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DEDICATED TO
Mr. Jamil Akhtar
i
ii
TABLE OF CONTENTS
Chapter No. Title Page No.

I. Acronyms vii
II List of Figures ix
III. Preface x
IV. Foreword xi
1. INTRODUCTION 1
2. FIXATION 3
2.1 Rationale of Fixation 3
2.2 Mechanism of Fixation 3
2.3 Effects of Fixation 4
2.4 Properties of an Ideal Fixative 4
2.5 Classification of Fixatives 4
2.6 Fixation Artifacts 6
2.7 Simple Fixatives 6
2.7.1 Formaldehyde 6
2.7.2 Mercuric Chloride 7
2.7.3 Osmium Tetraoxide 8
2.7.4 Picric Acid 9
2.7.5 Potassium Dichromate 9
2.7.6 Ethanol 10
2.7.7 Acetic Acid 10
2.7.8 Trichloro Acetic Acid 11
2.8 Osmotic Considerations in Fixation 11
2.9 Factors Affecting Fixation 12
2.10 Compound Fixatives 14
2.10.1 Microanatomical Fixatives 14
2.10.2 Cytological Fixatives 17
2.11 Removal of Pigments 18
iii
3. DECALCIFICATION 21
3.1 Decalcifying Agents 21
3.1.1 Formic Acid 22
3.1.2 Nitric Acid 22
3.1.3 EDTA 23
3.1.4 3HUHQ\O¶V)OXLG 23
(EQHU¶V)OXLG 23
3.1.6 Ion Exchange Resins 24
3.2 Confirmation of Decalcification 24
4. TISSUE PROCESSING 26
4.1 Completion of Fixation 26
4.2 Dehydration 26
4.2.1 Ethanol 27
4.2.2. Methanol 27
4.2.3 Acetone 28
4.2.4 Dioxane 28
4.2.5 Isopropanol 28
4.3 Clearing 29
4.3.1 Xylene 29
4.3.2 Toluene 29
4.3.3. Benzene 30
4.3.4 Chloroform 30
4.3.5 Carbon Tetrachloride 30
4.3.6 Petrol 30
4.3.7 Cedar Wood Oil and Clove Oil 30
4.4 Impregnation/Infiltration 31
4.4.1 Paraffin Wax 31
4.4.2 Paraplast 32
4.4.3 Carbowax 33
4.4.4 Celloidin 34
4.4.5 LVN 34
iv
5. EMBEDDING 35
5.1 /HXFNKDUW¶V0RXOGV 35
5.2 Steel Base Moulds 35
5.3 Watch Glasses 36
6. MICROTOME 37
6.1 Cambridge Rocking Microtome 38
6.2 Rotary Microtome 39
6.3 Base Sledge Microtome 40
6.4 Sliding Microtome 40
6.5 Freezing Microtome 40
6.6 Cryostat 41
6.7 Laser Microtome 42
6.8 Parts of a Microtome 42
6.9 Microtome Knives 42
6.10 Sharpening of Microtome Knives 44
6.11 Knife Angles 46
7. SECTIONING 48
7.1 Adhesive 48
8. STAINING 50
8.1 Factors Affecting Staining 50
8.2 Classification of Stains 51
8.3 Simple Benzene and Derivatives 52
8.4 Haemotoxylin 53
8.5 Eosin 54
8.6 H/E Staining Procedure 55
8.7 Metachromatic Staining 57
8.8 Toluidine Blue Staining 58
8.9 Nissl Substance Staining 59
8.10 Trichrome Staining 60
0DOORU\¶V6WDLQLQJ 61
v
8.12 Von Gieson Staining 62
0DVVRQ¶V6WDLQLQJ 63
8.14 Aldehyde Fuchsin Staining 64
8.15 Myelin Sheath Staining 65
8.16 Periodic Acid Schiff Reaction 67
%HVW¶V&DUPLQH6WDLQLQJ 71
8.18 Lipids; Histochemical Demonstration 73
8.19 Bromine-Standard Sudan Black-B Staining 75
9. FROZEN SECTIONING 77
9.1 Aim/Significances 77
9.2 Disadvantages 77
9.3 Principle of Frozen Sections 78
10. CRYOSTAT 79
10.1 Anti Roll Plate 79
10.2 Free Floating Sections 80
10.3 Merits of Cryostat 80
10.4 Pre-Requisites of Cryostat Sectioning 80
10.5 Cryostat Sectioning 82
10.6 Freeze Drying 83
11. REFERENCES 85
ABOUT THE AUTHORS 86
vi
ACRONYMS
C2H6O Ethanol
C6H3N3O7 Picric Acid
C6H6 Benzene
CaCl2 Calcium Chloride
CaO Calcium Oxide [Quicklime]
CCl3COOH Trichloro Acetic Acid
CH3COOH Acetic Acid
CH3OH Methanol
CNS Central Nervous System
CO2 Carbon Dioxide
CuSO4 Copper Sulphate
D/D Diastase Digestion
DPX Dibutyl Phthalate Xylene
EDTA Ethylene Diamine Tetraacetic Acid
EFMP Elastic Fibers Microfibrillar Protein
GER Granular Endoplasmic Reticulum
H/E Haemotoxylin/Eosin
H2O2 Hydrogen Peroxide
HCHO Formaldehyde
HCl Hydro Chloric Acid
HCOOH Formic Acid
HgCl2 Mercuric Chloride
HgO Mercuric Oxide
HI4 Periodic Acid
HP Histopathology
Hx Haemotoxylin
K2Cr2O7 Potassium Dichromate
KCO3 Potassium Carbonate
KMnO4 Potassium Permanganate
KOH Potassium Hydroxide
LVN Low Viscosity Nitrocellulose
MP Melting Point
OCT Optimum Cutting Temperature
OP Over Proof
OsO2 Osmium Dioxide
OsO4 Osmium Tetraoxide
PAS Periodic Acid Schiff
PMA Phospho Molybdic Acid
PNS Peripheral Nervous System
vii
PTA Phospho Tungstic Acid
RBCs Red Blood Cells
RNA Ribonucleic Acid
UP Under Proof
viii
LIST OF FIGURES
Figure No. Title Page No.
6.1 18th Century Microtome by Alexander Cummings 37
6.2 Cambridge Rocking Microtome 38
6.3 Rotary Microtome 39
6.4 Base Sledge Microtome 40
6.5 (a) Microtome Knives 43
6.5 (b) Microtome Knives 44
6.6 Direction of the movements in Honing 45
6.7 Direction of the movements in Stropping 46
10.1 Cryostat 79
ix
Preface
7KHLGHDRIGUDIWLQJDERRNRQµ+LVWRWHFKQLTXHV¶ZDVLQRXUPLQG since the time we were

studying at National Institute of Health. Infact it was impossible at that time and even now
to find a book on the subject which covers all the topics of the curriculum for BS Medical
Laboratory Technology. Fortunately we had the supervision of our much respected teacher
Mr. Jamil Akhtar from whom we learnt the basic of histotechniques. We, at that time
prepared lecture notes and this book represents an extended and thoroughly revised version
of a collection of lecture notes plus our practical experience. This book develops
understanding of basic principles and elaborates the procedures used in the histopathology
laboratory.
While writing this book the syllabus of BS Medical Laboratory Technology was kept in
mind so it would definitely assist the students in their degree exams. We would like to
acknowledge our colleagues and friends who supported us in fulfillment of this task
including Ishtiaq Ahmed, Najam Farooq, Abdul Hadi, AS Mirza, Shafqat Ali, Dr. Bashir
Ahmed, Ahmed Farooq, Ahmed Waqas, Asad Nawaz, Abdul Hameed, Humayun Shafique,
Qasim Ansari, Raza Waheed and Waqas Amir. Thanks to all friends for accompanying and
supporting us during the entire process since they helped us whenever we needed it.
The manual is prepared with the idea that revisions must be made regularly in order to have
an updated text for the students on regular interval of time. Your suggestions and feedback
are highly appreciated in this regard.
We are also humbly obliged to Dr. Haroon Khan and Dr. Hassan Abbas Zaheer whose
worthy support and encouragement was extremely useful during the compilation of this
monograph.
Last but not the least; we truly appreciate the support from our publisher, Lambert
Academic Publishing, Saarbrücken, Germany and Acquisition Editor, Mark Williams who
has provided us with an enthusiastic and intelligent assistance.
Usman WAHEED
Asim ANSARI
Islamabad, March 4, 2012
x
Forward
I DPYHU\SOHDVHGWRZULWHWKHIRUZDUGIRU³+LVWRWHFKQLTXHV´E\8VPDQ:DKHHGDQG$VLP
Ansari. The book is based on practical experiences and knowledge of the authors on
³7HFKQLTXHV XVHGLQ+LVWRSDWKRORJ\´ RQH RI WKHFUXFLDOGLVFLSOLQH LQODERUDWRU\ VFLHQFHV
The group of writers comprises of well educated experienced technologists, having a vast
experience in this field.
Although, a lot of educational matter is available on various disciplines of medical

laboratory sciences, but most of it is from the developed countries and the vocabulary and
language used is difficult to understand by our students. The writers of this book are
medical technologist and they have taken care to ensure that all the concepts are explained
in plain simple English which can easily be comprehended by all readers of this specialty.
Moreover, the procedures and methods are written in a very precise and compact manner
making it a great resource book. Since this book is written in such a practical manner it
can easily be used for developing the departmental (Histopathology) standard operation
procedures (SOPs) and in training schedules of histo-technicians/technologist.
I strongly believe that after reading this book one can easily absorb the philosophy behind
histopathology techniques and understand the essence of this very vital subject. This book
can easily form the basis for curriculum of medical laboratory sciences.
7KHDXWKRUV¶effort in producing this book is highly commendable and I am sure they will
continue to improve and update the material in subsequent editions.
Dr. Haroon Khan

MBBS, MSc, PhD
Associate Professor
Department of Pathology
Pakistan Institute of Medical Sciences
Islamabad.
March 3, 2012
xi

1. INTRODUCTION
Histology is the microscopic study of normal tissues while histopathology is the study of
diseased tissues. The techniques required for histological or histopathological microscopic
studies are termed Histotechniques (Histological Techniques or Microtechinques). Infact it is the
study of procedures or stages to reach the final stained slide of the specimen for
microscopic examination. The persons responsible for performing these procedures are
FDOOHGµ+LVWRWHFKQRORJLVWV¶DQGWKHILHOGRIVSHFLDOL]DWLRQLVWHUPHGas µ+LVWRWHFKQRORJ\¶
These techniques, employed in a histopathology laboratory, help a histo-pathologist to

determine whether a patient has malignancy or some other disease process. The
microscopic examination involves following steps:
Documentation:
Every specimen is given the histopathology [HP] number which is pen down at top corner
of the request slip, in a specified record register and on the top of container.
Fixation:
It is a process by which the constituents of cells are fixed in a physical and partly also in a
chemical state, so that they will withstand subsequent treatment with various reagents with
a minimum of loss, or decomposition. This is achieved by exposing the tissue to chemical
compounds called fixatives.
Gross Examination:
The specimen is examined grossly by noting anatomical features of it. Representative
sections are selected for further procedures while small biopsies are selected as a whole.
Decalcification:
In case of calcified tissue (bone), calcium salts are removed from the bony tissue. It is done
before gross examination.
Tissue Processing:
It involves a number of stages in which selected portions of tissue are passed through a
number of chemicals in a sequence. In this stage tissues are impregnated with a solidifying
medium to facilitate its fine sectioning. Following four stages are involved:
i. Completion of Fixation
ii. Dehydration
iii. Dealcoholization or Clearing
1
iv. Impregnation or Infiltration
Embedding:
In this step all processed tissues are blocked out in a solidifying medium (,.e.g. paraffin
wax) which is used in the impregnation stage.
Sectioning:
Paraffin blocks of tissues are then sectioned by means of a device termed as Microtome.
The sections which are produced are capable of transmitting light and are at a micron level
thickness.
Staining:
Tissue sections are picked onto slides and staining is performed in order to examine
different tissue components under the microscope.
Mounting:
Cover slip is applied on each section using appropriate medium to obtain permanent
preparation for microscope
2
2. FIXATION
As soon as the tissue is removed from the body, cells begin to undergo changes, which
result in their breakdown and ultimate destruction. These are referred to as post-mortem
changes, which may be either putrefactive or autolytic in nature. These changes are
prevented by decreasing the temperature or using chemicals called fixatives. So Fixation is
a process by which a tissue is safe-guarded against post-mortem changes.
x Putrefaction is due to the invasion of the tissue by bacteria, which generally disseminate
from the alimentary tract and spread quickly into the surrounding organs causing
decomposition.
x Autolysis is due to the action of enzymes from dead cells. This phenomenon mainly
occurs in the CNS and the endocrine system.
2.1 Rationale of Fixation:
There are number of reasons for which fixation is the first step in histotechniques. Most
cells consist of an outer complex membrane containing the fluid protoplasm, which is a
true, colloidal, mixed solution of salts, proteins, carbohydrates, lipids, organic acid and
enzymes. If the cells are not fixed, many of these substances would be lost by simple
solution, dialysis, osmotic swelling and rupture of the cells in the processing that must
precede the cutting and staining of sections. On microscopic examination, autolyzed tissues
show changes in the nuclei while the cytoplasm becomes cloudy and loses its staining
affinities. Fine intracellular structures such as mitochondria are lost in a very short period if
material is left unfixed.
2.2 Mechanism of Fixation:
Most fixatives act by denaturing or precipitating proteins. A fixative when acts on a protein
component of tissue, cross links them to form a sponge or meshwork due to which
structural proteins are stabilized and most of other free molecules, e.g. lipids, mucins etc.
are trapped inside meshes and so preserved. The amount of fixing fluid should be
approximately 10-20 times the volume of the specimen, except when osmium tetra oxide is
used.
3
2.3 Effects of Fixation:
2.3.1 Most fixatives produce some tissue hardening, which help in cutting of sections, but
this hardening effect will be reinforced by the action of alcohols used during the
process of dehydration.
2.3.2 Certain fixatives act as mordents for certain stains, e.g. after the use of mercuric
fixatives the staining of tissue constituents with many dyes is enhanced.
2.3.3 Fixatives usually increase the optical differentiation of cell and tissue components.
2.3.4 Fixatives render the cells insensitive to hypo and hypertonic solutions used
subsequent to fixation.
2.3.5 Micro-organisms are composed of proteins and they will also be fixed (killed)
preventing putrefactive changes in tissues.
2.4 Properties of an Idea Fixative:
A good fixative should be capable of fulfilling the following requirements:
2.4.1 It must penetrate the tissue rapidly and evenly.

2.4.2 It must be simple to prepare and economical to use.
2.4.3 It should impart sufficient hardness to facilitate sectioning.
2.4.4 It must inhibit bacterial decay and autolysis.
2.4.5 It must give good optical differentiation.
2.4.6 It must be non-irritant, non-toxic and non-corrosive.
2.4.7 It should not cause shrinkage, swelling or other distortion.
2.4.8 It should allow tissue to be stored for long periods of time.
2.4.9 It should permit the restoration of natural colour for photography and mounting as
museum specimens.
2.5 Classification of Fixatives
There are three classifications of fixatives as below:
2.5.1 Classification I:
Initially fixatives were classified as simple and compound fixatives.
2.5.1.1Simple Fixatives:
These fixatives comprise a single chemical substance, e.g. formalin, ethanol etc.
4
2.5.1.2Compound Fixatives:
These may be described as the product of two or more simple fixatives mixed together in
order to obtain the combined effect of their individual actions upon the tissue components.
([DPSOHV LQFOXGH &DUQR\¶V )OXLG HWKDQRO JODFLDO DFHtic acid) formal sublimate
(formaldehyde + mercuric chloride) etc.
2.5.2 Classification II:

Fixatives are also grouped according to their action upon the cell and tissue constituents.
2.5.2.1Microanatomical Fixatives:
These are those fixatives which preserve the tissue in a manner which permits the general
microscopical study of the tissue structures and allows the various layers of tissues to retain
their former relationship with each other.
2.5.2.2Cytological Fixatives:
These are employed for their specific action upon a specific part of the cell structure. They
are subdivided into nuclear and cytoplasmic fixatives depending upon which of the cell
inclusions they act.
Fixatives having glacial acetic acid or pH of 4.6 or less are termed nuclear fixatives.
Fixatives having pH above 4.6 or lacking glacial acetic acid are termed cytoplasmic
fixatives.
2.5.3 Classification III:

The most recent classification includes:
2.5.3.1Aldehydes:
These are organic compounds having functional group ±CHO. Fixation properties of
aldehydes are due to reactions of aldehydic group, e.g. formaldehyde, glutaraldehyde etc.
2.5.3.2Oxidizing Agents:
These bring about fixation through oxidation reduction reactions, e.g. chromic acid,
osmium tetraoxide and potassium dichromate etc.
2.5.3.3Fixatives of Unknown Mechanism:

This group includes certain fixatives whose mode of action in fixation process is still not
clear, e.g. mercuric chloride, picric acid.
5
2.5.3.4Protein Denaturing Agents:
These fixatives bring about precipitation of structural proteins and more over denature
lysosomal enzymes in the course of their fixing actions, e.g. ethanol, methanol, glacial
acetic acid.
2.5.3.5Physical Methods of Fixation:

In this group of fixatives, fixation is done by means of heat and microwave energy. Heat
and microwave energy enhances oscillation energy of polar molecules as higher as 2650 Hz
at which proteins in tissues are precipitated and so fixed. (Microwave energy is heat energy
used in a control chamber).
2.6 Fixation Artifacts:

Fixation is associated with a number of problems termed as fixation artifacts, e.g.
Formaldehyde fixatives give brown pigmentation to tissues.

