving a solid, liquid, or gaseous solute. A solvent is usually a
liquid, but can also be a solid or gas. The most common solvent
in everyday life is water. Most other commonly-used solvents are
organic (carbon-containing) chemicals.
▶ Mordant: A mordant or dye fixative is a substance used to set
dyes on fabrics by forming a coordination complex with the dye,
which then attaches to the fabric. It may be used for dyeing fabrics
or for intensifying stains in cell or tissue preparations.
▶ Progressive staining: It stain to a desired intensity and no more.
Therefore they do not require differentiation in a dilute acid alcohol.
▶ Regressive staining:It means that the tissue is deliberately
over stained and then de-stained (differentiated) until the proper
endpoint is reached.
▶ Accelerators: An accentuator is any chemical which facilitates
the staining process. Usually the purpose is to intensify staining,
and accentuation with this meaning is obviously the derivation
of the term. However, it should be noted that inhibition of
staining can also accentuate a structure in comparison to the
background staining.
▶ Metachromasia: It is a characteristical change in the color of
staining carried out in biological tissues, exhibited by certain
dyes when they bind to particular substances present in these
tissues, called chromotropes. For example, toluidine blue
becomes dark blue when bound to cartilage.
Unit-10
Cell
Cell:

▶ “A cell is defined as the smallest, basic unit of life that is

responsible for all of life’s processes.”
▶ Cells are the structural, functional, and biological units of all
living beings. A cell can replicate itself independently. Hence, they
are known as the building blocks of life.
▶ Each cell contains a fluid called the cytoplasm, which is enclosed by
a membrane. Also present in the cytoplasm are several biomolecules
like proteins, nucleic acids and lipids.
▶ Moreover, cellular structures called cell organelles are
suspended in the cytoplasm.
Structure of cell:
1. Cell Membrane
▶ The cell membrane supports and protects the cell. It controls the
movement of substances in and out of the cells. It separates the cell from
the external environment. The cell membrane is present in all the cells.
▶ The cell membrane is the outer covering of a cell within which all
other organelles, such as the cytoplasm and nucleus, are enclosed. It is
also referred to as the plasma membrane.
▶ By structure, it is a porous membrane (with pores) which permit the
movement of selective substances in and out of the cell. Besides this,
the cell membrane also protects the cellular component from damage and
leakage.
▶ It forms the wall-like structure between two cells as well as between
the cell and its surroundings.
▶ Plants are immobile, so their cell structures are well-adapted to
protect from them from external factors. The cell wall helps to
reinforce this function.
2. Cell Wall

▶ The cell wall is the most prominent part of the plant’s cell structure.
It is made up of cellulose, hemicellulose and pectin.
▶ The cell wall is present exclusively in plant cells. It protects the
plasma membrane and other cellular components. The cell wall is also
the outermost layer of plant cells.
▶ It is a rigid and stiff structure surrounding the cell membrane.
▶ It provides shape and support to the cells and protects them from
mechanical shocks and injuries.
3. Cytoplasm

