1. The document discusses the various steps involved in tissue processing for microscopic examination, which includes fixation, processing, embedding, sectioning and staining of tissues. 2. Key steps include fixation of tissues using formalin to preserve structure, dehydration using increasing concentrations of alcohol, clearing with xylene, impregnation and embedding in paraffin wax. 3. Thin sections are then cut from the paraffin blocks using a microtome and stained, usually with hematoxylin and eosin, for microscopic examination.

1. BY Dr SURYAGOPAN.P

2.  ►The most commonly used method of examining
tissues microscopically is by sectioning and with the
exception of frozen sections of tissues which will
permit thin sections to be cut easily.

 ► Tissue for study can be obtained from:
 Biopsies
 Autopsies

3.  Specimen recieved in pathology laboratory is first adequately
fixed.
 A brief gross description of shape, size,colour,and other
morphological details of specimen are recorded in the
register.
 One or more small representitive bits of the whole
specimen are taken.

4.  The number designated in the register for the specimen
during the receipt of specimen is written on a piece of thick
filter paper using a pencil.
 Tissue bit along with its designated number is then wrapped
well in a square piece of filter paper and placed in a plastic
or metal perforated embedding cassette.
 After grossing the remainder of the specimen is put back in a
original container.

5.  Describing the specimen and placing all/parts of it
into a small plastic cassette which holds the tissue
while it is being processed to a paraffin block.
 Initially, the cassettes are placed into a fixative.

6.  • Proper identification and orientation of the specimen.
 • Unlabelled specimen should never be processed.
 • A properly completed histopathology requisition form
 containing patient’s name, age, sex, relevant clinical data,
 surgical findings, nature of operation and name of tissue
 submitted.
 • Careful search and examination of all the tissue submitted
 in order.

7. ●Should be insoluble
 • Should not contaminate
 • Should not penetrate
 • Should not react
 • Clearly identifiable
 • Eg:India ink, silver nitrate, alcian blue,
graphite pencil

8.  SPECIMEN SIZE
 AGITATION
 HEAT
 VISCOSITY
 VACUUM
 ULTRASONICS

9.  1.FIXATION
 2.DEHYDRATION
 3.CLEARING
 4.IMPREGNATION
 5.EMBEDDING
 6.SECTIONING
 7.STAINING
 8.MOUNTING

10.  It is a process in which a specimen is treated by exposing
it to a fixative for a particular period of time in order to
facilitate the succeeding steps.
 The purpose of fixation is to preserve tissues permanently
in as life-like a state as possible.
 The fixative should be 15 – 20 times more in volume then
the specimen.

11.  1. Prevent autolysis and putrefaction
 2.Preserve cells and tissues
 3.Make the cellular component insoluble to
reagent used in tissue processing
 4.It should penetrate and fix tissues rapidly
and evenly.
 5.To mildly harden tissues
 6.Devitalize or inactivate infectious agent
 7.Solidification of colloid materials

12. 1.ACCORDING TO THE
COMPONENTS PRESENT
2.ACCORDING TO THE TYPE
OF FIXATION/MODE OF
ACTION
3.DEPENDING ON THE
NATURE OF
SPECIMEN/MATERIAL USED

13. SIMPLE
•FORMALDEHYDE
•GLUTARALDEHYDE
•ETHYL ALCOHOL
COMPOUND
•FORMALIN BASED
FIXATIVES.
•MERCURIAL
FIXATIVES.
•DICHROMATE
FIXATIVES.
•PICRIC ACID
FIXATIVES.
ALCOHOL
CONTAINING
•CARNOY’S FLUID
•ACETIC ACID
FORMALIN.

14. NATURE OF CHEMICAL EXAMPLES
CHEMICAL METHODS
Aldehydes(cross-linking) Formaldehyde,glutaraldehyde,acrolein.
Protein denaturing
(coagulating/dehydrating ) agents
Acetic acid,methyl alcohol,ethyl
alcohol.
Oxidizing agents Osmium tetroxide, potassium
permanganate, potassium dichromate.
Other cross-linking agents. Carbodimides
PHYSICAL METHODS Heat, microwave
Unknownmechanism Mercuric chloride, picric acid

15. FIXATIVES USED IN HISTOPATHOLOGY
MICROANATOMICALFIXATIVES
-FORMALIN BASED FIXATIVES
-BUFFERED GLUTARALDEHYDE
-MERCURIC FIXATIVE
-DICHROMATE FIXATIVES
-PICRIC ACID FIXATIVES.
HISTOCHEMICALFIXATIVES
-Formal saline
-Cold acetone
-Absolute alcohol
CYTOLOGICALFIXATIVES
●NUCLEAR FIXATIVES :
-Carnoy’s Fluid
-Clarke’s Fluid
-Newcomer’s Fluid
-Flemming’s Fluid
●CYTOPLASMIC FIXATIVES :
-Champy’s Fluid
-Regaud’s Fluid

16.  It is the process of removal of the calcium salts from
the specimen.
 The various agents used for decalcifying are;
 • Nitric acid
 • Hydrochloric acid
 • Formic acid
 • Picric acid
 • Acetic acid
 • Citric acid

17.  The most commonly used fixative is Formalin .
 It is prepared by mixing 40 % Formaldehyde gas in 100 w/v of
 distilled water.
 The resultant mixture is 100 % Formalin.
 Routinely, 10 % formalin is used which is prepared by mixing 10 ml of 100 %
formalin in 90 ml of distilled water.

