This document provides a comprehensive overview of histopathological techniques, focusing on tissue marking, fixation, decalcification, tissue processing, embedding, sectioning, and staining methods. It details processes such as the preparation of tissues, staining principles, and factors affecting the quality of stains, including the use of various dyes and mordants. Additionally, it outlines procedures for specific staining methods and the significance of mounting tissue sections for microscopic examination.

1. Histopathological Techniques
Tutorial for Exit Exam

2. Tissue marking
 Giving identification
(prevent loss of small
tissue)
 Cutting a notch
 Dye marking
( E.g. indian ink, Silver nitrate)
 Criteria for selection of
tissue marker
 Relatively insoluble
 Can’t cause reagent conta
 Remain on surface of
specimen
 Clearly identifiable

3. Samples for Histopathological Lab

4. Fixation
• Purpose is to preserve the
tissue structure
• First step in tissue preparation
• Aim:
 arrest autolysis
 Decomposition
 Coagulate the tissue ( make
tissue life-like state)
 Increase mechanical strength
• Fixatives can be classified
according to their;
1.Mechanism of action
2.Chemical nature
3.Combination

5. Determining fixation time of fixatives
• d= K x square root of T
Were d is depth of penetration
K is coefficient of diffusibility
T is period of immersion

6. Trimming
 After fixation
 To reach a sample size
 Hard tissues (such as
bones and teeth) must
be decalcified before
trimming.
Decalcification
 Complete removal of calcium
salt hard tissue following
fixation.
• Why decalcification?
1. Prevent tearing & ragging of
section
2. Prevent damage of microtome
knife/blades
3. Making bone soft
Achieved by:
• Dissolution of calcium by a
dilute mineral acid.
• Using Chelating agents EDTA
by eclectric current

8. Tissue processing
(Dehydration, clearing, impregnation)

13. Impregnation
• Make the tissue firm
• Facilitate easy sectioning
• 2 processes happen simultaneously
 Impregnation of the paraffin wax in to tissue
 Infiltration of clearing agent out of the tissue
• Also called internal embedding since medium penetrates
tissues & provides support from inside of tissue
• High melting point paraffin used

14. Embedding
• Gives external solid support
• Also called block preparation or external embeding
• Choice of paraffin depends on:
Nature of the tissue
Temp. at which tissues are sectioned
Thickness of the section

15. Question?
Q. 1. A senior lab technologist was performing
histopathological test in a histopathology lab. He
impregnated the dehydrated and cleared tissue with
molten paraffin wax before embedding. What
differentiates this impregnation process from
embedding?
A.Embedding for external support of tissue
B.Impregnation for external support of tissue
C.Impregnation is done by solid paraffin wax
D.Embedding is done by molten paraffin wax

16. Sectioning

17. Sectioning…
Water bath

18. Question?
• Q. 2. A fresh biopsy -5cm/hr was sent to a lab for
pathological examination. Lab tech. wanted to
make frozen section using cryostst. The cryostst
solidified the tissue in low temp.& produce
qualified thin tissue ribbons. Which activity can
the technologist perform by cryostat?
A. Dehydrating tissue with alcohols
B. Clearing tissue with clearing agents
C. Sectioning tissue to thin sections
D. Embedding tissue with paraffin medium

20. Microscopy

21. • Direct staining
Application of simple dye to
stain the tissue in varying
shades of colours
• Progressive staining
Stain applied to the tissue in
strict sequence and for
specific times.
• 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
• Indirect staining
It means use of mordant to
facilitate a particular
staining method or the use
of accentuator to improve
either the selectivity or the
intensity of stain.
• Decolourization
Partial or complete removal
of stain from tissue sections.
E.g. acid alcohol treatment

22. Dyes used in staining
• Dyes are classified in various ways :
1. According to source (Natural & Synthetic)
2. Affinity to tissues (Acidophilic & Basophilic)
3. Chemical composition (Thiazines, Azo-dyes &
Rosailins)

23. Factors controlling selectivity of tissue component
 Dyes or stains are not taken by every part of tissue
and this difference is called selectivity of stain
 Factors that determine selectivity of stain include;
1. Number and affinity of binding sites
2. Rate of reagent uptake
3. Rate of reagent loss
4. Presence of non-dye constituent
 Mordants and accentuators
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24. • 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. e.g. Ammonium
and Potassium alum for haematoxylin.
• Accentuators
Substances that causes increase in selectively or in
staining power of dye. e.g. Phenol in Carbol fuchsin,
KOH in Mehtylene blue