▶ The cytoplasm is a thick, clear, jelly-like substance present

inside the cell membrane.
▶ Most of the chemical reactions within a cell take place in
this cytoplasm.
▶ The cell organelles such as endoplasmic reticulum,
vacuoles, mitochondria, ribosomes, are suspended in this
cytoplasm.
4. Nucleus
▶ The nucleus contains the hereditary material of the cell, the
DNA.
▶ It sends signals to the cells to grow, mature, divide and die.
▶ The nucleus is surrounded by the nuclear envelope that
separates the DNA from the rest of the cell.
▶ The nucleus protects the DNA and is an integral
component of a plant’s cell structure.
5. Nucleolus
▶ The nucleolus is the site of ribosome synthesis. Also, it is involved in
controlling cellular activities and cellular reproduction
6. Nuclear membrane
▶ The nuclear membrane protects the nucleus by forming a
boundary between the nucleus and other cell organelles.
7. Chromosomes
▶ Chromosomes play a crucial role in determining the sex of an
individual.
Each human cells contain 23 pairs of chromosomes
8. Endoplasmic reticulum
▶ The endoplasmic reticulum is involved in the transportation of
substances throughout the cell. It plays a primary role in the metabolism
of carbohydrates, synthesis of lipids, steroids, and proteins.
9. Golgi Bodies
▶ Golgi bodies are called the cell’s post office as it is involved in the
transportation of materials within the cell
10. Ribosome
▶ Ribosomes are the protein synthesisers of the cell
11. Mitochondria
▶ The mitochondrion is called “the powerhouse of the cell.” It is called
so because it produces ATP – the cell’s energy currency
12. Lysosomes
▶ Lysosomes protect the cell by engulfing the foreign bodies entering the cell
and helps in cell renewal. Therefore, it is known as the cell’s suicide bags.
13. Vacuoles
▶ Vacuoles stores food, water, and other waste materials in the cell
Function of Cells
A cell performs these major functions essential for the growth
and development of an organism. Important functions of cell are
as follows:
▶ Provides Support and Structure
All the organisms are made up of cells. They form the structural
basis of all the organisms. The cell wall and the cell membrane are
the main components that function to provide support and
structure to the organism. For eg., the skin is made up of a large
number of cells.
▶ Facilitate Growth Mitosis
In the process of mitosis, the parent cell divides into the
daughter cells. Thus, the cells multiply and facilitate the growth
in an organism.
▶ Allows Transport of Substances
Various nutrients are imported by the cells to carry out various chemical
processes going on inside the cells. The waste produced by the
chemical processes is eliminated from the cells by active and passive
transport.
Small molecules such as oxygen, carbon dioxide, and ethanol diffuse across
the cell membrane along the concentration gradient. This is known as
passive transport. The larger molecules diffuse across the cell membrane
through active transport where the cells require a lot of energy to transport
the substances.
▶ Energy Production
Cells require energy to carry out various chemical processes. This energy
is produced by the cells through a process called photosynthesis in plants
and respiration in animals.
▶ Aids in Reproduction
A cell aids in reproduction through the processes called mitosis
and meiosis. Mitosis is termed as the asexual reproduction where
the parent cell divides to form daughter cells.
Meiosis causes the daughter cells to be genetically different from
the parent cells. Thus, we can understand why cells are known as
the structural and functional unit of life.
This is because they are responsible for providing structure to the
organisms and performs several functions necessary for carrying
out life’s processes.
Cells division
There are two types of cell division:
1. Mitosis
2. Meiosis.
▶ Mitosis: It is a fundamental process for life. During mitosis, a cell
duplicates all of its contents, including its chromosomes, and splits to
form two identical daughter cells.
Because this process is so critical, the steps of mitosis are carefully
controlled by a number of genes. When mitosis is not regulated
correctly, health problems such as cancer can result.
▶ Meiosis: The other type of cell division, meiosis,
ensures that humans have the same number of chromosomes
in each generation.
It is a two-step process that reduces the chromosome number
by
half—from 46 to 23—to form sperm and egg cells.
When the sperm and egg cells unite at conception, each
contributes 23 chromosomes so the resulting embryo will
have the usual 46.
Meiosis also allows genetic variation through a process of DNA
shuffling while the cells are dividing.
 Unit-11
Exfoliative Cytology
Exfoliative Cytology:

▶ It is the study of cells that have been shed or removed from

the epithelial surface of various organs.
▶ Cells from all organs, which communicate with the exterior of
the
body, are suitable for study.
▶ These cells can be recovered either from natural secretions
such as urine, sputum and vaginal or prostate fluids or by
artificial means such as paracentesis or lavage.
▶ The cells can be collected from the epithelial surfaces by
lightly scraping the surface, by swabbing, aspirating or washing
the surfaces.
Collection and Processing of specimen
for cytology:
▶ 1. Cervical smears:-
Cervical smears are made from material collected with help of a speculum
( a metal or plastic device ) which is inserted into the vagina and allows the
uterine cervix to be readily visible. A specialized spatula known as the Ayre
spatula or cervical spatula is used for collection. The collection is made at
the junction of the columnar epithelium by visualizing the cervix, the spatula
is inserted via the speculum into the cervical os and rotated through 360
degrees.smeared over a pre-labelled microscope slide and fixed immediately.
It is ideal for detection of cervical carcinoma.
▶ 2. Aspiration from the posterior fornix:- With the aid of a
speculum, cellular material is collected from the posterior fornix,
using a disposable plastic pipette with a suction bulb. Following
aspiration, smears are prepared and fixed immediately.
▶ 3. Vaginal smears:- Vaginal smears are valuable for the assessment of
hormonal function. Cellular material is collected by scraping the
upper third of the lateral wall of the vagina with a wooden spatula.
The cells are evenly and thinly smeared over a clean pre-labelled
microscope slide and fixed.
▶ 4. Endocervical smears:- This is used mainly for follow up
cases where a surgical treatment has been used after a cone
biopsy has been taken for assessment of dysplasia and
malignancy or as a curative procedure. A cotton tip swab is
inserted into the endocervix and rotated gently to cover a wide
area of the endocervix. The material collected is smeared on a
clean pre- labelled microscope slide and fixed.
▶ 5. Endometrial aspiration:- This procedure has to be performed
under strict aseptic conditions so as not to introduce infection
into the patient. A cannula is inserted into the uterine cavity and
the cellular material is aspirated using a syringe. Thin smears are
made on clean pre-labelled slides and fixed.
NON.GYNAECOLOGICAL CYTOLOGY:-
This aspect of cytology involves the study of cells suspended in body
fluids. The specimen are varied and taken from various parts of the
body.
▶ 1. Sputum:-
 Sputum specimen is valuable for the study of respiratory tract
disorders. It is used in the dingnose of the following
abnormal conditions:
A) Malignant disease of the lower respiratory tract.
B) Pulmonary asbestosis.
C)Pulmonary inflammatory conditions due to fungal
infection, bacterial infection, viral infection or parasitic
infection.
▶ It is normally collected as early morning deep cough specimens and
is preferably submitted on three consecutive days. It is not advisable
to collect sputum specimen after a recent bronchoscopy has been
done.
▶ Preparation of smears:-
 Sputum must be processed in a biological safety cabinet. Purulent
or blood stained particles are selected from the sputum with a
microbiological wire loop and used to make thin smears.
 Bronchial washings are usually submitted in sterile containers. They
are centrifuged without delay and smears made from the sediment.
They can also be spun at 150 rpm for 10 minutes in a cytocentrifuge
directly onto a clean prelabelled microscope slides and fixed
immediately.
▶ Fixation:- Fixation should be carried out while the smear are still
▶ 2) Pleural fluid and ascitic fluid:-
 These are serious fluids that normaly lubricate the wall of pleural,
pericardial fluid, synovial fluid. CSF and peritoneal cavities. They
increase in volume and contain cells under certain pathological
conditions. Cytological examination of these fluids reveal malignant
cells which may arise from tumours of the surrounding
mesothelium or they be metastatic deposits.
▶ Collection and preparation of smears:
 By means of a needle or canula with an attached syringe, the
specimens are aspirated from the pleural or peritoneal cavities. The
aspirated material is transferred into a sterile container and sent to the
laboratory. The specimens are centrifuged at 800 rpm for 10 minutes or
cytospun at 1500 rpm for 10 minutes and thin smears made, at least two
smears from each specimen. Any clots that are formed are fixed and
histological lab.
▶ Fixation:- The choice of stain is Romanowskv, then smear should be air
dried and then fixed with methanol.
▶ 3. Urine:-
 Urine cytology is of great value in the diagnosis of urethral
tumours, urinary bladder carcinoma, carcinoma of the kidney
and carcinoma of the prostate in males. Normal urine
contains few or no cells; but under certain pathological conditions,
the urine contains many abnormal cells. Early morning
specimens of urine are preferred because they give larger
concentration of cells due to relatively long residence in the
bladder.
▶ Fixation:- Urine tends to wash of slide during fixation and staining
due to the low protein content.
 Unit-12
Fixation (Cytological
Specimen)
Fixation of Cytology Specimens
▶ Fixation means prevention of degeneration of cells and tissue by
the autolytic enzymes present in the cells and preservation of cells
as close as possible to the living state.
▶ To achieve this smears are placed in the fixative solutions for
specific periods of time before the staining procedure is started.
▶ Fixation changes the physical and chemical state of the cells and
determines the subsequent staining reactions that could be carried
out on the smears.
VARIOUS TYPES OF CYTOLOGICAL
FIXATIVES:
▶ These fixatives can be subdivided
into:
1. (A) Nuclear fixative and
2. (B) Cytoplasmic fixatives.
1. Nuclear fixatives:
▶ 1. Carnoy's fluid
a) Absolute alcohol = 60 ml
b) Chloroform 30 ml
c) Glacial acetic acid = 10 ml
▶ Specific features :-
 It penetrates very rapidly and gives excellent nuclear fixation.
 Nissl substance and glycogen are preserved.
 Good fixative for carbohydrates.
 It causes considerable shrinkage.
 It destroys or dissolves most cytoplasmic elements.
 It causes haemolysis of erythrocyte.
 It is used for urgent biopsy.
 Fixative is usually complete in 12 hours. ( small pieces 23 mm
thick require 15 minutes for fixation).
▶ 2) Clarke's fluid :-
 Absolute alcohol = 75 ml
 Glacial acetic acid = 25 ml
▶ Specific features :-
 This fixative penetrates rapidly, gives good nuclear fixation
and effects preservation of cytoplasmic elements.
 Itis an excellent fixative for smears or coverslip preparations
of cell cultures for general fixation and chromosome
analysis.
▶ 3. Newcomer's fluid:
 Isopropanol = 60 ml
 Propionic acid = 40 ml
 Petroleum either = 10 m)
 Acetone = 10
 Dioxane = 10 ml
▶ SPECIFIC FEATURES
 This fixative penetrates rapidly and preserves
the chromatin better than Carnoy's fluid.
 It is a good fixative for the preservation
of mucopolysaccharides.
▶ 4. Alcohol — ether ( Equal volumes of 95% alcohol
and ether):-
 This is the routinely used cytological fixative for wet smears.
 It is specially recommended for use with the Papanicolaou
staining technique.
 Smears are fixed within 30 minutes but can be left in the
fixative for longer period.
 Smears are rinsed in water before staining.