18. MECHANISM OF ACTION:
 It forms cross links between amino acids of
proteins thereby making them insoluble.
 It fixes 4 mm thick tissue in 8 hours.

19.  ADVANTAGES :
 1. Rapid penetration
 2. Easy availability & cheap
 3. Does not overharden the tissue
 4. Fixes lipids for frozen sections
 5. Ideal for mailing
 DISADVANTAGES:
 1. Irritant to the nose,the eyes and mucous membranes
 2. Formation of precipitate of paraformaldehyde which
can be prevented by adding 11- 16 % methanol.
 3. Formation of black formalin pigment , Acid
formaldehyde hematin.

20.  It is the process in which the water content in the
tissue to be processed is completely reduced by
passing the tissue through increasing concentrations
of dehydrating agents.

21. DEHYDRATING AGENTS
 Ethanol- fast acting, electron microscopy specimen, clear colourless,
flamable liquid.
 Industrial Methylated Spirit -methonol 1%+IPA, Same physical property
as ethanol.
 Methanol - subsituted for ethanol,toxic, inflammable.
 Propan-2-01,Isopropyl alcohol - Microwave processing schedule,.
 Butyl alcohol - plant and animal histology, slow dehydrant
 Acetone -Rapid in action with poor penetration, clear colorless &
Inflammable liquid, Removes lipids & causes brittleness of the tissue.
 Universal Solvents -Tertiary butanol, Tetra hydro furan,
Dioxane,dehydrate+clear, not for delicate tissues.

22.  The duration of the procedure can be noted down as;
 1. 70 % alcohol – 1 hour
 2. 70 % alcohol – 1 hour
 3. 95 % alcohol – 1 hour
 4. 95 % alcohol – 1 hour
 5. Absolute alcohol – 1 hour
 6. Absolute alcohol – 1 hour
 7. Absolute alcohol – 1 hour

24.  It is the procedure where in the alcohol in the tissue is
replaced by a fluid which will dissolve the wax used for
impregnating the tissues .
The various clearing agents used are
Cedar wood oil : The best agent but
is expensive.
Benzene : It is carcinogenic.
Xylene : It is most commonly used.
Chloroform: Toxic and expensive.
Carbon tetrachloride

25. Rapid removal of dehydrating agent
Flammability Ease of removal by
melting paraffin
Disposal
Toxicity Minimal tissue damage

26.  Volume of clearing agent should be 50-100 times the volume of
tissue.
 In chloroform or carbon tetrachloride tissues are left over night for
clearing .
 In Xylene,benzene,toluene tissues are given one change after 30 to 60
minutes.
 Technique for cedar wood oil is different.

27.  TECHNIQUE:-
 Cedar wood oil poured into specimen jar & same quantity of absolute
alcohol superimposed on it .
 The specimen is placed into alcohol , it floats at the interface of two
fluids.
 As tissue is cleared it sinks into cedar wood oil.
 The alcohol is removed & specimen is transferred to fresh cedar wood
oil

28.  In processing for electron microscopy clearing
is done in 1,2 epoxy propane,2 changes 15 min
in each.
 ELECTRON MICROSCOPY
1,2 epoxy
propane
1,2 epoxy
propane
15 min15 min

29.  •Most common reagent used in processing of
CNS specimens.
 ADV: Tissue can be left in this with out any
damage for longer
 time, non-flammable.
 •DIS ADV:-
 ►Slower in action, Highly toxic.
 ► Does not effect refractive index of tissue.
 ► End point of clearing can not be determined.
 ► Expensive compared to other clearing agents.

30.  Is toxic.
 Similar action as chloroform.
 •Much cheaper.
 •Non inflamable.

31.  •Permeating the tissue with a supporting medium is
called Impregnation.
 The various waxes which are used are,
 1. Paraffin wax
 2. Paraplast
 3. Paraplast plus
 4. Gelatin
 5. Celloidin

32.  Impregnation with paraffin wax is carried out in a
oven heated to 54-56 degree.
 •The temperature depends upon the melting point of
wax used for impregnation.
 •Types of impregnation oven:
 Electric heated oven.
 Vacuum embedding oven.
 Gas heated oven.