Mercuric chloride fixatives leave a black precipitate of mercury in tissues.
Some fixatives produce shrinkage in tissues and similarly some produce swelling.
Due to poor penetration of fixatives biochemical molecules like glycogen diffuse from
unfixed parts giving false localization or considerable loss. This is termed streaming
artifact and is mostly observed in case of glycogen.
2.7 SIMPLE FIXATIVES
2.7.1 Formaldehyde
Commercially available formaldehyde is a saturated solution of formaldehyde (HCHO) gas

in water, about 40% gas by weight. The formaldehyde available commercially is 40% but it
is considered 100% and then 10% is prepared for fixing laboratory specimens.
2.7.1.1 Advantages:
i. Formalin is cheaper, easy to prepare and stable.

ii. It fixes the tissue in natural colour.
iii. Shrinkage and brittleness are not caused by formalin fixatives.
iv. It is best fixative for nervous tissue.
v. Frozen sections can be prepared with great ease from formalin fixed material.
vi. Different staining techniques can be used on tissue fixed in formalin
vii. It is a good fixative for lipids and proteins. (Mucins are well preserved)
6
viii. Compound fixatives of formalin can easily be prepared for special work on tissues.
ix. The blocks fixed in formalin do not require washing before processing.
2.7.1.2 Disadvantages:
i. These fixatives are toxic, irritant to skin and may cause dermatitis due to prolonged
handling.
ii. Formalin vapors are irritant to nasal mucosa and may cause sinusitis. This can be
prevented by handling the specimen in a ventilated area.
iii. It may also cause asthma in some allergic individuals.
iv. When kept for longer periods especially in the cold, formalin fixatives develop a
white precipitate of paraformaldehyde which is a polymer of formaldehyde. The
polymer does not change the fixing ability of formalin and can be removed by
filtration. All commercial formaldehydes contain 11-16% methanol, which tends to
inhibit the formation of paraformaldehyde. Paraformaldehyde commonly has a
slight odor of formaldehyde due to decomposition.
v. Methanol has a denaturing effect on proteins, and this factor makes formalin
unsuitable for the fixation required in electron microscopy. However, pure
formaldehyde is suitable.
vi. Traces of formic acid are normally present in commercial formalin due to which it is
acidic. This acid can be neutralized by the addition of a small quantity of magnesium
carbonate or a few drops of sodium hydroxide (NaOH). The acidity usually reduces
the quality of staining, particularly nuclear staining.
vii. In tissues containing much blood (, e.g. spleen), unbuffered* formalin leads to
formation of dark-brown artifact pigment granules. The granules consist of acid
formaldehyde, haematin and are doubly retractile.
viii. Formalin is medium speed fixative and complete fixation requires 12-24 hours.
*formal saline + handful calcium carbonate + well shaken
2.7.2 Mercuric Chloride
It belongs to the class of fixatives of unknown mechanism. Mercuric chloride HgCl2 is a

metallic salt and included in many fixatives. At room temperature its solubility in water is
about 7% and this saturated aqueous solution is used for fixation purposes.
2.7.2.1 Advantages:
i. These fixatives are mainly used as secondary fixatives in order to enhance the final
staining.
7
ii. These act as a mordant due to metallic mercury, so staining reactions are excellent.
iii. These give good staining results in case of special stains, e.g. trichrome stains, which
are used for connective tissue elements and muscle fibers differentiation. Similarly
metachromatic staining for cartilage matrix and mast cell granules, and chromaffin
reaction.
i. These fixatives produce a lot of shrinkage in tissues.
ii. Fixatives containing mercuric chloride leave a black precipitate of mercury in tissues
and it must be removed.
iii. Mercuric chloride fixatives are not stable and easily deteriorated.
iv. HgCl2 is corrosive and should not come into contact with metal surfaces. It is very
poisonous and must be handled with care.
2.7.3 Osmium Tetraoxide
It belongs to the class of µoxidizing DJHQWV¶ fixatives. It is also known as osmic acid and is a
pale yellow powder having solubility about 6% at 20° C. Osmium tetraoxide OsO4 is
extremely volatile and is easily reduced by contact with organic matter or by exposure to
day light. It should therefore be kept in a dark, chemically clean bottle.
2.7.3.1 Advantages:
i. It is the only fixative that can permanently fix lipids (Golgi bodies and mitochondria
are also preserved which are cytoplasmic organelles).
ii. It is seldom used alone as a fixative, but is usually combined with chromium salt. Its
vapors can be used for fixation.
iii. It can also be used for electron microscopy work.
i. One of its lower oxides, i.e. osmium dioxide OsO2 can become deposit in the cornea
resulting in blindness.
ii. The fixative is expensive and dangerous and penetration is also poor.
iii. Tissues fixed in OsO4 require washing out, i.e. they are placed in running tap water
for hours before taking them to reducing alcohols. If vapors are used for fixation,
washing out is not required and vapors penetration is also better than solution.
8
2.7.4 Picric Acid
It belongs to the class of fixatives of unknown mechanism. Its solubility is about 1% in

water. It is always kept in saturated aqueous solution form. Picric acid C6H3N3O7 is
explosive when dry, therefore for fixation purposes, yellow coloured solution is used.
2.7.4.1 Advantages:
i. It is recommended for histochemical demonstration of glycogen.
ii. Picric acid imparts a yellow colouration that acts as a merit in case of handling tiny
biopsies which have a chance of misplacing.
i. The yellow colouration is a disadvantage because it impairs final staining and so it
requires several washes in alcohol or xylene to remove yellow colour.
ii. It precipitates all proteins and combines with them to form picrates. These picrates
are soluble in water, and the tissue must not come in contact with water until the
picrates have been rendered insoluble by treatment with alcohol.
2.7.5 Potassium Dichromate
Potassium Dichromate K2Cr2O7 is one of the oldest and is most widely used simple fixative.
Two entirely different forms of fixation can be produced depending upon the pH of the
solution. If pH is below 4.6, the result is similar to those produced by chromic acid. If pH is
above 4.6 the cytoplasm is homogenously preserved and the mitochondria are fixed.
2.7.5.1 Advantages and Disadvantages:

i. It is recommended for myelinated nerve fibers in which it fixes myeline lipids. This
property is lost if mixed with glacial acetic acid.
ii. Tissues fixed in K2Cr2O7 UHTXLUH³:DVKLQJRXW´ (to remove oxidizing agent).
iii. It is a metallic salt so act as a mordant and may be used as a secondary fixative.
iv. Formaldehyde fixed tissues are placed in 3% potassium dichromate for about 8
KRXUV7KLVWUHDWPHQWLVQDPHGDV³3RVW-FKURPLQJ´DQGHQKDQFHVILQDOVWDLQLQJ
v. It is prepared by dissolving crystals of the anhydride CrO3 in distilled water, and is
stored as a 2% stock solution. It is a strong oxidizing agent so not combined with
reducing agents such as alcohol and formalin. It is a strong protein precipitant, and
SUHVHUYHVFDUERK\GUDWHV7LVVXHVUHTXLUH³ZDVKLQJRXW´WRDYRLGWKHIRUPDWLRQRIWKH
insoluble sub-oxide.
9
2.7.6 Ethanol
Ethyl alcohol C2H6O belongs to the class of fixatives of protein denaturing agents. It is a
colourless liquid that is readily miscible with water. Earlier it was used as a simple fixative
but now a day its use as a simple fixative is confined to histochemical methods. It is
frequently incorporated into compound fixatives.
2.7.6.1 Advantages and Disadvantages:

i. As a simple fixative it is used at a concentration of 70-100% which preserves
glycogen but GRHVQ¶W fix it.
ii. It is a reducing agent and should not be mixed with chromic acid, osmium tetraoxide
and potassium dichromate.
iii. It produces a lot of hardening and shrinkage of tissues.
iv. It is also a highly flammable solution.
v. It is good for enzyme demonstration.
2.7.7 Acetic Acid

Acetic Acid CH3COOH is a colourless solution with a pungent smell. At 17°C it solidifies,
which accounts for its name glacial acetic acid. It belongs to class of protein denaturing
agents.
2.7.7.1 Advantages:
i. It is often used by cytologists in studying chromosomes.
ii. It is very useful for nuclear studies because of its chromatin-precipitating properties.
iii. It is a powerful precipitant of nucleoprotein.
iv. It destroys mitochondria, golgi bodies and also RBCs so acts as a lytic agent.
v. The lytic agent action is a merit in case of cytological smears mixed with a lot of
blood which is hiding details of other cells and so when fixed in CH3COOH the red
cells are lyzed and details of other cells become visible.
i. The lytic action is a demerit when handling bone marrow biopsy because in that case
we have to preserve RBC series.
ii. When used alone it causes swelling so it is used in certain compound fixatives to
counteract the shrinkage produced by other components, e.g. mercuric chloride and
acetic acid.
iii. If it is mixed with K2Cr2O7, the lipid fixing ability of K2Cr2O7 is lost.
10
2.7.8 Trichloro Acetic Acid
Trichloroacetic acid [CCl3COOH] is sometimes incorporated into compound fixatives. It is

a general protein precipitant but has a swelling effect on many tissues to counter the
shrinkage produced by other simple fixatives. It can be used as a slow decalcifying agent
and the softening effect which it has on dense fibrous tissue is found to facilitate the
preparation of sections from blocks of this nature.
Box-1
Important Notes:
Apart from the size and thickness of the piece of tissues to be fixed, certain other considerations are
of importance:
Tissues containing a large amount of mucus fix slowly and poorly because the mucus prevents
penetration of the fixative. Whenever possible, e.g. in the case of pseudo-mucinous cysts of the
ovary, it is advisable to remove as much of the mucus as possible by washing with normal
saline. The same procedure may be applied to tissues containing blood, or organs containing
very large amount of blood e.g. lungs.
Fatty and lipomatous tissues fix slowly and blocks of such tissues should be thin and may
require longer time than average in fixative.
Like all chemicophysical reactions, fixation is accelerated by agitation, and this is one of the
advantages of the mechanical tissue processors.
Moderate heat 37-56°C will accelerate fixation but it also hastens and accentuates autolytic
changes in the deeper parts of the tissues block before the fixing agent gains access to these
regions.
2.8 Osmotic Considerations in Fixation
An ideal fixative should not cause swelling or shrinkage of cells due to osmotic factors. In
practice, only a few fixatives are isotonic. In general, heavy metal fixatives and those that
act mainly as protein precipitants, e.g. picric acid causes shrinkage whatever the osmotic
pressure of their solutions is. Acetic acid 5% is markedly hypertonic in relation to body
fluids leads to swelling of tissues, especially of collagen. Formaldehyde because of its
small, freely diffusible organic molecule and polymerizing action, must be used in as nearly
isotonic solution as possible. If the solution is hypotonic, swelling will be the result.
11
2.9 Factors Affecting Fixation
There are number of factors which can affect the fixation process. These factors are briefly
described below:
2.9.1 pH:
Tissues are composed of different types of proteins which are fixed or precipitated at
different pH values. Generally fixation is done by using fixatives having pH range of 5-8.
However sometimes a specific pH is required for proper fixation, e.g. gastric mucosal
biopsy is properly fixed at a pH value of 5.5. Similarly for chromosomal studies, nucleus
require proper fixation so a pH of 4.6 or below is recommended because at such low pH
nuclear proteins are well precipitated. Change of pH will change number of ions so reaction
will either increase or decrease. So the increase or decrease in rate of reaction will have
detrimental effect or simply harmful effect causing problems in microscopy.
2.9.2 Temperature:
Generally rise in temperature will increase rate of fixation, e.g. we use heated formalin
(60°C) in case of 2-3mm biopsy so the reaction between fixative and proteins will enhance
but diffusion of molecules and autolysis are also enhanced with rise in temperature. The
diffusion of molecules is a disadvantage for biochemical studies and therefore for such
studies tissues are fixed at 0-4° C to avoid diffusion of the molecules. Another aspect is that
we are using various inflammable chemicals in histopathology laboratory. So heating is a
very risky process. At the end, taking conclusion of all this we can say that if there is no
HPHUJHQF\DQGWLPHGRHVQ¶WPDWWHUIL[DWLRQDWURRPWHPSHUDWXUHLVSUHIHUUHG
2.9.3 Penetration of Fixatives:

The penetration of fixatives is usually slow because cells are bounded by semi-permeable
membrane. The intercellular substance also resist with penetration of the fixative.
Penetration of fixatives can be shown by the following relationship with time:
d Į t
d=K t
Where K is constant of proportionality and is known as Coefficient of Diffusibility. By re-

arranging the equation,
K=d/t
,I ³G´ LV WDNHQ LQ PLOOLPHWHUV DQG WLPH LQ KRXUV WKHQ ³&RHIILFLHQW RI Diffusibility´ LV
defined as penetration in mm/hours.
12
7KHµCoefficient of Diffusibility [.@¶ is different for different fixatives, e.g.
Fixatives 9DOXHRI³.´
10 % Formalin at room temperature 0.78 mm/hour
01 % Picric acid C6H3N3O7 0.5 mm/hour

Ethanol C2H6O 01 mm/hour
03% Potassium dichromate 1.33 mm/hour
So, potassium dichromate K2Cr2O7 is highly penetrating fixative whereas picric acid
C6H3N3O7 is a slow penetrating fixative. In this way, penetration affects the fixation
process.
2.9.4 Concentration of Fixatives:

Generally, concentration will have a positive effect. By concentrating the fixative, the
fixation process will be enhanced because in that case more number of molecules will be
present. But there are a few limitations to this:
a. Firstly, some of the fixatives are very expensive and we cannot use them in
concentrated form.
b. Secondly, maximum solubility of fixatives determines their concentration at which
they should be used, e.g. 1% picric acid is used for fixation because at this
concentration a saturated solution is obtained. If we want to use it at 10%
concentration, it will not be possible because to prepare it more heat is required and
that has many adverse effects.
Sometimes a fixative can be effective even at a low concentration, e.g. 3% glutaraldehyde is

a fixative normally used at 3% concentration but it will be effective even when the
concentration is reduced to 0.25%.
2.9.5 Time of Fixation:

Different fixatives are employed for different periods of time depending upon their reaction
and penetration. When tissues biopsies are kept in different fixatives for prolonged periods,
certain physical changes like shrinkage, hardness and brittleness of tissue biopsies is
observed.
13
In addition to these changes, certain chemical changes are also brought about by fixatives,
e.g. prolonged preservation in oxidizing agents result in oxidative cleavage of proteins. By
this cleavage, there is a considerable loss of peptides and amino acids.
2.9.6 Osmolarity of Fixatives:

Hypertonic fixatives cause shrinkage whereas swelling is observed in case of hypotonic
fixatives. There are no such effects in case of isotonic fixatives but such fixatives are
usually slow penetrating giving poor fixation. Normally a slight hypertonic fixative is used.
2.10 COMPOUND FIXATIVES