▶ 1) Alcohol-ether fixative :-
 Absolute ethyl alcohol — 50 ml
 Ether 50 ml
Mix and place in a jar with a tight stopper. Fixation is carried out for
about 30 minutes, Followed by a rinse in alcohol and then the section
is taken to water.
▶ 5. Schaudinn's Fluid:-ltis prepared as
follows:
 Saturated mercuric chloride solution 60 ml

 Absolute alcohol 33ml

 Glacial acetic acid I ml
Fixation is carried out for about 2 minutes.afterwashing in
distilled water, the mercuric chloride black clumps are
removed by adding a few drops of saturated alcoholic iodine
solution. After rinsing in water the smear is taken for
staining
2. Cytoplasmic fixative:
▶ 1) Champy's fluid :-
3 g/dl potassium dichromate = 7 ml
 1% (v/v) chromic acid = 7 ml
2 g/dl, osmium tetroxide = 4 ml
▶ Specific features :-
 This fixative cannot be stored, hence should be
prepared fresh before use. It penetrates poorly and
unevenly.
 It preserves mitochondria, fat, yolk and lipids.
 Tissue must be washed overnight after fixation.
▶ 2) Flemming's fluid :-
 Osmium tetroxide= 2gm
Distilled water = 100 ml
 Chromium trioxide = 1 gm
Distilled water
= 100 ml
▶ Working solution:-
 Solution A = 16 ml
 Solution B =60ml
 Acetic acid = 4
ml
▶ This perhaps is the most widely used fixative for the preservation
of nuclear structures, especially chromosomes.
▶ the omission of acetic acid, the solution becomes a cytoplasmic
fixative. Small pieces of tissue not more n 2 mm in thickness,
are adequately fixed in 12-24 hours.
▶ It preserves fat permanently. It is a costly fixative.
▶ Flemmings fluid minus acetic acid is very good for
mitochondria and other cytoplasmic structures.
 Unit-13
Cytological Staining
Hematoxylin and eosin stain