25. Types of commonly used stains
 Most staining reaction involve chemical interaction b/n dye and
substrate through salt linkage or hydrogen bonds
 Staining with dyes result in predictable color pattern based on
acid/base characteristics of tissue and dye
 Based on these stains can be divided in to
 Acidic
 Basic
 Neutral
 NB: This classification is not based on the pH of stains
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26. Types………..
1. Acid dyes:
 Exists as an anion in solution
 has affinity for cytoplasm
• Stains the acidophilic structure
 Most commonly used acidic stain is Eosin
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27. Types………..
2. Basic dyes:
 Exists as a cation in solution
 has affinity for nuclei & ribosome
• Stains the basophilic structure
 Most commonly used basic stain is Hematoxylin
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28. Types………..
3. Neutral dye:
 Combining aqueous solutions of acid and basic
dyes
 Capable of staining cytoplasm and nucleus
simultaneously and differentially
– This is called methachromatic staining
e.g
• Giemsa’s stain
• leishman’s stain etc.
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29. • Haematoxylin and Eosin staining
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
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30. Results of tissue staining
• Nucleus - blue
• Cytoplasm and background - pink
Causes of poor quality of staining
1. Poor or inadequate fixation of tissue.
2. Over or under-ripened Haematoxylin.
3. Overused or worked out Haematoxylin.
4. Over or under differentiation of haematoxylin
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31. 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.
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32. SPECIAL STAINING
VAN GIESON METHOD- staining of connective
tissue
• Principle: In the routine staining method collagen,
elastic fibres 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
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33. 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 33

34. • Results
1. Collagen Red
2. Muscle and Cornified epithelium -Yellow
(B) Nuclei -Blue to Black
34

35. McMANUS FOR GLYCOGEN (PAS)
Staining and identification of the various types of
carbohydrates
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. 35

36. Procedure
1. Deparaffinize and hydrate to distilled water.
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.
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37. 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.
37

38. • 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).
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39. Mounting of sections
 After staining the section is mounted under a
cover slip using a syrup fluid
 Why mounting?
1. To protect the specimen from physical injury
2. To protect the section from deterioration
3. To facilitate easy handling.
4. Preserving the slides for permanent storage

40. Mounting medium
 A syrup fluid applied between the section and the cover slip
Characteristic of a good mounting medium
1. It should not dry quickly
2. It should not dissolve out or fade tissue sections
3. It should not cause shrinkage and distortion of tissues
4. It should set hard thereby producing permanent mounting of sections
5. RI should be as near as possible to that of the glass which is 1.518.

41. Mounting medium……….
There are two types of mounting media
1.Aqueous media
2.Resinous media
Aqueous media
 designed to mount water miscible preparations
I. Glycerin –RI is 1.47
II. Farrant’s medium –RI of 1.43
III. Apathy’s medium –RI of 1.52

42. Mounting medium……….
Resinous media
 For preparations that have been dehydrated
 Sections cleared by Toludine/ xylene
1. Canada balsam - Canadian tree Abus Balsamea.
• Mount most type of tissue sections and also for thicker tissues
• refractive index is 1.54
2. DPX - for small tissue sections
• A mixture of distyrene (polystyrene), plasticizer (tricresyl
phosphate), and xylene
• refractive index is 1.53
3.XAM
• synthetic resin mixed with Xylene
• Its refractive index is 1.52.

43. Mounting
 Make quite sure the sections are clear, do not let the section go dry before
mounting
1. Hold the slide b/n thumb and forefinger and wipe both ends of the slid
2. Clean carefully around section and lay on a clean blotting paper with
section uppermost with appropriate cover slip
3. Place a drop of mountant on the cover slip
4. Invert the slide over the cover slip and lower it so that it just adhere to
the cover slip
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44. Mounting…………
5. Quickly turn slide over, then lay it on flat
surface to allow the mountant to spread
6. Wipe around the covers lip for neatness
N.B:
There should be no air bubbles, if any it should be returned to xylene to
remove cover slip
 No attempt should be made to pull the cover slip
Slight warming of the slide from below will make small air bubbles to
escape.
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45. • When preparing a tissue for Histopathology
and light microscopy , which method precedes
clearing the specimen with an organic solvent,
a. Fixation
b. Clearing
c. Staining
d. Dehydration
e. Embeding