The compound fixatives are divided into Micro-anatomical and Cytological Fixatives.
2.10.1Micro-anatomical Fixatives
2.10.1.1 Formol-Saline 10%:

Formal-saline is a micro-anatomical fixative, but not a compound one. It is recommended
for fixation of CNS material. It is very safe and is the basis of all museums fixatives
because it is the only fixative that allows the natural colour to be restored to the specimen.
This is a slow fixative and tissues fixed in it are liable to shrink during dehydration in
alcohol. Rubber gloves must be worn when handling specimen fixed in formal-saline.
Formula
Formalin 40% 100 ml
NaCl 8.5 g
Distilled water 900 ml
2.10.1.2 Neutral Buffered Formalin 10%:

This is recommended for storage of surgical and research specimens. The period of fixation
is 24 hours or longer. 7KLVIL[DWLYHGRHVQ¶WJLYHEURZQSLJPHQW to tissues because formic
acid formation is inhibited. It is also recommended for bone marrow biopsies where cellular
details are of importance. It is safe and fixes the bone marrow biopsy smoothly. However, it
cannot be used in routine busy laboratories because it is costly and difficult to prepare.
Formula
Sodium dihydrogen phosphate 3.5g NaH2PO4
Disodium hydrogen phosphate 6.5g Na2HPO4
Formalin 40% 100ml
Distilled water 900ml
14
2.10.1.3 Alcoholic Formaldehyde 10%:
The advantage of using this fixative is that the dehydration process in also initiated, so
tissues can directly be taken to absolute alcohol rather than taking them to lower
concentration of alcohol. The formula involves the mixing of two fixatives.
Formula
Formalin 40% 100ml
95% Alcohol 900ml
Calcium Acetate 0.05 g (for neutrality)
2.10.1.4 Formol Calcium:

This fixative is recommended for lipid fixation.
Formula
Formalin 40% 100ml
CaCl2 10% 100ml
2.10.1.5 Formol Sublimate:

This is recommended for routine post-mortem material. 7KHF\WRORJLFDOGHWDLOVDQG5%&¶V
are well preserved. No hardening or shrinkage is cause by this fixative.
Formula
Saturated HgCl2 900ml
Formalin 40% 100ml
2.10.1.6 %RXLQ¶V Fluid:

It is a compound fixative of picric acid. It is poorly penetrating fixative.
Formula
Saturated picric acid 75ml
Formalin 40% 25ml
Glacial acetic acid 05ml
Advantages:
i. This fixative fixes nuclei properly due to presence of glacial acetic acid.
ii. %RXLQ¶VIOXLGimparts a yellow colouration to tissues which is helpful in handling of
tiny fragmentary biopsies, e.g. rectal biopsy or needle biopsy from kidney.
15
iii. It is also recommended for glycogen demonstration.
iv. It is also recommended for delicate tissue biopsies, e.g. embryos or testicular biopsy.
2.10.1.7 *HQGUH¶V)OXLG:
This fixative is widely used for glycogen preservation because of the combined effect of
picric acid and alcohol.
Formula
Glacial Acetic Acid 5ml
Picric acid in 95% alcohol 80ml
Formalin solution 40% 15ml
2.10.1.8 =HQNHU¶V6ROXWLRQ:
This is recommended for the fixation of small pieces of liver and spleen.
Formula
Mercuric Chloride 5g
Potassium dichromate 2.5g
Sodium Sulphate 1.0g (optional)
Add 5ml of glacial acetic acid just before use.
Merits:
i. It is an excellent fixative for nuclei and of connective tissue fibers.
ii. It is recommended for tissues which are to be stained by one of the trichrome
techniques.
Demerits:
i. ,WUHTXLUHV³ZDVKLQJRXW´
ii. Prolonged immersion causes brittleness.
iii. It is not recommended for frozen sections.
2.10.1.9 Zenker-Formol:
This fixative is recommended for pituitary tissue and bone marrow. It gives excellent
nuclear fixation and cytoplasmic organelles are well preserved.
16
Formula
Mercuric Chloride 5.0g
Potassium dichromate 2.5g
Sodium Sulphate 1.0g (Optional)
7KLVIL[DWLYHDOVRUHTXLUHV³ZDVKLQJRXW´
2.10.2 Cytological Fixatives

The cytological fixatives are divided into two groups as follows:
Nuclear Fixatives Cytoplasmic Fixatives

i. FlemPLQJ¶V Fluid L )OHPPLQJ¶V Fluid without Acetic
Acid
LL&DUQR\¶V)OXLG LL+HOO\¶V)OXLG
iii. Formalin with Post Chroming
2.10.2.1 )OHPPLQJ¶V)OXLd:
This fixative is recommended for the preservation of nuclear structure, e.g. chromosomes. It
is the only fixative which can permanently preserve fats. This fixative is relatively costly
but small volumes are required for fixation. The fixative is poorly penetrating so only be
used for small biopsies. It must be prepared immediately before use because it deteriorates
rapidly.
Formula
Chromic acid 1% 15ml
OsO4 Aqueous 04ml
Glacial Acetic Acid 01ml
2.10.2.2 &DUQR\¶V)OXLG
This fixative is best for lymph glands and urgent biopsies but excessive shrinkage is caused
E\WKHVROXWLRQDQG5%&¶VDUHDOVRKDHPolyzed.
Formula
Absolute alcohol 60ml
Chloroform 30ml
Glacial acetic acid 10ml
17
2.10.2.3 )OHPPLQJ¶VFluid without Acetic Acid:
It LVUHFRPPHQGHGIRUPLWRFKRQGULD7KHIRUPXODLVVLPLODUWR)OHPPLQJ¶VIOXLGEXWDFHWLF
acid is not added and the merits and demerits are also similar.
2.10.24 +HOO\¶V)OXLG
This is same like Zenker-Formol.
2.11 Removal of Pigments

When stained sections are examined microscopically, a deposit or pigment is frequently
observed. This may be either artificial or natural in origin.
a. The artificial pigments are of two main types as follows.
x Mercuric chloride precipitate
x Formaldehyde precipitate
b. The natural pigments are also of two main types:
x Exogenous pigments, which consist of foreign matter absorbed by the body during
life. The most commonly encountered is carbon which occurs as a jet-black pigment
in sections of lung and bronchial glands. Other example is tattooing ink.
x Endogenous pigments, which are produced within the organism, e.g. haemosiderin
which can be demonstrated by Prussian blue reaction.
2.11.1 Mercuric Chloride Precipitate:

The removal of HgCl2 pigment is done as follows:
Solution 1: Solution 2:
Potassium iodide 02g Sodium thiosulphate 05g
Iodine 01g Distilled water 100ml
Procedure:
i. Bring sections to water and immerse in solution 1 for 10 minutes to remove HgCl 2
deposit
2HgCl + I2 Æ HgCl2 + HgI2
ii. Rinse in water and place in 5% sodium hyposulfite for 1-2 minutes to remove iodine.
2Na2SO2O3 + I2 Æ 2NaI + Na2S4O6
iii. Wash sections in running tap water for 2-5 minutes to remove the sodium
thiosulphate crystals, and then stain.
18
2.11.2 Formalin Precipitate:
This removal of formaldehyde precipitate is achieved by:
9HURFD\¶V0HWKRG
Formula
KOH, 1% aqueous solution 01ml
Ethyl alcohol, 80% 100ml
Procedure:
i. Bring sections to 80% alcohol.
ii. Treat with alcoholic hydroxide solution for 10 minutes.
iii. Wash with 2 changes of water.
iv. Transfer to 80% alcohol for 5 minutes.
v. Wash in water and continue with staining.
%DUUHWW¶s Alcoholic Picric Acid Method:

i. Deparaffinize with Xylene and wash in absolute alcohol.
ii. Immerse in saturated alcohol picric acid (85%) for 30 minutes.
iii. Wash in absolute alcohol to remove the picric acid.
iv. Bring section to water and continue staining in the normal way.
Box-2
What is Secondary Fixation?
Following fixation with formalin, it is sometimes advantageous to refix the tissue for a further 4
hours in a second fixative. The fixatives usually selected for this purpose are picric acid or osmium
tetraoxide or mercuric chloride. Formal-VXEOLPDWH IRUPDOLQ PHUFXULF FKORULGH DQG =HQNHU¶V
formol can also be sued for secondary fixation. It has following advantages:
a. This secondary fixation has the advantage of imparting a firm texture to the tissue.
b. In many cases, secondary fixation improves the subsequent staining results.
What is Post Chroming?

In order to facilitate certain staining procedures, fixed tissues are immersed in 3% K2Cr2O7 for
several hours prior to staining. This procedure is termed post chromatization or post-chroming and
is used mainly with tissues fixed in formalin. This procedure should be followed by washing out
because K2Cr2O7 is a oxidizing agent.
19
What is Washing Out?
Tissues are placed in running tap water before taking them to reducing alcohols. This is done after
FHUWDLQIL[DWLYHVKDYHEHHQXVHGDQGLVWHUPHG³ZDVKLQJRXW´7KLVPD\EHGRQHLQVHYHUDOZD\V
but whatever the method, it is very important to ensure that the specimen is washed in a constant
stream of fresh water. The purpose of washing out is to remove oxidizing agents such as K2Cr2O7
and OsO4 to prevent reduction when coming into contact with the alcohol. This procedure is also
important in removing all traces of formaldehyde from tissues to be embedded in gelatin.
It is very important to make sure that the water surrounding the tissues is constantly being changed,
preferably by means of a siphon system. Failure to observe this point may result in the fixative being
inadequately removed from the tissue.
20
3. DECALCIFICATION
Bone and teeth normally contain inorganic salts which comprises 65% of bone composition.
Of this amount 85% is calcium phosphate, 10% calcium carbonate and remaining 5%
include chlorides and fluorides of calcium and magnesium. The organic part which makes
35% of bone composition comprises collagen fibers and different proteoglycans. Thus
rigidity of bones is due to the high proportion of deposited minerals. This deposition of
minerals in the matrix occurs during bone formation by a process name as calcification.
³Decalcification is opposite to calcification. It is a process by which

calcium salts are removed from a calcified biopsy by means of decalcifying
DJHQWV´
Certain tissues are pathologically calcified, e.g. thyroid, larger blood vessels in old people,
cancerous biopsies and different abscesses. When we receive a bony biopsy or
pathologically calcified biopsy, it is very difficult to section it due to the presence of
calcium salts and so decalcification of such biopsies is necessary.
By decalcification, the tissue biopsy is softened and its sectioning is facilitated.

Decalcification is preceded by fixation and followed by tissue processing. It is very
necessary to fix a tissue biopsy before taking it to decalcifying agents. This is because the
agents are acids so have a detrimental effect on unfixed biopsies, e.g. organic matter
(collagen) is first destroyed followed by nucleic acids destruction by acids.
3.1 Decalcifying Agents
The chemicals used for decalcification process are termed as decalcifying agents. An acid is
the essential part of a decalcifying agent and a second substance is usually used to prevent
distortion of tissue. Buffer solutions of pH 4.4 ± 4.5 and chelating agents, e.g. EDTA can
also be used.
The four acids most commonly used for removing calcium salts from tissues are formic,
nitric, trichloroacetic and hydrochloric acids. The speed at which the calcium salts are
dissolved out of the tissue is dependent upon the strength, temperature and volume of the
decalcifying agent.
21
Box 3
An increase in either the concentration of acid or the temperature at which decalcification takes
place, can decrease the time required for decalcification but this results in poor staining and partial
digestion of tissue. These effects do not apply to EDTA which may be used at 40 ± 60o C
successfully.
3.1.1 Formic Acid

Formic acid [HCOOH] is recommended for post-mortem and research tissue. It take 2 ± 7
days for decalcification so not suitable for urgent tissue biopsies.
Merits:
i. By using this solution excellent staining results are obtained which make it excellent
for research work.
Demerits:
i. At 5% strength, it is slow in action, so unsuitable for urgent work; however the
process can be speeded by increasing the formic acid content upto 25ml in the given
IRUPXODDVLQ*RRGLQJDQG6WHZDUW¶VIOXLG
ii. But by increasing the concentration in excess of 8%, disturbs the opacity of solution.
Formula
Formic Acid (specific gravity 120 gravity) 5ml
Formalin 40% 5ml
3.1.2 Nitric Acid

Nitric Acid is recommended for urgent tissue biopsies (3 ± 4mm thickness). The time
required for decalcification is from 1 ± 3 days.
Merits:
i. This is a rapidly acting decalcifying agent and permits good nuclear staining.
Demerits:
i. Good nuclear staining is obtained but it is not as good as that obtained by formic
acid.
ii. It is very reactive so biopsy may be impaired if kept for longer periods than required.
iii. In addition to this, it develops a yellow colour due to formation of nitrous acid. This
enhances decalcification but impairs staining. To avoid this 0.1% urea is added
which does not affect the efficiency of the acid.
22
Formula
i. Nitric Acid Formaldehyde
Nitric acid (specific gravity 1.41) 10ml
Formalin 40% 5 ± 10 ml
Distilled water upto 100ml
ii. Aqueous Nitric Acid
Nitric Acid (specific gravity 1.41) 5 ± 10 ml
Distilled water upto 100ml
3.1.3 E.D.T.A.
This is a very slow decalcifying agent recommended for detailed microscopical studies
where time is not an important factor. The time required for decalcification is 3 weeks,
during which the solution must be changed at intervals of 3 days and 1 day in final stage.
Merits:
i. Histological artifacts are minimized by the solution.
ii. The staining results are also excellent.
Demerits:
i. It is slow in action and chelating agent so tends to harden the tissue.
Formula
EDTA disodium salt 5.5g
10% neutral formalin 100ml
3.1.4 3HUHQ\O¶V)OXLG
This solution was introduced as a fixative for ova, but it has gained popularity in recent
years as a good decalcifying agent. It is mostly used for softening of tissues in case of dense
fibrous tissues.
Formula
Nitric Acid 10% 40ml
Absolute Ethanol 30ml
Chromic Acid 0.5% 30ml
3.1.5 (EQHU¶V)OXLG
The use of this fluid is recommended for teeth and staining results are good but staining
results of nucleus are comparatively poorer. Various formulae have been given for this, but
following gives good results:
23
Formula
Saturated NaCl 36% 50ml
HCl 08ml
3.1.6 Ion Exchange Resins

The use of an ion exchange resin into the decalcifying solution has been claimed to speed
up the process of decalcification, and to improve staining. The principle is that the calcium
ions are removed from the solution by the resin, thereby increasing the rate of solubility of
the calcium from the tissue.
3.2 Confirmation of Decalcification

The process of decalcification can be assessed by the following ways:
3.2.1 X-rays:
X-ray examination is the most satisfactory method depending upon the facilities available.
These are used because bone is radio-opaque due to presence of calcium salts. When the
calcium salts are removed, tissue becomes transparent for such rays. When mercuric
chloride has been used as a fixative, x-rays cannot be applied because this fixatives renders
tissue radio-opaque.
3.2.2 Chemical Test:

A chemical test is a simple and reliable method when x-rays are not available. It is a two
stage test which depends on the detection of dissolved calcium in the decalcifying fluid. A
positive result at either stage indicates that further decalcification of tissue in fresh fluid is
required and the test should be repeated after a suitable interval.
3.2.2.1 Method:
Decant 5ml of the used decalcifying fluid into a clean test tube and add a small piece of
litmus paper.
Add strong ammonia of specific gravity 0.88 drop by drop while agitating the tube until
the litmus paper just turnss blue, indicating alkalinity.
If the solution becomes turbid at this stage, calcium is present in considerable amounts
and the tissue should be transferred to fresh decalcifying fluid.
If the solution remains clear proceed with the second stage of the test. Add 0.5ml
saturated aqueous ammonium oxalate, mix and allow to stand for 30 minutes. Any
turbidity developing during this period indicates the presence of calcium and re
immersion of the tissue in fresh decalcifying fluid is necessary.
24
If the solution remains clear, it may be assumed that decalcification is complete.
3.2.3 Decalcification can also be assessed by:

a. Weighing the biopsy before and after decalcification.
b. Bending the bony biopsy.
c. Putting a pin into the biopsy.
Box 4
R It is important that sufficient time is allowed between tests to ensure dissolution of calcium by
the fresh decalcifying fluid. Intervals of 3 ± 4 hours are considered adequate for most
decalcifying solutions.
R When using the chemical test to control the degree of decalcification, it is essential that the
decalcifying fluid is prepared with distilled water.
R Failure to observe this precaution may result in false positive readings being produced by the
presence of calcium cons in tap water.
25
4. TISSUE PROCESSING
Any treatment with a tissue that is required to impregnate it with a solidifying medium to
facilitate its fine sectioning for microscopic examination is named as tissue processing
which involves following four steps:
Completion of Fixation
Dehydration
Dealcoholization or Cleaning
Impregnation or Infiltration
4.1 COMPLETION OF FIXATION