▶ Hematoxylin and eosin staining technique functions to

recognize different types of tissues and their morphological
changes, especially in cancer diagnosis.
▶ Hematoxylin has a deep blue-purple color and stains
nucleic acids by a complex, incompletely understood
reaction.
▶ Eosin is pink and stains proteins nonspecifically. In a
typical tissue, nuclei are stained blue, whereas the cytoplasm
and extracellular matrix have varying degrees of pink
staining.
▶ Hematoxylin and eosin are both dyes have a high affinity for
tissues, depending on the Acidity and/or alkalinity of the
dyes.
▶ Principle
Eosin dye is acidic dye hence it as a negative charge. Therefore it stains the
basic structures of a cell (acidophils), giving them a red or pink color, for
example, the cytoplasm is positively charged, and therefore it will take up
the eosin dye, and appear pink.
Hematoxylin dyes are basic dyes, hence they are positively charged.Therefore
it will stain the acidic structures of tissues and cell structures purplish-blue.
Hematoxylin is not basic by itself. It has to be conjugated with a mordant
(aluminum salt) before it is used so as to strengthen its positive charge
for efficiency in binding to the tissue components.
The mordant, which also defines the color of the stain, will bind to the
tissue, then the hematoxylin will bind to the mordant to form a tissue-
mordant- hematoxylin complex link. This will stain the nuclei and chromatin
bodies purple.
▶ Reagents
▶ Distilled water
▶ Alum hematoxylin
▶ Acid alcohol
▶ Scott’s tap water
▶ Eosin dye
▶ Procedure
1. Clean the sections to distilled water.
2. Then stain nuclei with the alum hematoxylin (Mayer’s) to fix the
tissue, for about 5 minutes.
3. Rinse the stain with smoothly running tap water
4. Using the differentiator, 0.3% acid alcohol, and note the endpoint i.e the
correct endpoint is after bluing up, the background becomes colorless.
5. Rinse the stain in smoothly running tap water.
6. Rinse the satin in Scott’s tap water substitute which shortens the
time for the correct end-point.
7. Rinse with running tap water
8. Flood the smear with eosin for 2 mins, and since eosin is highly
soluble in water, use enough quantify of it. The over stained eosin can
be removed or washed off with running tap water.
9. Dehydrate the smear, clear, and mount using a clean coverslip.
▶ Results and Interpretation
 Nuclei are stained blue
 cytoplasm and extracellular matrix have varying degrees
of pink staining.
MGG stain
▶ Introduction
May Grunwald Giemsa stain is one of many stains under the Romanowsky
staining procedure. It is a combination of two stains, May Grunwald stain
and Giemsa stain. Like other Romanowsky stains, the principle is the
same. It is used for bone marrow smear staining.
▶ Materials
May Grunwald dye
Absolute methanol
Giemsa dye
Glycerol
Phosphate
buffer pH
▶ Method
Preparation of May Grunwald stain
 Dissolve 0.3 g of May Grunwald dye in 100 mL absolute
methanol in a 250 mL conical flask.
 Warm the mixture to 50°C in a water bath for a few hours and
allow it to cool to room temperature.
 Stir the mixture on a magnetic stirrer and leave it stirring
for 24 hours.
 Filter the mixture and stain is ready for use.
▶ Preparation of Giemsa stain
 Add 1.0 g of Giemsa dye into 66 mL of glycerol and warm
the mixture in a conical flask for 1-2 hours at 50°C.
 Cool the mixture to room temperature and add 66 mL
of absolute methanol.
 Leave the mixture to dissolve for 2-3 days, mixing it
at intervals.
 The stain is then ready for use after filtering.
▶ Staining
1. Fix BM smears in absolute methanol for 10-15 minutes.
2. Prepare an equal volume of May Grunwald stain and phosphate buffer pH
6.8. Mix well and pour onto the slides to fully flood the slides. Stain for
10 minutes.
3. Prepare a 1:10 dilution of Giemsa stain with phosphate buffer pH 6.8.
Mix well.
4. After 10 minutes of May Grunwald staining, pour away the May Grunwald
stain off the slides.
5. Then pour the Giemsa mixture onto the slides and stain for another
15 minutes.
6. After 15 minutes, pour off stain and flush the slides with running tap
water.
7. Clean excess stain with kim wipes.
8. Air dry the slides. Place the long cover slip on the area of interest.
9. The slide is now ready for examination.
▶ Results :
1. methylene blue stains blue the acidic components of the
cell
2. eosin stains orange-red the alkaline components of the cell
3. Azure stains red and purple the basic cellular components
PAP stain
▶ Papanicolaou stain (also Papanicolaou’s stain or PAP stain) is the
most important stain utilized in the practice of Cytopathology. It
is a polychromatic stain containing multiple dyes to
differentially stain various components of the cells.
▶ This technique was developed by George Papanicolaou, the
father of Cytopathology. This method is used to differentiate cells
in the smear preparation of various gynecological specimens (pap
smears), materials containing exfoliative cells and material from
fine needle aspiration.
Principle of PAPANICOLAOU stain