33.  •Size & type of tissue.
 •Clearing agent employed.
 •Use of vacuum embedding oven.

34.  •After clearing the tissue is transferred to oven
maintained at 56 degree temperature.
 •Volume of wax should be 25-50 times the
volume of tissue.
 •Wax must be changed at least once during the
process.

35.  •Should be free from dust & foreign
 particles.
 •Should not contain water.
 •Melting point should be 54degrees.
 •Soft wax(45 dg) for foetal & areolar
 tissue.
 •Hard wax (60 dg) for hard & fibrous
 tissue

36.  It is done by transfering the tissue which has been
cleared of the alcohol to a mould filled with molten
wax & is allowed to cool & solidify.
 After solidification, a wax block is obtained
which is then sectioned to obtain ribbons.
EMBEDDING

37.  A. Leuckhart’s Moulds: L- shaped brass pieces
which is placed in opposing positions & can be
manipulated to increase or decrease the size of the
block to be prepared.
 B. Glass or Metal petri dishes :
 C. Watch glass
 D. Paper boats .

38.  It is the procedure in which the blocks which have
been prepared are cut or sectioned and thin strips of
varying thickness are prepared.
 The instrument by which this is done is called as a
 Microtome.
 TYPES OF MICROTOMES:
 • Sliding
 • Rotary
 • Rocking
 • Freezing
 • Base sledge

39.  •Mixture of purified paraffin &
plastic polymers.
 •Greater elasticity than paraffin .
 •Wrinkle free serial sections can
be cut.
 •Does not need ice application
while sectioning

40.  Staining of the section is done to bring out the
particular details in the tissue under study .
 The most commonly used stain in routine practice
is Haematoxylin & eosin stain.

41.  Procedure :
 1. Deparaffinization with xylene.
 2. Hydration
 3. Wash under water
 4. Stain with Haematoxylin for 15 min
 5. Wash with water
 6. Differentiate with 1 % acid alcohol
 7. Wash with water for 10 min
 8. Stain with 1% Eosin for 2 min
 9. Wash with water.
 10. Dehydration
 11. Clearing with xylene
 12. Dry
 13. Mount

42.  Adhesives used for fixing the sections on the
slides :
 Albumin solution ( Mayor’s egg albumin)
 Starch paste
 Gelatin

43.  Mountants :
 DPX ( Distrene Dibutyl phthalate Xylene ).
 Canada Balsam
 Colophonium resin
 Terpene resin

44.  Procedure:
 • Place the solution and paraffin in respective
 beakers of the equipment.
 • The timing leaver is set at zero and the machine
 at started at require time
 • The basket with the cassettes Automatically
 change position and takes a bath in different
 reagents kept in beakers in order to accomplish,
 dehydration, clearing, infiltration. the final dip in
 the warm paraffin.
 • Cassettes are opened next day morning for
 embedding.

45.  It is purified nitrocellulose.
 ADV:
BASE MOLDS FOR EMBEDDING TISSUE EMBEDDING CASSETTES

46.  Normally, the preparation of serial sections of celloidin-
impregnated tissue is a tedious procedure because the
sections will not adhere to one another in the same manner as
paraffin embedded material.
 Double embedding is a technique in which tissues are first
impregnated with celloidin and subsequently blocked in
paraffin wax.

47.  ADVANTAGES:
 1) serial sections can be easily prepared.
 2) an extra degree of resilience is given when
cutting hard tissues.
 DISADVANTAGES:
 It is tedious method.

48. •All the before mentioned procedures upto the impregnation step can be
done automatically in a single, unmanned instrument , which is the
Automated Tissue processor.

49.  •Transfer tissue both by day & night.
 •Reduce time in each fluid by continual
agitation.
 •The 24hr clock is provided.
 •Allows rapid processing.
 •Racks to carry 25 cassettes.
 •Eliminates human error.

50.  •Tissue containers:
 Has 24 containers subdivided into 2
 Compartments.
 •Beakers &Wax baths:
 Provided with 10 beakers for reagents & 2
 wax baths maintained at 56 degree temp.
 •Stirring Mechanism: One arm supports
stirring mechanism.

51.  • Tissue processing is a very much critical step that
needs to be monitored with utmost care.
 • Since it takes longer time to process the tissue, any
mistakes alters the tissues requiring repetition.
 • The tissues should not be under processed or over
processed that may hamper the tissue details.
 • It is essential that the tissues are processed with
proper techniques and rendered them for subsequent
steps..

52.  •Hand book of histopathologic techniques:
C.F.A. CULLING.
 Theory & practice of histological techniques:
JHON.D.BANCROT
 Histopathology technique and it’s
management: RAMADAS NAYAK