In automatic tissues processors one or two jars of fixative are kept in the processing
schedule and processing is started from these jobs to ensure complete fixation.
4.2 DEHYDRATION
Tissues contain large amount of water, both intracellular and extracellular. This water must
be removed so that it may be replaced by wax. This process of water removal is called
dehydration. This process is done because the solidifying media, e.g. paraffin wax is not
miscible with water. Tissue biopsies contain two types of water; one coming from aqueous
fixative solution and secondly the water present inside the tissue as its component.
In dehydration two mechanisms are involved as follows:

a) Hydrophilic Mechanism:
In this mechanism the dehydrating agent shows affinity for water and so attaches the water
from the tissue biopsy. This affinity is due to polar character of water molecules and
dehydrating agent used.
b) Dilution Mechanism:
The dilution mechanism is very simple, i.e. when biopsies are transferred from one jar to
another the dehydrating agent dilutes out water and finally biopsies become completely free
from water.
Different types of dehydrating agents are being used in histopathology department. The best
agent is ethyl alcohol which has the advantage that it is not poisonous. Less common agent
are methanol, acetone, dioxane and isopropyl alcohol etc.
26
4.2.1 Ethanol:
Ethanol C2H5OH or ethyl alcohol is commonly called alcohol and obtained by the process
of fermentation of sugars which is a biochemical change brought about by certain micro-
organisms (bacteria and yeast). Initially complex sugars like sucrose and starch are
FRQYHUWHGWRVLPSOHVXJDULQWKHSUHVHQFHRIDQHQ]\PH³,QYHUWDVH´SURGXFHGE\yeast. The
glucose formed is then converted to ethyl alcohol by the action of an enzyme termed as
³=\PDVH´ which is also produced by micro organisms. The process is illustrated by the
following chemical reactions:
C12H22O11 invertase C6H12O6 + C6H12O6

C6H12O6 zymase 2C2H5OH + 2CO2
The ethanol (C2H6O) obtained in this way is RQO\ EHFDXVH WKH PLFUREHV FDQ¶W DFW DV
catalyst beyond this concentration. By fractional distillation ethanol of 95.6% concentration
LVREWDLQHGZKLFKLVWHUPHGDV³UHFWLILHGVSLULW´7RREWDLQDEVROXWHalcohol, the quicklime,
i.e. Cao is added which absorbs water and so after distillation absolute alcohol is obtained.
Properties of Ethanol:
Ethanol is an excellent dehydrating agent and shows a strong affinity for water. It is also
non-toxic but it is costly and bears a high excise duty. It is quicker in action but has a
hardening effect on tissue. So it is used in graded strengths, beginning from 70%, Transfer
of tissue directly from formalin to higher grades of alcohol, e.g. 85 or 95% is risky because
it will lead to distortion of tissues. In case of delicate tissue biopsies, e.g. brain and embryo,
dehydration should be started with 50% alcohol.
Normally ethanol obtained by suppliers is not 100% and contains different qualities of
water, so poorly dehydrating the tissues. The purity is checked by using CuSO4 which exists
in 2 forms Anhydrous CuSO4 is white and when it absorbs water, hydrous form is obtained
which is blue in colour.
4.2.2 Methanol:
Methanol CH3OH is also known as wooden alcohol or wooden spirit and is obtained by
cracking a pyrolysis of wood at a temperature of 160oC. Methanol is very toxic and
inflammable and has an unpleasant odor. It is rarely used as a dehydrating agent due to its
toxicity.
27
4.2.3 Acetone:
It belongs to ketone family. It is cheap and quick in action as compared to ethanol but it
causes a lot of harness. Moreover, more volume is required for dehydration. It is highly
volatile and flammable so never be used in vicinity of open flame. It is not recommended
for automatic issue processors due to its volatility. It is potentially dangerous and there is
always a hazard of fire. It is used for quick dehydration in manual processing.
4.2.4 Dioxane:
Dioxane or diethylene dioxide is a unique reagent which has the unusual property of being
miscible with both water and molten paraffin wax. It is also miscible with alcohol and
xylene. It is simple to use and produces little shrinkage. It is also very toxic and so no
longer recommended.
4.2.5 Iso Propanol:

If alcohol is not available isopropanol can be used. So it can act as a substitute for ethanol.
,W GRHVQ¶W KDYH DQ\ KDUGHQLQJ HIIHFW %XW ODUJH YROXPHV are required and so takes a long
time for complete dehydration. Isopropanol is also termed as isoprophyl alcohol.
[Ethylene glycol monoethyl ether or cellosolve can also act as dehydrating agents]
Box 5
The purchase and use of absolute ethanol is subject to many restrictions for customs and excise
o
purpose. Therefore, its substitute, i.e. 74 OP spirit (absolute industrial methylated spirit) is
normally used in laboratories. 3URRIVSLULWLVOHJDOO\GHILQHGDV³WKHVSLULWZKLFKDWWKHWHPSHUDWXUH
of 51oF weighs exactly 12 ± 13th SDUWV RI DQ HTXDO YROXPH RI GLVWLOOHG ZDWHU´ $W o F it has a
specific gravity of 0.919 and contains 57.1% v/v or 49.2% w/w of ethanol. Spirits are described as
so many degrees over proof (OP) or under-proof (UP). Proof spirit is the standard and referred to as
100o. A spirit stated as 70o would therefore be 30o UP (100o ± 70o). A spirit stated as 160o would be
60o OP (100o + 60o). 95% alcohol is equivalent to 60 OP, which means that 100 volumes of this
would contain as much ethanol as 166 volumes of proof spirit.
As proof spirit (100) contains 57% ethanol, 74 OP (174) would contain:
57 X 174/100 % ethanol = 99%
100o=57%, 1o=57/100, 174o=57/100x174 = 99.18%
28
4.3 CLEARING
The removal of dehydrating agent from a tissue biopsy is termed as cleaning or
dealcoholization. The chemicals used for the purpose are termed clearing agents. The term
clearing is used because it was found initially that most of the chemicals, e.g. xylene and
toluene increase the refractive index of tissue biopsy and make them transparent. However,
now a days it is proved that certain other chemicals, e.g. carbon tetrachloride and
chloroform have no such effects on biopsies but can be used to remove the dehydrating
agent. The term dealcoholization is also a misnomer because certain dehydrating agents are
not alcohols, e.g. acetone.
In histopathology clearing can also be used for the purpose of making tissues, embryos and
parasites transparent so that their internal structure is demonstrable to the naked eye. This is
done by impregnating them with a clearing agent whose refractive index is close to that of
the tissues themselves. For this purpose oil of wintergreen (methyl salicylate) and xylene
are used.
Clearing Agents:
The clearing agents used must be miscible with both alcohol and paraffin wax. The most
common clearing agents are xylene, toluene, chloroform, cedar wood oil, carbon
tetrachloride, e.g., petrol.
4.3.1 Xylene:
It is an aromatic compound and benzene derivative. It is good clearing agent and increases
the transparency of tissue biopsies. It is comparatively cheaper and highly flammable.
Prolonged immersion of tissues (>3 hour) makes tissues hard and sectioning is difficult to
perform. It also causes brittleness of tissues. Tissue blocks 3mm in thickness are cleared in
15 ± 30 min but certain material, e.g. brain and lymph nodes become brittle if immersion is
prolonged. Such biopsies are best cleared by chloroform. When dehydration is not complete
and biopsy is kept in xylene jar, xylene become milky.
4.3.2 Toluene:
,Q JHQHUDO SURSHUWLHV WROXHQH LV VLPLODU WR [\OHQH EXW LW LV SUHIHUDEOH EHFDXVH LW GRHVQ¶W
harden the tissues. It is comparatively slower in action but somewhat expensive than
xylene. Its fumes may be toxic. It is flammable and has clearing property like xylene.
29
4.3.3 Benzene:
It is similar to xylene and toluene. Initially, benzene C6H6 was considered as a very good
clearing agent but now a days its use has been restricted due to its known carcinogenic
properties.
4.3.4 Chloroform:
It is recommended for nervous tissues, lymph nodes and embryos as it causes little
VKULQNDJH DQG GRHVQ¶W KDUGHQ WKH WLVVXHV ,W LV UHODWLYHO\ H[SHQVLYH DQG QRQ-flammable.
Unlike Xylene and toluene, thick tissue biopsies upto 1cm can be cleared whereas in case of
xylene and toluene the thickness should not be more than 3 ± PP ,W GRHVQ¶W KDYH WKH
transparency effect on tissues. Traces of chloroform in tissues cause a problem in
sectioning. Its vapors are anesthetic and WR[LF2QKHDWLQJLWUHOHDVHDWR[LFJDV³3KRVJHQH´
Moreover, it may have a deleterious effect on the rubber sealing ring of the vacuum
impregnating bath.
4.3.5 Carbon Tetrachloride:

7KLV FOHDULQJ DJHQW VKRZV VLPLODU SURSHUWLHV DV OLNH FKORURIRUP ,W GRHVQ¶W KDYH
transparency effect and just like chloroform releases phosgene gas.
4.3.6 Petrol:
Its properties are similar to xylene but are not recommended because different additives are
present in petrol and moreover it cannot be used as a clearing agent when methanol is used
as a dehydrating agent due to their non-miscibility.
4.3.7 Cedar Wood oil and Clove Oil:

These are rarely used as clearing agents because of their cost and slow action. So can be
used for delicate biopsies. But more time is consumed in clearing stage and later on in the
impregnation stage. It is due to high viscosity of oils. It is of great importance in research
labs and in embryological procedure. These oils impart consistency to tissues like skin and
dense fibrous material which facilitate subsequent sectioning.
These oils can tolerate small traces of water. This property is helpful in humid climate areas
where it is difficult to keep the dehydrating agent completely free of water. Cedar wood oil
for histological purpose is a thin, colourless, slightly yellow fluid distinct from the more
viscous type used for oil immersion objectives and which is unsuitable for clearing.
30
4.3.8 Carbon disulphide paraffin oil and methyl benzoate are less commonly used
clearing agents.
Box 6
In certain cases of tissues processing the clearing stage can be omitted because we can use such a
dehydrating agent which is miscible both with water and paraffin wax. Two such examples are:
a. Dioxane (diethylene dioxide)
b. Cellosolve (ethylene glycol monoethyl ether)
These two dehydrating agents are miscible with paraffin wax and so it is not necessary to use any
clearing agent. However these two chemicals are very costly, slow penetrating and their vapours
are toxic. Therefore, the tissue processing involving clearing step is preferred. On the other hand,
advantage of dioxane and cellosolve is that these reagents can be used again and again for
dehydration purpose. These can themselves be dehydrated by CaCl2 and calcium oxide or
quicklime (CaO).
4.4 IMPREGNATION OR INFILTRATION

After clearing, tissue biopsies are transferred to some suitable solidifying medium which
replaces the clearing agent and then completely fills the natural cavities, spaces and
interstices of the tissues. Later on this medium is solidified and biopsies are internally
supported. An impregnating media must enter the tissue spaces in liquid form and then
solidify to support the tissues or cells. Apart from giving support, handling of tiny biopsies
is also facilitated by the step of impregnation and then by embedding.
Impregnating Media:
A wide range of impregnating media is available. Some of which are listed below:
i. Paraffin Wax
ii. Paraplast
iii. Celloidin
iv. L.V.N
v. Carbowax
Generally speaking, the volume of the impregnating medium should be at least 25 times the
volume of tissue.
4.4.1 Paraffin Wax

It is the most widely used impregnating medium obtained by the cracking of mineral oil and
is a mixture of saturated hydrocarbons. Various varieties of paraffin wax having melting
point (MP) in the range of 40 ± 70o C are available which can be sued according to the
31
temperature of laboratory. Normally a paraffin wax of melting point (MP) 54 ± 58o C is
used.
Nowadays different additives are added by the manufacturer, to get a proper MP and
moreover to increase the stickiness of the wax. Lard, beeswax, satiric acid, rubber and
ceresin are various examples of the additive used. Histopathological handling of tissues
XVLQJ SDUDIILQ ZD[ DV D VXSSRUWLQJ PHGLXP LV WHUPHG DV ³3DUDIILQ ZD[ VHFWLRQLQJ
WHFKQLTXH´.
Advantages:
i. Paraffin wax is cheaper, free of colouration and non-reactive to tissue
components.
ii. Paraffin wax sectioning techniques require minimal supervision during the
embedding and infiltration stages.
iii. A large number of specimens can be processed by this technique because it is easier
in handling of specimens.
iv. Paraffin wax blocks and sections can be stored at room temperature.
v. Paraffin wax sections can be transported without any special treatment.
vi. Very thin sections can be obtained from paraffin wax blocks.
vii.A multitude of staining techniques can be applied, after paraffin wax impregnation
and embedding.
viii. Serial sections can also be easily produced by this technique.
ix. Paraffin wax is suitable for tissue blocks of different consistency, i.e. soft as well as
tough blocks can easily be support by paraffin wax.
Disadvantages:
i. The most important disadvantage is that heat is involved in melting of paraffin wax
and sometimes overheating results in causing a lot of harness and brittleness to
tissues.
ii. Paraffin wax undergoes shrinkage on cooling due to which cells in tissues may
disrupt. Normally 10% shrinkage is caused on paraffin wax cooling.
iii. Sometimes very tough tissue material is received in the laboratory that can only be
supported by celloidin not by paraffin wax.
4.4.2 Paraplast:
Paraplast is a mixture of purified paraffin wax and several synthetic plastic polymers. Its
MP is 56 ± 57o &%\ XVLQJSDUDSODVWULEERQVHFWLRQLQJLVHDV\DQGLWGRHVQ¶WFUDFN OLNH
paraffin wax. In case of paraplast sudden cooling is not required during embedding and
32
while sectioning it is not necessary to keep the blocks on ice cubes. The blocks obtained are
more uniform and it is more resilient than paraffin wax and allows the sectioning of large
blocks and dense bone blocks with great ease.
4.4.3 Carbowax
Higher alcohols, e.g. those with 12 or more carbon atoms are solid at ordinary room
temperatures. Those with 18 ± 22 carbon atoms in their molecules have physical
characteristics that are suitable for tissue embedding. Such alcohols are polyethylene glycol
DQGDUHJLYHQWKHQDPH³&DUERZD[´
For routine tissues, four changes of carbowax, i.e. 70%, 90% and two times in 100% at the
56 o C are used. The length of time is 30 minutes, 45 minutes and 1 hour respectively.
Carbowax is soluble and miscible with water, so from aqueous fixative, tissues can directly
be taken to carbowax and there is no need of dehydration and clearing stages. It does not
remove substances like lipids and neutral fats, thus allowing these substances to be
demonstrated in thin sections. This technique is also good for many enzyme histochemical
studies. Moreover, it reduces shrinkage and distortion.
Disadvantages:
i. The blocks of carbowax must not be allowed to come into contact with water or ice,
EHFDXVHFDUERZD[LVYHU\³K\JURVFRSLF´
ii. During the use of carbowax, blocks and unstained sections must be stored in dry
airtight containers coated with paraffin wax in a cool atmosphere. Otherwise
moisture at room temperature softens the blocks.
iii. &DUERZD[ LV ZDWHU VROXEOH VR VHFWLRQV FDQ¶W EH IORDWHG RXW RQ ZDWHU )RU WKLV
purpose special mixtures are used, e.g. Pearse
Pearse:
Diethylene glycol 40 parts
Distilled water 50 parts
Formalin 40% 10 parts
Blank and McCarthy:

0.02% gelatin equal parts
0.02% potassium dichromate equal parts
Boil for 5 minutes, cool and filter.
33
4.4.4 Celloidin
Celloidin is the trade name given name to a purified form of nitro-cellulose. It is available
in an amorphous yellowish wood, like substance dampened with alcohol. The working
solution is prepared in equal parts of diethyl ether and ethyl alcohol (solvents). The strength
of working solutions are 2, 4 and 8%.
During embedding, solidification of celloidin occurs by simple evaporation process or can

be hastened by dipping the blocks in chloroform. It is very good for neuropathological
biopsies. It is of particular value as a histological embedding medium for sectioning hard
tissues of a mixed consistency. Moreover sectioning of very thick sections is also facilitated
by celloidin.
Disadvantages:
i. By using celloidin, thin and serial sections are difficult to obtain and only thick
section of 5 ± 10 u can be produced.
ii. Evaporation is a constant problem when using celloidin and the working solutions
should always be stored in bottles fitted with ground-glass stoppers.
iii. It must be remembered that one of the solvent, i.e. ether is dangerous because of its
vapours and therefore celloidin should never be used in the vicinity of open flame.
4.4.5 L.V.N.
Low Viscosity Nitrocellulose is similar to celloidin but due to low viscosity, thinner
sections are easily produced. Moreover a harder block is formed with L.V.N than with
celloidin. The L.V.N sections have a tendency to crack, but plasticizers can be incorporated
into the medium to overcome this problem.
34
5. EMBEDDING
In this stage processed tissue blocks are now externally supported by the same supporting
medium as used in the impregnation stage. Tissues are embedded using supporting medium
in molten state with the help of different moulds. It gives an external support which then
facilitates sectioning on a microtome. Different types of moulds are used for making blocks.
Most commonly used are briefly described below:
5.1 LeXFNKDUW¶V0RXOGV
These are convenient moulds for routine work and are widely used. They consist of two L-
shaped pieces of metal, usually brass, and may be purchased in a variety of sizes. They are
arranged on a glass or metal plate to forma mould (socket) of the desired size. When a
socket is obtained tissue specimen is put at the base of socket and then molten paraffin wax
is dispensed. The tissue block is pressed with a warm forcep. Then more wax is poured and
block is allowed to cool for solidification. For cooling, blocks can be kept in refrigerator or
on cold plates if available.
Advantages:
i. These moulds give even blocks with parallel sides.
ii. They are adjustable to give a wide variety of block sizes.
On the other hand, they are somewhat cumbersome and too slow for a busy
laboratory.
5.2 Steel Base Moulds

The system using steel base moulds and tissue embedding cassette is given the name
³7LVVXH 7HN 6\VWHP´ 7KH ELRSVy is placed in depression of the base mould and then
paraffin wax is dispensed. The tissue biopsy is properly centered and oriented with warm
forcep. The tissue embedding cassette is applied and wax is poured in its cavity. Then the
steel base mould with fixed embedding cassette is kept on a cold plate for quick, even
solidification of the embedding medium to get a paraffin block. Steel base mould can be
reused whereas embedding cassette gives a permanent paraffin, block and fits in chuk
adapter of microtome. The steel base moulds are pre-coated with a release compound in
alcoholic solution so that the wax does not adhere to them.
35
Advantages:
There are various advantages of the Tissue Tek System. Some of which are:
i. Handling of tissue block is very easy. Embedding cassettes are directly fixed in
block holder (Chuk Adapter) of the microtome.
ii. Embedding cassettes can also be used for processing of tissue biopsy.
iii. Numbering of blocks is very easy; as a separate specified area is present in the
cassette.
iv. Transportation of blocks in the form of embedding caste is very easy.
5.3 Watch Glasses

These are ideal for embedding fragmentary biopsies. It is not essential to smear them with
glycerol, but it is a sensible precaution because sometimes blocks are difficult to remove.
There are not commonly used in the laboratory.
36
6. MICROTOME
The microscope is designed to facilitate the study of animal tissue by transmitted light and
for this purpose the tissue must be sliced into thin lamellae or sections. To obtain such thin
VHFWLRQVRIXQLIRUPWKLFNQHVVDGHYLFHLVXVHGWHUPHGDV³0LFURWRPH´The first microtome
was invented by George Adams and Alexander Cummings in 1770. Another scientist from
Czech Republic, -DQ (YDQJHOLVWD 3XUN\QČ, who was a physiologist, further developed the
device which was the first to be used practically.
Figure 6.1 18th Century Microtome by Alexander Cummings

Adapted from Journal of the Royal Microscopical Society, 1910, pages 779-782. The Royal
Microscopical Society, Oxford, England.
There are different designs of microtomes but all possess some basic requirements. All
microtomes must have a rigid support for tissue block as well as for microtome knife. After
each cutting stroke, advancement of the tissue block towards the knife edge with respect to
predetermined microns. The means of movement of tissue block across a fixed knife or
sometimes movement of knife across a fixed tissue block. The flywheel in many
microtomes can be operated by hand. This has the advantage that a clean cut can be made,
as the relatively large mass of the flywheel prevents the sample from being stopped during
the sample cut. The flywheel in newer models is often integrated inside the microtome
casing.
Different types of microtomes are now available commercially. Some impotent types are
briefly described below:
37
6.1 Cambridge Rocking Microtome
This microtome is very simple in design. It is not manufactured now a days but remains a
favourite microtome for many workers because of its simplicity of operation and
maintenance and its ability to produce sections of high quality.
Principle:
In this microtome the tissue block is swung across a fixed knife edge in the form of an arc.
A spring is attached with the operating hand which controls the movements of advancement
as well as of block across the knife. Sections prepared on the Cambridge rocking microtome
are cut in a slightly curved plane.
The Cambridge rocking microtome was one of the first instruments to be incorporated into
a cryostat by British manufacturers for the preparation of sections from unfixed tissue at a
temperature of about -20oC. This was because of its simple mechanism and small number of
moving parts. However, nowadays rotary microtome is used in cryostat.
Figure 6.2 Cambridge Rocking Microtome

Adapted from http://www.radicalscientific.com
Advantages:
i. This microtome is very simple in design and easy to operate.
ii. It is also cheaper and free from high custom duty.
iii. Small and soft tissue blocks can easily be sectioned.
Disadvantages:
i. This microtome is only suitable for small sized blocks (1-2cm2).
ii. The sections obtained are slightly curved, so not so much uniform in thickness and
therefore disturbs focusing under the microscope.
iii. Tough material cannot be sectioned by this microtome.
38
6.2 Rotary Microtome
It is the most widely used sectioning device in routine histopathological laboratories. This
was invented by Minot in 1885-1886 and independently by Pfeiffer in 1886. Pfeiffer was a
mechanic at Johns Hopkins.
Principle:
In this microtome the Tissue block moves across a fixed knife edge in a vertical plane, i.e.
by vertical rise and fall movement perpendicular to knife edge. The vertical movement and
advancement movements are controlled by an operating handle which is in the form of a
rotor or wheel on one side of the machine. Due to this shape of operating handle, the rotary
microtome is given this name. The block holder of rotary microtome is equipped with
adjusting screws to ensure that the block is parallel to the microtome knife in all planes.
Figure 6.3 Rotary Microtome

Adapted from http://www.radicalscientific.com
Advantages:
i. The rotary microtome is very useful for serial sectioning.
ii. This microtome is more rigid then Cambridge rocking microtome.
iii. Sectioning of somewhat tough material is also possible.
iv. Tissues blocks of size 3-4 cm2 can also be sectioned easily.
v. This microtome is used in cryostat, although other types were initially used.
vi. It is recommended for research labs because it gives sections of superior quality.
vii. It is also recommended for laboratories with high workload because it is easy to
operate and tissues of uniform thickness can be obtained.
Disadvantages:
i) This microtome is complex in design and construction and this complexity leads to its
high cost.
39
ii) It is not suitable for large blocks or very tough material embedded in celloidin or LVN.
The knife is also dangerously placed (blade up)
6.3 Base sledge Microtome

The base sledge microtome is heavy duty rigidly constructed machine adaptable for
sectioning specimens embedded in all forms of media.
Principle:
In this microtome the tissue block resting on a type of sledge is pushed horizontally beneath
a knife edge that can be slanted. So, larger sections can more easily be cut with the knife set
at an angle. By changing the slant angle, less resistance is offered by block in sectioning.
This microtome is excellent for cutting sections from blocks of tough tissue, especially if
the blocks are large and offer marked resistance to the knife. This microtome may be
adapted for frozen section cutting by replacement of the paraffin wax object holder with
either a CO2 freezing stage or a thermo module.
Figure 6.4 Base Sledge Microtome

Adapted from http://websites.labx.com
6.4 Sliding Microtome

The fundamental difference between the sliding microtome and those described on previous
pages is that with this instrument the block remains stationary while the microtome knife
moves during the process of sectioning.
The main value of sliding microtome is the ease with which it cuts sections from tissue
embedded in celloidin. A number of instruments of varying designs are produced
FRPPHUFLDOO\ 2QH RI WKH PRVW SRSXODU DPRQJ WKHP LV WKH ³5HLFKHUW 8QLYHUVDO 6OLGLQJ
0LFURWRPH´
6.5 Freezing Microtome

Freezing microtome is one of the methods for obtaining frozen sections. It is used primarily
for cutting sections of fixed tissue:
a. When speed is of the utmost importance.
40
b. When a cryostat is not available.
It is also required to demonstrate fat histologically, and when certain neurological structures
are to be studied.
Principle:
In this microtome representative portion of a biopsy is rapidly frozen in a circular stage.
This stage is perforated at the periphery and is supplied with a cylinder of CO 2 under
pressure. As CO2 escapes freely through the perforated stage, lowering of temperature
occurs and tissue block is frozen at the top surface of the stage. Then sectioning is
performed by a movable knife which can move across the surface of the fixed frozen tissue
block.
This is a crude method in which there is no temperature control and one can suffer a lot of
problems during the sectioning. The freezing microtome differs markedly from those
machines used for preparation of paraffin wax sections.
Thermo Modules:
Thermoelectric cooling units may be used in place of CO2 gas to freeze the tissue and cool
the knife. These units referred to refrigeration capacity and function by a phenomenon
NQRZQDVWKH³3HOWLHU´HIIHFW7KHVHXQLWVDUHSURGXFHGFRPPHUFLDOO\DQGDUe designed to
fit a large number of microtomes. But on the other hand they are now less commonly used
because of the widely used cryostat. For freezing microtome, optimum cutting temperature
for the tissue is usually about -20o C.
6.6 Cryostat
The best method of preparing sections from unfixed tissue is by use of a cryostat. The
cryostat consists of a microtome housed in a deep freeze, cabined, maintained at a
temperature of approximately -15 to -30o C. A thermostat controls the temperature of
cabinet. The optimum working temperature of cryostat is -18 to -20o C. The microtome is
handled by on operating handle outside the cabinet.
There is a variety of cryostats manufactured; the fundamental difference between them is

the type of microtome employed. The earliest models manufactured in UK incorporated the
Cambridge rocking microtome. However, nowadays rotary microtome is commonly used.
Several models fitted with base sledge microtomes, suitable for cutting larger and tougher
tissue blocks, are also available. Wedge Profile knife is used in Cryostat necessary.
Cryostat, are usually provided with a rapid freezing attachment for this purpose and for
attaching tissue blocks to the block holder. This latter is extremely valuable when the
41
instrument is to be used for preparing urgent sections from biopsies. The tissue blocks not
to be sectioned immediately should be quenched and stored at a temperature of -20o C in
airtight containers or aluminum foils. In order to avoid the formation of large disruptive ice
crystals when freezing fresh tissue, rapid freezing (quenching) is necessary.
The maintenance and procedure of using cryostat is described in chapter 10s.
6.7 Laser Microtome

Laser Microtome operates using a cutting action of an infra-red laser. It is used for contact
IUHHVOLFLQJ7KHXVHGRHVQ¶WUHTXLUHSUHSDUDWLRQRIWKHVDPSOHWKURXJKHPEHGGLQJIUHH]LQJ
or chemical fixation, thereby minimizing the artifacts from preparation methods.
6.8 Parts of a Microtome

The basic parts of a microtome are as follows:
Operating handle, this is rotated to move the tissue block for cutting and advancement
movements
Tissue block holder, with fixing screws
Thickness gauge, to set the selected microns
Knife clumps with screws, for knife support
Operating handle lock
Knife safeguard
Feedback mechanism: It is used to move the block towards or away from the knife edge.
When in operation, it controls the regular advancement movement of the block after
each cutting stroke for the predetermined microns.
6.9 Microtome Knives

The microtome knives are of two main types as follows:
6.9.1 Those which require sharpening and stropping; and
6.9.2 Disposable knives which are actually disposable blades with disposable blade
holder.
The first class of knives is further classified according to their cross-section (profile) into 4
classes as follows:
6.9.1.1 Wedge Profile Knife:

This knife is wedge shaped in its cross-section, i.e. broader at the base and tapering towards
the edge. This knife is plane on both surfaces. It is rigid because its edge is well supported.
It is widely used for sectioning of tissue blocks embedded in different media. It is also used
42
for frozen sections in a cryostat. Disadvantage is that during the sharpening a knife back is
required which keep the surfaces of knife apart from the hone.
6.9.1.2 Planoconcave Profile Knife:

It is plane on one surface and has a hollow ground on other side. This knife is usually
recommended for celloidin or paraffin was embedding blocks. It is less rigid than wedge
profile knife because its base is weaker due to concavity. When plane surface of the knife is
used to sharpen the edge, knife back is required while it is not required when edge is
sharpened from concave side. Concave side is helpful in picking slightly curved sections in
rocking microtome.
6.9.1.3 Biconcave Profile Knife:

Its both surfaces are hollow ground. It is used for softer tissue blocks like paraffin wax
blocks. Its edge is weakly supported and is not vibration free while sectioning. Knife back
is not required during sharpening of this knife. The popular Heiffer knife has a biconcave
profile. This knife with its distinctive integral handle was designed for use with the
Cambridge rocking microtome.
6.9.1.4 Tool Edge Profile Knife:

This knife is plane on both surfaces with a steep cutting edge. It is used for cutting extra
hard materials such as un-decalcified bone. With the exception of Heiffer knife, most
knives have detachable handles fitted at one end.
Figure 6.5 (a): Microtome Knives

Adapted from http://train-srv.manipalu.com/wpress/?p=1368
43
Figure 6.5 (b): Microtome Knives
Adapted from Baker FJ, Silverton RE. Introduction to Medical Laboratory
Technology. © Butterworth & Co (Publishers) Ltd.
6.9.2 Disposable Knives:

Nowadays disposable blades which eliminate the use of sharpening are now in common
use. These can be fitted in disposable blade holders and use for sectioning of a number of
blocks depending on the degree of hardness.
When such a blade becomes blunt, it can be replaced by a new one. The blade holder
permits easy loading and replacement when loaded. The holder is placed on microtome in
usual way. Use of disposable blades saves a worker from the fatigue of sharpening and
moreover, thin, fine sections are easy to obtain.
6.10 Sharpening of Microtome Knives:

A microtome knife requires sharpening whenever its cutting edge becomes blunt or
damaged. The process of sharpening is divided into two stages, honing and stropping, and
each of these operations may be performed either by hand or by means of automatic knife ±
sharpening machines.
Honing is the grinding of metal from the knife edge with an abrasive substance until all
nicks have been removed, and the edge is sharp and straight.
Stropping is cleaning or polishing the knife edge on a softer material, usually leather͘
6.10.1 Manual Honing

The honing is the grinding of metal from the knife edge with an abrasive substance until all
nicks have been removed and the edge is sharp and straight. The hone is a rectangular block
of natural or synthetic stone, graded coarse, medium or fine according to the degree of its
abrasiveness. It is also called oil stone because oil is used as a lubricant. A widely used
natural stone of medium grade is the Belgian yellow stone, which gives good results at
reasonable speed. Other stones in common use are:
44
Arkanas which is natural stone of clear white to pale yellow colour. It is less abrasive than
Belgian and slower in action.
Aloxite which is a series of composite stones ranging in abrasiveness from coarse to
superfine.
Procedure:
The knife is placed at one end of the hone and is pushed diagonally forward in such a way
that cutting edge is leading. At the end of stroke, the knife is turned over on its back and
with cutting edge leading again (with back trailing), it is pulled back along the hone towards
the operator. Pressure on the knife needs to be just sufficient to maintain its edge in contact
with the surface of the hone. The number of strokes required depends on the condition of
the knife, but honing is complete when all the major nicks have been removed.
Figure 6.6: Direction of the movements in honing