▶ Papanicolaou stain includes both acidic and basic dyes. Acidic dye
stains the basic components of the cell and basic dye stain the acidic
components of the cell.
▶ The polychromatic PAP stain involves five dyes in three solutions.
▶ Hematoxylin : Natural dye hematoxylin is the nuclear stain which
stains cell nuclei blue. It has affinity for chromatin, attaching to
sulphate groups on the D.N.A. molecule. Harris’ hematoxylin is
the commonest cytologically although Gills’ hematoxylin and
Hematoxylin S can be used.
▶ Orange Green 6 : This is the first acidic counterstain (cytoplasmic
stain) which stains matured and keratinized cells. The target
structures are staine d orange in different intensities.
▶ Eosin Azure : This is the second counterstain which is a polychrome
mixture of eosin Y, light green SF and Bismarck brown. Eosin Y gives
a pink colour to cytoplasm of mature squamous cells, nucleoli, cilia and
red blood cells. Staining solutions commonly used in cytology are EA
31 and EA 50, while EA 65
▶ Light green SF stains blue to cytoplasm of metabolically active
cells like parabasal squamous cells, intermediate squamous cells and
columnar cells.
▶ Bismarck brown Y stains nothing and sometimes it is often
omitted.
Composition and preparation of reagents
▶ Harris’ hematoxylin :
Hematoxylin = 5g Ethanol =
50ml Potassium alum
= 100g
Distilled water (50°C) =
1000ml
Mercuric oxide = 2-5g Glacial
acetic acid = 40ml
▶ Orange G 6 :
Orange G (10% aqueous) = 50ml
Alcohol = 950ml
Phosphotungstic acid = 0-15g
 ▶ EA 50 :
0.04 M light green SF = 10ml
0.3M eosin Y = 20ml
Phosphotungstic acid = 2g
Alcohol = 750ml
Methanol = 250ml
Glacial acetic acid =
20ml
▶ Filter all stains before use.
Procedure of PAPANICOLAOU staining
1. 95% Ethanol 15 minutes (fixation)
2. Rinse in tap water
3. Harris or Gill Hematoxylin 1-3 minutes (Time vary with selection
of hematoxylin solution)
4. Rinse in tap water or Scott's tap water
5. 95% Ethanol 10 dips
6. OG-6 stain for 1.5 minutes.
7. 95% Ethanol 10 dips
8. EA-50, or Modified EA-50, or EA-65 stain for 2.5 minutes.
9. 95% Ethanol 10 dips, 2 changes
10. 100% Ethanol 1 minute
11. Clear in 2 changes of xylene, 2 minutes each
12. Mount with permanent mounting medium
Results and interpretation of PAPANICOLAOU
Staining
 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
 Unit-14
Role of Laminar airflow and
cytotechnician in cytology