Adapted from Frank Burr Mallory and James Homer Wright's Pathological
Technique: a Practical Manual for Workers in Pathological Histology and
Bacteriology, W.B. Saunders, Philadelphia 1918; page 27
6.10.2 Stropping
After honing, if edge is seen under a low-power microscope the edge will be seen fine but
regularly serrated. These serrations are due to the abrasiveness of stone and are removed by
the polishing process termed stropping.
Stopping is done in a manner similar to honing from toe to heal but unlike honing, back is
leading and edge is trailing. Strops may be either flexible or rigid. Good quality leather such
as horsehide is recommended due to its durability and effectiveness.
45
Figure 6.7 : Direction of the movements in stropping
Adapted from Frank Burr Mallory and James Homer Wright's Pathological
Technique: a Practical Manual for Workers in Pathological Histology and
Bacteriology, W.B. Saunders, Philadelphia 1918; page 27
6.10.3 Automatic Sharpening Machines

These machines offer tremendous saving in time and relatively inexperienced personnel can
produce well sharpened knives with a uniform level. This machine is provided with a glass
or copper plate and is incorporated with rotary and oscillatory movements. In this machine
a main spindle is provided having the knife holder. The plate is lapped with an abrasive
material and knife is fixed in the holder in such a way that only edge machine is switched
on, the plate moves beneath the knife edge and it is sharpened. After a specific interval of
time, the spindle rotates and the knife turns over at its back and then sharpening of the other
edge of the knife takes place. The machine is provided with a digital pad where all the
knobs for controlling speed of movement and time of sharpening etc. are given.
Knife Parts:
a) Foot b) Edge c) Base d) Heel
Knife Back:
It is U-shaped piece placed at base of knife so that knife is lifted and only edge is rubbed on
a bone.
6.11 Knife Angles

There are three types of angles that can be observed using a microtone knife.
a. Slant Angle
b. Clearance Angle
c. Bevel Angle
46
6.11.1 Slant Angle
In case of base sledge microtome the knife is horizontally placed and the angle produced is
called slant angle. Thus resistance is decreased and tough tissue material can be sectioned
easily. The cutting surface is always different from main surface.
6.11.2 Clearance Angle

The surface of blocks should be parallel to cutting facet of bevel and not to main surface of
knife. Therefore, the knife is set at a tilt on the microtome to allow a clearance angle
between the cutting facet and block surface. Clearance angles between 1 and 6 degrees have
proved to the most satisfactory. Biconcave knife requires a smaller clearance angle than
wedge-type knives.
6.11.2 Bevel Angle

The angle between two cutting facets of microtone knife is called bevel angle as shown in
the following figures.
47
7. SECTIONING
Paraffin blocks of tissues are sectioned by means of a Microtome. The sections which are
produced are capable of transmitting light and are at micron level thickness. Black
bottomed water both is used in sectioning because there is no interference of light.
The sectioning procedure includes following steps:
1. After embedding paraffin blocks are kept on a cold plate with their surfaces upside
down. Just before sectioning blocks are transferred to ice cubes.
2. A suitable knife is inserted in microtome and knife edge is kept slightly away from
the block holder by using feedback mechanism.
3. Then paraffin block is fixed in the block holders.
4. Knife edge is brought towards the block surface by using feedback mechanism when
block just touches the knife edge, microns are set for trimming of the block.
5. Trimming is done at 10-15u but in case of tiny biopsies trimming is rarely done.
6. After trimming fine sectioning is done at 3-4u and ribbon of sections is floated on
the surface of cold tap water in a kidney tray and wrinkles are removed by using
forceps to flatten the ribbon of sections.
7. Then 2 or 3 sections from the ribbon are transferred to warm water in a water bath
having temperature 5-10oC less than the melting point of paraffin wax. Here sections
are completely flattened due to expanding of paraffin wax.
8. Then sections are picked on slides already smeared with adhesive. (Mayer¶s albumin
glycerol adhesive).
9. The slide is then properly numbered and kept on a hot plate having temperature of
about 5-10oC above than the melting point of the wax. This step is meant for drying
of sections and melting of paraffin wax surrounding the sections.
7.1 Adhesive
1RUPDOO\0D\HU¶V$lbumin glycerol adhesive is used. No adhesive is used when tissues are
of uniform thickness. It is used when:
i. Tissues are not of uniform thickness.
ii. Tough tissues material is received in laboratory
iii. There are prolonged staining techniques, e.g in immune-histochemistry.
48
7.1.1 Preparation:
1. Egg white is highly viscous.
2. Glycerol is a form of alcohol so termed as glycerin.
3. Glycerin is also viscous so 50% of it is added to 50% egg white to form Adhesive.
4. Fungus may grow in the adhesive so preservatives like thymol can be added.
49
8. STAINING
When an unstained section is observed under a microscope, different cellular components

transmit the white light almost equally. In such a case, no sharp contrast is present and we
can observe very little details of the cells. Staining is preformed to colour the cellular
components differently so producing a contrast or differentiation. By this way different
cellular components become more prominent for microscopic studies. The colour is infact a
perception of eye and determined by the specific wavelength that is perceived by a normal
eye.
Staining is helpful to produce a contrast because different cellular components possess

affinities for different stains, e.g. nuclei show a high affinity for basic dyes like
Haemotoxylin whereas cytoplasm components shown a high affinity for acidic dyes like
Eosin.
8.1 Factors Affecting Staining

In staining, different physical and chemical factors are involved. The most important factors
are:
Acidic and Basic Reaction
Cellular components are acidic or basic in nature, so as the stains. Very clearly therefore,
acidic components of cell are stained with basic dyes and vice versa.
Adsorption
In adsorption certain small molecules of dyes are attached and fixed on the surface of larger
molecules of the tissues components e.g. staining of dextrons with iodine.
Differential Solubility
In differential solubility, a stain or dye bears different solubilities into different solvents,
e.g. in lipids staining the dyes like Sudan Black B or Oil Red O are alcoholic solutions.
When these solutions are applied to lipids, due to higher solubility in lipids the dyes
gradually leave the original solvent (alcohol) and enter the lipids and so staining is brought
about.
50
8.2 Classification of Stains/Dyes
Stains are coloured materials and are such chemicals which react with different components
like tissue components with different affinities to give them different colours. Stains are
classified by two ways:
8.2.1 1st Classification

Stains are classified as natural stains and synthetic stains.
i. Natural Stains:
Natural stains are extracted from a natural source and they constitute a small group of dyes.
Examples are haemotoxylin, Orcein, Litmus, Saffron and Carmine.
ii. Synthetic Stains:
6\QWKHWLFG\HVFRQVWLWXWHDODUJHJURXSRIVWDLQV7KHVHDUHUHIHUUHGWRDV³&RDOWDUG\HV´
because these are synthesized from substances obtained from coal tar. Examples are Eosin,
basic fuchsin, toluidine etc.
8.2.1 2nd Classification

Stains are classified as acidic stains, basic stains and neutral stains.
i. Acidic Stains:
In acidic stains, e.g. eosin, the colouring substance is contained in the acid component while
basic component is colourless. These stain the basic components of cell. The base is usually
sodium.
ii. Basic Stains:
In basic stains, such as haemotoxylin, the colouring substance is contained in the basic part
of compound. The acidic radical is colourless. These stain the acidic parts of the tissues.
iii. Neutral Stains:
These are obtained by combining aqueous solutions of basic and acidic dyes. The resulting
dye is insoluble in water but soluble in alcohol e.g. LeisKPDQ¶V VWDLQ 7KHVH VWDLQ ERWK
nuclei (acidic part) and cytoplasm (basic part).
51
8.2.3 3rd Classification
i. Microanatomical Stains:
These stains are used for demonstrating the general relationship of tissues to each other.
Nuclei and cytoplasm are differentiated but their included structures are not necessarily
differentiated.
ii. Cytological Stains:
Cytological stains demonstrate the minute structures in the nucleus and cytoplasm of cells
without the general differentiation of various tissue types.
8.3 Simple Benzene and Derivatives

Benzene C6H6 and its other simple derivatives are colourless in visible spectrum of light
because their adsorption band lies in the UV region. There are certain atomic groups which
when added to colourless benzene molecules, the adsorption is shifted from UV region to
YLVLEOHUHJLRQ6XFKSURGXFWVDUHNQRZQDV³&KURPRJHnV´DQGWKHDWRPLFJURXSWKDWVKLIWV
WKHEDQGLVJLYHQWKHQDPH³&KURPRSKRUH´
A chromogen is a coloured product but it differs from a stain or dye because any colour
which it imparts to a tissue is easily washed off and is not retained by the tissue. To convert
FKURPRJHQLQWRDG\HZHQHHGDQRWKHUDWRPLFJURXSNQRZQDV³$X[RFKURPH´
A dye or stain bears acidic and basic parts and so it shows salt forming properties. The
retention of a dye by any tissues component is its basic feature. So a synthetic dye may be
described as a benzene derivative, to which a chromophore and an auxochrome have been
added.
Box 7
Direct and Indirect Staining
Staining brought about by the aid of a mordant is called indirect staining and without mordant, the
staining is termed as direct staining.
Mordants
Mordants are metallic substances which act as a link between the stain and the tissue to be stained.
Accentuators
The substances which increase the staining intensity of staining solution without acting as mordant
are termed accentuators and are not necessary like mordant.
52
Vital Staining
The staining of inclusions in live cells is referred to as vital staining. Living cells may be stained
after removal from organism (supravital staining), or while still part of the body (intravital
staining).
8.4 HAEMOTOXYLIN
It is natural dye derived by the extraction of the wood of a Mexican tree Haemotoxylon
Campechianum. Haemotoxylin has poor staining properties and is normally used in
conjunction with a mordant. If the staining solution is oxidized, the staining intensity and
SURSHUWLHVDUHHQKDQFHG7KLVSURFHVVRIR[LGDWLRQLVNQRZQDV³5LSHQLQJ´,WLVGRQHLQWZR
ways. The first way of doing ripening is spontaneous or natural. The staining mixture is
placed in air openly and atmospheric oxygen brings ripening. It is very lengthy process and
takes 3 ± 4 months.
Other way of ripening is by chemical oxidants, i.e. some chemical agents are used for
ripening e.g. mercuric oxide. An exact amount of chemical oxidant is used with respect to
the amount of haemotoxylin. When the oxidant is in excess, over ripening result. The exact
quantities of oxidants are:
HX OXIDANT
1g 0.5 g(HgO)
1g 0.175 g (KMnO4)
1g 0.05 g (KIO4)
1g 2mL (H2O2)
Mordant is substance which interlinks dye molecules and tissue components. Haemotoxylin
is called indirect stain because it requires mordant. The mordant is indispensible for staining
reaction. In haemotoxylin staining, divalent or trivalent metallic salts are used, although
simple salts of metals like aluminum (Al), Iron (Fe) Chromium (Cr) can also be used but
conventionally alum salts are used in this staining.
K2SO4.Al2 (SO4)3.24H2O (Potassium Alum)
(NH4)2 SO4. Al2 (SO4)3.24H2O (Ammonium Alum)
(NH4)2 SO4. Fe2 (SO4)3.24H2O (Iron Alum)
Simple chloride and sulphate salts of aluminium can also be used. Use of alum slats is just a
tradition because initially these were cheaper.
53
8.4.1 Preparation
In routine, Harris haemotoxylin is used. In this type ripening is done by mercuric oxide
(HgO) potassium and aluminium is used as a mordant.
8.4.2 Ingredients
Hx Stain Powder 2.5 g
Absolute Alcohol 25 ml
Potassium Alum 50 g
Distilled H2O 500 ml
Mercuric Oxide 1.25 g
Glacial Acetic Acid 20 ml
Significance of Ingredients
R Hx is a stain /dye.
R Absolute alcohol is solvent for Hx powder.
R Alum is mordant.
R Dist. H2O is solvent for alum.
R Mercuric oxide brings ripening process.
R Glacial acetic acid enhances staining intensity.
8.4.3 Procedure
1. Dissolve Hx powder in absolute alcohol and alum in distilled H2O with gentle heat.
2. Mix both solutions and bring to boil rapidly. Then remove container from flame and
add HgO.
3. Cool the mixture rapidly and add glacial acetic acid.
4. Filter the stain and store in dark coloured bottle.
8.5 EOSIN
Eosin is commonly used as a countersstain along with haemotoxylin. Eosin is a synthetic
dye and belongs to xanthenes dyes group of stain. Eosin is a type of acidic dye so
cytoplasm, muscle fibers, RBCs and connective tissue fibers etc show affinity for eosin.
Eosin has an excellent property of staining different tissues components in different shades
ranging between orange to deep red. Different brands of eosin are available, eosin Y, B, S.
Eosin Y is commonly used. Orange G phloxine is a substitute.
8.5.1 Preparation
1. Eosin powder 1g Dist H2O 100 ml
2. K2 Cr2 O7 1g Dist H2O 100 ml
54
Prepare separately and then mix both solutions.
8.6 H/E Staining Procedure

Reagents Time Rationale
Xylene I 5-10 min De-waxing or
Xylene II 5-10 min Deparaffinization
Abs. Alcohol I 2-3 min Dexylenation

Abs. Alcohol II 1-2 min
90% Alcohol 1-2 min

70% Alcohol 1-2 min Hydration
50% Alcohol 1-2 min
Running tap water 1-2 min
Hx Stain 1-2 min Nuclear Staining

Progressive
3-5 min
Regressive
1% Acid water 1-2 dips Differentiation

(only for
regressive)
Alkaline 2-3 min Bluing

Running tap water
or (1% lithium
carbonate)
Eosin Stain 3-5 min Cytoplasmic Staining
Running tap water 1-2 min Excess Stain Removal
70% Alcohol 1-2 min

90% Alcohol 1-2 min Dehydration
Abs. Alcohol 1-2 min
Xylene I 3-5 min Clearing

Xylene II 3-5 min
Mounting of sections by a mounting media (Canada Balsom).

55
8.6.1 Results
Nuclei Blue
Cytoplasm Pinkish Red
Muscle fibers, Fibrin Deep Red
Collagen Fibers Pink
RBCs Orange
8.6.2 Bluing
In Hx staining dissociation of Al2 (SO4)3 takes place in aluminium ions and sulfate ions
whereas some molecules of H2O dissociate into OH- and H+ ions.
Al2 (SO4)3 Æ 2Al3+ + 3SO4-2

H2O ÆÅ H+ + OH-
Aluminium ions combine with OH ions to form insoluble aluminium hydroxide which
combines with dye molecules to form a complex known DV³/$.(´7KLVODNHWKHQOLQNV
with tissue components to bring about Hx staining. There are different sources of H+ ions in
Hx solution like dissociation of water, dissociations of acetic acid and H+ ions from
differentiators. When H+ ion are in excess, they suppress the concentration of OH- ions
which are then not available for the formation of Al (OH)3 necessary for Hx staining. In
bluing, excess of H+ ions are neutralized by some alkaline medium to make OH- ions
available, for Al (OH)3 formation.
Thus bluing is a neutralization reaction because Hx solutions having H+ ions in excess are
reddish and when H+ neutralized, Hx solution turns blue from red. The alkaline medium can
be running tap water (alkaline), 1% lithium carbonate or 1% ammonia water.
8.6.3 Differentiation
Nuclei show affinity for basic dyes and cytoplasm for acidic dyes but these affinities are not
absolute, i.e. certain part of the nuclei are stained with acidic dyes and so is in the case with
cytoplasmic parts which are stained with basic dyes. In Hx staining relative removal of the
dye is performed which means it is removed from cytoplasmic parts while nuclei still
remain strongly stained with Hx stain. This selective removal is known as differentiation. It
is performed in 1% acid water or 1% acid alcohol. The staining is known is Regressive
Method of staining.
56
Sometimes differentiation is not required and Hx staining is controlled by reducing this
time. This way of staining is called Progressive Method of staining. So Hx can be used
regressively or progressively.
8.7 METACHROMATIC STAINING

Metachromasia is a property by which a certain tissue component is stained by a certain dye
in shade or colour totally different from the original colour of the staining solution. The
stains which stain the tissue components in different colour than their original colour are
FDOOHGµMHWDFKURPDWLF6WDLQV¶. The metachromatic property depends on:
x Nature of Dye
x Nature of Tissue Component
Nature of Dye:
All metachromatic stains are basic in nature and contain large cationic Radicals. These
PDLQO\ EHORQJ WR ³$QLOLQH *URXS RI '\HV´ 7KHVH G\HV KDYH WKH SURSHUW\ RI
Polymerization. Their metachromatic property is totally dependent on polymerization. For
example, original solution of toluidine blue comprises of monomeric units and is blue in
colour. When it is applied to tissue, dimers and trimers are initiated to form due to which
violet colour is produced. As polymerization is completed, a complete metachromatic
colour appears, i.e. Red.
Nature of Tissue Component:

About tissue components it is found that these are composed of large anionic radicals like
phosphates (PO4), sulphates (SO4) carboxylic groups etc. These are acidic in nature.
Formerly such tissues components were called Acid polysaccharides. However, now a new
name Glycosaminoglycans is given to these tissue components. Example of such
components are heparan sulphate (heparin) concentrated in mast cell granules, chondroitin
sulphate richly present in cartilage matrix and sometimes dermatan sulphate and keratan
sulphate etc.
In all these substances, anionic radicals are very close to each other (0.5 nm apart) and
when stain is applied to such tissue components, basic cationic radicals are primarily linked
with acidic anionic radicals to form salts. At the same time a secondary linkage between the
dye molecules take place which is due to the proximity of anionic radicals of the tissue
component and facilitate the polymerization. Glycosaminoglycan joins with protein to form
proteoglycans, so proteoglycans and sulphated glycosaminoglycan both show
metachromatic property because these are Acid Mucopolysaccharides.
57
In metachromatic staining two types of reactions takes place:
Acid Base Reaction (Primary Reaction)
Polymerization, i.e. reaction between the dye molecules within the tissues (Secondary
Reaction)
8.7.1 Principle
The principle is dependent on two types of reactions (dual bonding). In first step, basic dyes
are bonded with acidic tissue components and in second reaction, dye molecules themselves
are polymerized to give a secondary bonding. It is due to proximity of tissue components.
8.8 TOLUIDINE BLUE STAINING FOR MAST CELL GRANULES

Mast cells are counterpart parts of basophils. These are large, round cells with a nucleus
centrally placed. The cytoplasm is full of coarse granules which usually obscure the
nucleus. These granules show metachromasia. The mast cells may be oval in shape and
their function includes the secretion of heparin (anticoagulant) and histamine (potent
vasodilator).
8.8.1 Reagent
1% toluidine blue in 50% isopropanol, which is standard toluidine blue stain
8.8.2 Procedure
i. Dewax and hydrate the sections.
ii. Remove pigments where necessary by neutralizing stain with acids or alkali.
iii. Stain the sections with toluidine blue staining solution for 15-20 min at 37oC.
iv. Differentiate the sections in absolute isopropanol till the background is clear (1
min). It is needed for removing the excess stain.
v. Dehydrate, clear and mount the sections with DPX [Dibutyl Phthalate Xylene] or
Canada balsam.
vi. Observe under microscope.
8.8.3 Results
1. Mast Cell granules, Purple to Red

cartilage matrix, (metachromatically
mucopolysaccharides stained)
2. Background Colourless
58
3. Some Abnormal Blue
Glycoproteins e.g. (orthochromatically
Amyloids stained)
8.9 NISSL SUBSTANCE STAINING

In neurons, there is abundant basophilic material in the form of clumps and aggregates. This
material is chemically ribonucleoprotein which display or manifest granular endoplasmic
reticulum (GER) found in protein synthesis. This basophilic material preset in the
cytoplasm of nerve cells is called Nissle substance or Nissl Body. Nissl substance is present
in nerve cell body (soma) and dendrites but absent in axon-hillock and axon itself. It was
named after Franz Nissl, German neurologist. It is believe that Nissle substance is involved
in the synthesis of neurotransmitters such as acetylcholine.
8.9.1 Demonstration
Nissl substance comprises acidic anionic radicals or molecules (RNA) and so can be
demonstrated by basic cationic dyes like those of aniline group. Nissl substance can be
stained by the toluidine blue staining solution or thionine etc. However, best results are
REWDLQHGZLWK³&UHV\OH)DVW9LROHW´
8.9.2 Principle:
The principle of this staining technique is very simple i.e. an acid base reactions. Cresyle
fast violet stains the acidic Nissle material along with nuclei which contain DNA and RNA.
8.9.3 Reagent:
0.1% aqueous solution of Cresyle fast violet, acidified with 0.7 ml of 10% acetic acid for
each 100 ml of solution to get a pH 3.5 -3.8.
8.9.4 Procedure
ii. Stain with Cresyle fast violet solution for 30 minutes at room temperature.
iii. Differentiate in 95% ethanol.
iv. Clear and mount the sections.
v. Observe under microscope.
59
8.9.5 Results
Nissle substance Violet
Nuclei Violet
Background Colourless
8.10 TRICHROME STAINING

Trichrome staining technique is not the name for a single method of staining but this term is
used for a group of staining techniques which are:
x 0DOORU\¶VVWDLQLQJ7HFKQLTXH
x MaVVRQ¶V6WDLQLQJ7HFKQLTXH
x Von Gieson Staining Technique
All these staining methods are called differentiation methods because these are used for
differential diagnosis of connective tissue fibers (collagen) from other tissue structures
mainly muscle fibers.
8.10.1 Significance
Trichrome staining techniques are required in cases, where muscle tumors, e.g.
Rhabdomyosarcoma needs to the differentiated from connective tissue tumors. This is
because in routine H/E staining, both collagen and muscle fibers are similarly stained in
case of malignant tissue. However in normal tissue stained with H/E, these two elements
can easily be differentiated with the help of staining intensities and morphological features
which are lost in case of malignancy.
8.10.2 Principle
In Trichrome staining different acidic dyes comprising molecules of different sizes are
used. Trichrome staining mainly depends on physical characteristics like Relative
Molecular Size of the dye molecules and permeability of different tissue components like
collagen, muscle, RBCs, fibrin etc.
Permeability of different tissue components depends upon the lattice structure of protein
acquired after fixation because during fixation cross linkage of protein results. Sometimes a
very loose extended lattice structure of protein is obtained as in case of collagen fibers.
Such a lattice structure is highly porous or permeable. In other case a lattice of medium
sized meshes is obtained as in case of muscle proteins. Some proteins produce a very tight
lattice structure and thus giving the least permeability or porosity, e.g. RBCs proteins.
60
In Trichrome staining, dimolecular sizes are important. And always the large dye molecule
stains the collagen and smaller one stain the muscle, irrespective of nature of the dye.
8.10.3 Role of PMA or PTA

It is common practice that role of PMA or PTA in trichrome staining is considered as a
mordant. But this concept is totally wrong. Actually these two acids which can be used as
substitute for each other, clear or free the meshes of collagen protein from small sized dyes
molecules like those of acid fuchsin. Later on molecules of these acids are themselves
replaced by larger sized dye molecules like Aniline blue.
8.11 0$//25<¶667$,1,1*
,Q0DOORU\¶VFRQQHFWLYHWLVVXHVWDLQLQJG\HVOLNH2UDQJH*$FLGIXFKsin and Aniline blue
are used along with phosphotungstic acid (PTA) a phosphomolybdic acid (PMA). When
Orange G is applied on sections, tight lattice proteins like those of RBCs are strongly
stained with the dye because small sized dye molecules are firmly fitted in small sized
meshes of the lattice structure. Muscle and collagen fibers are also stained but here the dye
molecules are loosely placed. Next Acid fuchsin stains muscle replacing the orange G
molecules. Collagen is also stained with acid fuchsin but again molecules of dye are free
and loosely placed in meshes of extended lattice of collagen. Then PMA or PTA smoothly
replaces acid fuchsin dye molecules from the collagen lattice network and finally acid
molecules are replaced by aniline blue dye molecules.
8.11.1 Reagents
1. 0.5% aqueous Acid Fuchsin
2. Orange G, Aniline Blue staining solution:
a. Orange G 02 grams
b. Aniline blue 0.5 grams
c. PMA/PTA 1 gram
d. Distilled H2O 100 ml
x Dissolve PMA in distilled water and then add the two dyes. Mix well, filter and use.
The original sWDLQGRHVQ¶WFRQWDLQDQ\QXFOHDUVWDLQVRQRZDGD\VLURQhaemotoxylin is used as a

nuclear stain.
8.11.2 Procedure
ii. Stain with solution± 1 for 5 min.
iii. Rinse in water.
61
iv. Stain with solution ± 2 for 10-15 min.
v. Differentiate in 95% ethanol.
vi. Dehydrate, clear and mount the sections.
vii. Nuclear staining is optional and if required, then iron haemotoxylin is used as an
initial step.
8.11.3 Results
Muscle and Fibrin Deep Red
Collagen Fibers Blue
Reticular Fibers Blue
5%&¶V Orange
Nuclei Black
8.12 VONGIESON STAINING

VonGieson staining technique is also one of the trichrome stains.
8.12.1 Reagents
A. Von Gieson Staining Solution:
a. Saturated picric acid 100 ml
b. 1% Acid Fuchsin 10 ml
B. Iron haemotoxylin staining solution
8.12.2 Procedure
ii. Nuclear staining is performed by iron haemotoxylin for 3-5 minutes (regressive
method)
iii. Perform differentiation and bluing.
iv. Stain with solution ± 1 for 5-10 minute.
v. Dehydrate, clear and mount the sections.
vi. Observe under the microscope.
8.12.3 Results
Nuclei Black
Muscle Fibers Yellow
Collagen and Reticular Fibers Red
62
8.13 0$6621¶6STAINING
0DVVRQ¶Vtrichrome stain is also included in the trichrome stains.
8.13.1 Reagents
i. 0.5% Acid Fuchsin:
a. Acid fuchsin 0.5g
b. Glacial acetic acid 0.5ml
c. Distilled H2O Upto 100ml
ii. PMA solution:
a. PMA 1g
b. Distilled H2O Upto 100ml
iii. 2% methyl blue / Light green:
a. Methyl Blue / Light green 2 grams
b. Glacial Acetic Acid 2.5 grams
c. Distilled H2O Upto 100 ml
iv. Iron Haemotoxylin solution
8.13.2 Procedure
ii. Nuclear staining is performed by iron haemotoxylin regressively.
iii. Differentiate and blue the sections.
iv. Stain with solution ± 1 for 3-5 min.
v. Rinse in water.
vi. Flood the sections in PMA solution.
vii. Drain the PMA solution.
viii. Stain with solution -3 for 5-10 minutes.
ix. Differentiate in 1% acetic acid.
x. Dehydrate, clear and mount the sections.
8.13.3 Results
Nuclei Black
Collagen and Reticular Fibers Blue or Light green
Muscle and Elastic Fibers Red
Box 8
Iron haemotoxylin is used in Trichrome staining because it is more resistant as compared to Harris
haemotoxylin. It is not easily washed off.
The PAP stain used in Cytology has the same principle as that of Trichrome staining technique.
Dye and mordant cRPSOH[LVFDOOHG³/$.(´
63
8.14 ALDEHYDE FUCHSIN STAINING
Elastic fibers are the connective tissues fibers and are thin, elongated fibers composed of a
protein called Elastin. These are branched and show a straight course and form loose
network. In fresh state these appear yellow and so also known as yellow fibers. These fibers
are highly resistant to boiled water, acid, alkali and enzymes, e.g. trypsin. However these
are digested by a pancreatic enzyme Elastase. These are also highly refractile fibers. In
routine H/E staining, elastic fibers are eosinophlic. Chemically these are composed of two
types of proteins:
Elastin, which forms core of the elastic fiber and is present as an Amorphous
material.
Elastic Fibers Microfibrillar Protein (EFMP) which is present in peripheral part. This
protein is fibrillar in nature but not amorphous.
Differential staining of elastic fiber is possible and one of the staining techniques is
Aldehyde Fuchsin Staining Technique.
8.14.1 Principle
EFMP protein is composed of cystine amino acids. Disulphide linkages of this amino acid
of EFMP are oxidized to sulfonic acid groups (-S03H) due to which elastic fibers become
basophilic and are stained with basic fuchsin.
8.14.2 Reagents
1. 1% KMnO4 (oxidizing agent)
2. 1% Oxalic Acid
3. Aldehyde Fuchsin Staining Solution:
i. Basic fuchsin 1gm
ii. 70% Ethanol 100ml
iii. Conc. HCI 1ml
iv. Para Aldehyde 2ml
4. Counterstain (Eosin or Neutral Red)
Procedure of Reagents Preparation

i. Dissolve basic fuchsin in 70% ethanol with heat.
ii. Cool and filter the mixture.
iii. Then add concentrated HCI and paraldehyde.
64
iv. Keep the solution for 2 days at room temperature during which ripening of the
staining solution takes place.
v. In the ripening process, an aldehyde fuchsin complex is obtained and there is change
in colour from red to purple.
vi. Ripening period can be shortened by rise in temperature, i.e. keeping at 50-60oC.
vii. Store at 4oC in dark coloured bottle. The solution is stable for several months.
8.14.3 Procedure of Staining

ii. Treat with 1% KMnO4 (for oxidation of disulphide linkage of cystine to form
sulfonic acid) for 3-5 minutes.
iii. Wash in water and treat with 1% oxalic acid to wash off colouring of KMnO4.
iv. Wash in water and rinse in 70% ethanol.
v. Stain with aldehyde fuchsin staining solution for 20 minutes.
vi. Wash in water and countersstain with a suitable dye like eosin or neutral red.
vii. Dehydrate, clear and mount the sections.
viii. Observe under microscope.
8.14.4 Results
Elastic fibers, Mast Granules,
Cartilage Matrix, Sulphated Blue / Purple or Red
Mucopolysaccharides
Other structures, e.g. collagen Countersstain used
8.15 MYELIN SHEATH STAINING

The myelin sheath is an insulating layer wrapping up nerve cell fibers, which are then called
myelinated nerve fibers. The myelin sheath is made up of a lipoprotein called Myelin, in
which lipids and proteins are present in the proportion of 80% and 20% respectively. The
myelin is highly refractile in nature due to which the myelinated nerve fibers appear white
on gross examination. This sheath is structure less but develops from supporting cells
present in nervous tissue. In the CNS, oligodendrocytes are the cells responsible for the
formation of myelin sheath whereas in PNS 6FKZDQQ¶V cells are involved in the formation
of sheath. The plasma lemma of the cell stretches and wraps up the nerve cell fibers in the
form of concentric layers as myelin sheath develops.
65
8.15.1 Demonstration
There are different techniques for myelin sheath demonstration. Some of them demonstrate
nerve fibers leaving the myelin sheath invisible while others demonstrating the myelin
sheath and leaving nerve fibers invisible e.g. Nerve fibers can be demonstrated by silver
impregnation and supravital stains like new methylene blue. In these techniques myelin
sheath remains unclear. Other techniques like haemotoxylin staining using iron alum as
mordant and treatment with osmium tetraoxide can be used for the staining of myelin sheath
leaving nerve fibers unstained.
8.15.2 :HLO¶V6WDLQLQJ0HWKRG
In this method myelin is stained with haemotoxylin having iron alum as a mordant. This
technique uses the mordant and stain in the same solution.
Fixative Choice:
Formol calcium is recommended because lipids are preset in the myelin sheath. Moreover,
for nervous tissue biopsies always thicker sections are required because nervous tissue is
very brittle and crumbling results when this sectioning is attempted (10-15 um). To avoid
crumbling celloidin can be used which has rubbery consistency and prevents crumbling.
8.15.3 Reagents
x 4% Iron Alum 50ml
x 1% Hx (prepared from 10% alcoholic Hx) 50ml
Mix both solutions to form iron Hx.
8.15.4 Differentiators
x 4% Iron Alum
x :HLJHUW¶V ERUD[ IHUULF\DQLGH VROXWLRQ VRGLXP ERUDWH J 3RWDVVLXP IHUULF\DQLGH
2.5g + Distilled water 200ml)
8.15.5 Procedure
ii. Stain with staining solutions for 10-45 min at 50-60oC.
iii. Wash in running tap water.
iv. Differentiate in 4% iron alum for 1-2 min.
v. :DVKLQZDWHUDQGFRPSOHWHGLIIHUHQWLDWLRQLQZHLJHUW¶Vdifferentiator.
vi. Wash in tap water.
vii. Dehydrate, clear and mount the sections.
viii. Observe under microscope.
66
8.15.6 Results
Myelin Black
Background Yellow to colourless
8.15.7 Interpretation
In WHLO¶VPHWKRGVHFWLRQVDUHUHJUHVVLYHO\VWDLQHG6RWZRVWDJHGLIIHUHQWLDWLRQLVDSSOLHG
i. First is iron alum differentiator. It is a mordant differentiator, i.e. law of mass action
is applied in these differentiators.
ii. 6HFRQG LV ZHLJHUW¶V GLIIHUHQWLDWRU ZKLFK LV D IRUP RI R[LGL]LQJ DJHQW WKDW performs
differentiation by converting the dye into a colourless form called Leucoform.
8.16 PERIODIC ACID SCHIFF REACTION

P.A.S. is a useful reaction for the demonstration of tissue carbohydrates especially
Glycogen which is of diagnostic importance in many benign and malignant tumors like
those of urinary bladder, kidneys and ovaries etc. PAS reaction is also useful for the
demonstration of fungi and mucins. PAS reaction is also useful for the diagnosis of
erythroblastic leukaemias because erythroblasts leukaemias are PAS +ve whereas
normoblasts are PAS ±ve. It is also one of the procedure for staining of basement
membrane.
PAS is mainly used for glycogen demonstration. Glycogen is a polymer of glucose. Several
units of x-D-glucose linked together in straight and branched chains to give a molecule of
glycogen which resembles a branched tree. In a straight chain, glucose molecules are linked
together by 1-4 glycolytic linkage whereas at branched points, molecules are linked
together through 1-6 glycolytic linkages. In PAS reaction, 1-2 glycol groups are required
which are present in different tissue carbohydrates like Glycogen.
8.16.1 Principle
In PAS reaction, periodic acid (HIO4) brings about oxidative cleavage of 1-2 glycol groups
to produce dialdehyde structure. This dialdehyde structure reacts with schiff reagent to
produce a magenta colour complex.
8.16.2 Schiff Reagent

In the preparation of this reagent basic fuchsin dye is dissolved in water. A deep magenta
coloured solution is obtained. This colour is lost by sulphuration process done by addition
of some sulphuric containing compounds like potassium metabisulphite. It is actually due to
disturbance of chromophonic group of the stain, i.e. quinonoid groups.
67
The solution obtained in this way is called schiff reagent or Leucosulphonic acid. Magenta
colour of the schiff reagent is restored if it is allowed to react with dialdehyde structure
obtained by the oxidative cleavage of 1,2 glycol groups of glycogen molecule.
8.16.3 Regents
i. Periodic Acid (HIO4) 0.5 ± 1%
a ± Periodic acid 1gram
b ± Distilled H2O 200ml
ii. Schiff Reagent
a ± Basic fuchsin 1g
b ± Distilled H2O 200ml
c ± Potassium metabisulphite 2g
d ± Conc. HCI 2ml
e ± Activated Charcoal 0.2g
Reagent Preparation:
i. Dissolve basic fuchsin stain in boiling distilled water and then removing the burner
to avoid spilling.
ii. Cool down the solutions to 50oC and add potassium metabisulfite.
iii. Bring solution to room temperature and add concentrated HCI.
iv. Keep the solution in dark overnight and then shake with activated charcoal.
v. Finally filter the staining solution, transfer it to dark coloured brown bottle and store
at 4oC.
vi. This solution is called scKLII¶VUHDJHQWDQGLWLVcolourless or pale yellow.
Sulphuration speeds up in acidic medium due to which concentrated HCI is added. Charcoal
is a carbon containing compound which is obtained by anaerobic pyrolysis of wood. It is
heated to 800-100oC and then it is given the name activated charcoal. Activated charcoal is
used for adsorption of different molecules, e.g. during sulphuration some sulphur remains
unbound and sometimes basic fuchsin shows impurities. This unbound sulphur or impurities
are absorbed by activated charcoal.
8.16.4 Diastase Digestion (D/D) Stage:

This stage is diastase digestion. Importance of this stage in PAS reaction is when PAS
positivity requires to be confirmed only for Glycogen. Glycogen is digested by different
amylases like alpha-amylase, beta-amylase, salivary amylase (Ptyalin) and diastase, which
contains both alpha and beta types of amylases. For confirmation of glycogen in a section,
duplicate slides, i.e. a control section slide and a test section slide are first treated with
68
enzymes. If glycogen is present in the test section slide, it is digested by the enzymes into
glucose and maltose. These sugars are washed off in the running tap water and after this
treatment PAS reaction is performed on both slides. Presence of glycogen is evidenced by
loss of stain i.e. ±ve PAS staining. Fungi are PAS +ve but D/D stage is not required because
PAS positivity of fungi is due to carbohydrates other than glycogen, e.g. cellulose, chitin
which are resistant to amylases.
8.16.5 Procedure
i. Dewax and hydrate the sections which are duplicate, i.e. two control and two test
slides.
ii. Treat one control and one test with 1% solution of diastase for 1 hour at 37oC or
cover both the sections with saliva and keep them at room temperature for 20-30
min.
iii. Wash both the enzyme treated slides with running tap water.
iv. Keep all the four slides in 1% periodic acid solution for 10-15 min.
v. Wash all the slides in running tap water for 5-10 min.
vi. Keep the slides in 6FKLII¶V reagent for 10-15 min.
vii. Wash the slides in running tap water for 5 min.
viii. Staining of nuclei can be performed with Harris haemotoxylin as an optional step.
ix. Dehydrate, clear and mount the sections.
x. Observe under microscope.
8.16.6 Results
Glycogen and other PAS +ve Carbohydrates Magenta Colour
The enzyme treated sections show loss of staining if glycogen is present.
8.16.7 Precautions
1. Reagents should not be expired and in accurate quantity.
2. Proper time must be given for staining.
3. Positive and ±ve controls should be run parallel.
4. D/D stage must be given proper time.
8.16.8 Fixatives for Glycogen

Glycogen is a stored form of glucose. It is normally intracytoplasmic and exists in a protein
environment. Glycogen can be demonstrated normally in different tissues structures. The
main sites are liver, skeletal muscle and cardiac muscle, hair follicles, endometrial glands,
cervical and vaginal epithelium, thyroid colloid, cartilage matrix, megakaryocytes and
69
neutrophils etc. Abnormal glycogen is of diagnostic importance in different benign and
malignant tumors especially those of urinary bladders, kidneys, ovaries and testes etc.
Fixation of a tissue in which glycogen is to be demonstrated must be prompt to avoid the

action of enzymes. So it is recommended that fixation must be as quick as possible and
moreover this fixation should be done at low temperature, i.e. 4°C. Low temperature is
required to slow down the autolysis. Moreover, in case of glycogen, streaming artifact is
very common. In an unfixed tissue glycogen molecules readily diffuse about and give false
localization when the tissue is kept at room temperature or higher temperature. Therefore,
fixation is done at low temperature.
8.16.9 Ideas for Glycogen Fixation

For the fixation of glycogen, there are two different theories as follows:
One theory describes that fixation of glycogen is brought about by condensation of
water molecules from the glycogen molecule by the action of fixative. This theory is
upheld by the fact that the 70 ± 80% ethyl alcohol is used as fixative for glycogen.
Other theory negates this upper idea and narrates that proteins associated with
glycogen are cross linked and glycogen molecules are preserved due to entrapment
in the protein lattice. This theory is widely accepted and is supported by glycogen
fixatives like picric acid fixatives
8.16.10 Interpretation of Results
Slides P.A.S Interpretations

Control with Diastase Solution -ve Glycogen is digested by
Diastase
Test treated with Diastase +/- If +ve, it means carbohydrates
Solution other than glycogen are present.
If ±ve, it means glycogen is
present.
Control without Diastase +ve Glycogen is present
Solution
Test without Diastase Solution +/- If +ve, it means carbohydrates
are present.
If ±ve, it means no
carbohydrates are present.
70
8.17 %(67¶6&$50,1(67$,1,1*
It is another staining technique for demonstration of glycogen. This technique is selective
for glycogen but not specific because some other substances like fibrin, neutral mucins are
also stained in this method.
8.17.1 Principle
In this staining technique, the rationale that is involved is hydrogen bond formation between
OH groups present in the glycogen molecule and H+ ions of the Carminic Acid.
8.17.2 Carmine Stock Solution

,PSRUWDQWLQJUHGLHQWVRIVWRFNVROXWLRQWREHXVHGLQ%HVW¶VFarmine staining are as follows:
Carmine:
It is the actual staining extract. Carmine contains about 56% carminic acid which is the
actual staining ingredient. In addition to carminic acid some proteins and ions like those of
aluminum and calcium are also present.
Potassium Carbonate and Chloride:

Potassium carbonate [KCO3] and potassium chloride [KCl] are also added in the stain. If
these salts are not available, equal amounts of ammonia or sodium salts can be used.
Carmine also shows tendency to stain the background proteins due to electrostatic linkage.
These ionic salts inhibit this staining by neutralizing electrostatic charges and hence non
specific background staining is checked.
Ammonia:
It has a dual role; it provides a high alkaline pH (10 ± 11). At this pH, non-specific
background staining is prohibited by the potassium salts. It also acts as the solvent for
carmine.
Preparation:
i. Take 2.0 grams of carmine, 1.0 gram of potassium carbonate and 5.0 grams of
potassium chloride.
ii. Dissolve all these ingredients in 60 ml of water by boiling for 5 minutes to obtain a
carmine metallic ion complex which then acts as the staining ingredient. The boiling
is done in a large flask to avoid spillage.
iii. Cool the mixture and then add 20 ml of concentrated ammonia.
iv. Filter the solution and store in a dark coloured bottle tightly screwed at 4°C. This is
the stock carmine solution
71
Working Solution of Carmine:
Carmine stock solution 15ml
Concentrated Ammonia 12.5ml
Methyl Alcohol (Methanol) 12.5ml
8.17.4 Differentiators
,Q %HVW¶V FDUPLQH VWDLQLQJ GLIIHUHQWLDWLRQ RI WKH VWDLQ FDQ EH GRQH by using one of the
following:
1. %HVW¶V'LIIHUHQWLDWRU
Methanol 40 parts
Ethanol 80 parts
Dist. H2O 100 parts
2. 74° O.P Spirit

It contains a methanol content of 99.18%.
8.17.5 Procedure
i. Dewax and hydrate the sections. Duplicate sections may be run and D/D stage may also
be performed (optional)
ii. Stain the nuclei with Iron Hx by regressive method.
i. Differentiate and blue the sections.
ii. Stain with working carmine solution for 5 ± 15 minutes (it depends upon the age of the
stock solution. Older the stock solution more will be the time)
iii. 'LIIHUHQWLDWHLPPHGLDWHO\LQ%HVW¶VGLIIHUHQWLDWor or in 74° O.P Spirit.
iv. Wash in fresh alcohol, clear and mount the sections.
v. Observe under microscope.
8.17.6 Results:
Glycogen Deep Red
Fibrin and Neutral Mucins Pale Red
Nuclei Blue ± Black
72
Box 9
Failure in staining may be encountered due to following reasons:
R ,IVHFWLRQVDUHZDVKHGLQZDWHUDIWHUVWDLQLQJLQVWHDGRIGLIIHUHQWLDWLQJLQ%HVW¶VGLIIHUHQWLDWRURU
74° O.P. spirit.
R If the stock solution is deteriorated.
R By filtering the stock solution just before preparing the working stain.
R By performing the staining of the sections in the closed containers like coplin jar.
2QHRWKHUSUREOHPLQ%HVW¶VFDUPLQHVWDLQLQJLVWKDWDSUHFLSLWDWHLVOHIWRQWKHVHFWLRQZKLFKPD\
be checked by the following precautions:
a) Perform staining in closed containers.
b) Filter the working solution just before use and after staining immediately perform
differentiation.
8.18 LIPIDS; HISTOCHEMICAL DEMONSTRATION

Lipids are histochemically demonstrated in different pathological disorder mainly related to
lipid metabolism. Different tumors of lipids including lymphomas and lyposarcomas are
encountered in a histo laboratory where histochemical studies of lipids are helpful in the
diagnosis.
Lipids are considered as heterogeneous organic compounds composed of carbon, hydrogen

and oxygen. The oxygen content of lipids is less than that present in carbohydrates due to
which in oxidation process lipids require more oxygen which results in the release of
greater amount of energy. Normally lipids are considered as amorphous greasy material
which are hydrophobic and so dissolve in organic solvents. But exceptions are there like
cholesterol which is not amorphous and is in crystalline form. Its melting point is 144°C.
Similarly phospholipids are hydrophilic in nature and glycolipids are moderately
hydrophilic. Lipids are considered as derivatives of fatty acids which are their building
blocks.
8.18.1 Choice of Fixatives

Different fixatives like osmium tetraoxide, chromic acid and potassium dichromate can fix
the lipids but all these fixatives alter the chemical reactivity of lipids for their stains like Oil
Red O, Sudan dyes etc.
Formol Calcium is considered as best fixative for histochemical demonstration of lipids.

)RUPDOGHK\GHLWVHOIFDQ¶WIL[WKHOLSLGVEXWLWLVDJRRGSUHVHUYDWLYe of lipids due to cross
linking of this fixative with structural proteins which results in formation of a lattice in
73
which lipid molecules are trapped and so preserved. Moreover, on prolonged storage,
formalin fixatives produce formic acid on oxidation which is conducive to hydrolysis of
lipids. Formation of formic acid is checked by using buffered formalin or formalin solution
having some alkaline salts like calcium acetate or CaCI2. Formol calcium is preferred to
buffered formaldehyde because calcium salts not only check the formation of formic acid
but also helps in the formation of lattice structure.
8.18.2 Choice of Sectioning Technique

Frozen sections are obligatory because in routine sectioning techniques like paraffin
sectioning technique, there is considerable loss of lipids in different organic solvents used in
the dehydration and clearing steps.
Normally unfixed frozen sections are preferred but practically fixation of the tissue is more
or less required for good sectioning, so frozen sections are produced form the lipid material,
short fixed in formol calcium or frozen sections are taken and post fixed in formol calcium.
8.18.3 Staining of Lipids

Staining of lipids can be done by using stains like Oil Red O or Sudan dyes. Of these two,
Sudan Black B stain is widely used for the demonstration of most of the lipids. In Sudan
staining of lipids, there are two theories. One theory holds the idea of adsorption and other
theory favors the idea that staining is accomplished on the idea of differential solubility.
8.18.4 Differential Solubility

In differential solubility principle, the stain is dissolved in a solvent to get a staining
solution. Sudan Black B stain is prepared in 70% ethanol. The staining solution is then
applied to a tissue component like Sudan B staining solution is applied to lipids. Staining is
affected due to much higher solubility of the stain in tissue components than that in the
original solvent. In Sudan Black B staining, solubility of the dye is much higher in lipids
than that in 70% ethanol.
8.18.5 Adsorption
The other idea about lipid staining is adsorption. In this process, Sudan Black B molecules
are attached and fixed by lipid molecules.
8.18.6 Application of Controls

In lipid staining positive and negative controls are important to confirm staining of different
types.
74
Extraction of Lipids for Negative Control:
To obtain negative controls extraction of lipids is done from the section which is called
³'HOLSLGLzDWLRQ´. For extraction of lipids chloroform, acetone, ether and other organic
solvents may be used. For bound lipids, extraction can be done by using HCl where as
water can be used for the extraction of hydrophilic lipids (phospholipids). The mixture used
for delipidization is as follows:
Chloroform 66ml
Methanol 33ml
Water 04ml
Conc. HCl 01ml
Extraction of Lipids for Positive Control:

For positive controls, nowadays different types of lipids are commercially available. These
lipids may be applied on a piece of filter paper or may be suspended in gelatin. The pieces
of filter papers having applied with lipids or sections from gelatin material are mounted on
slides and then stained. In this way positive control slides are obtained.
8.19 Bromine-Standard Sudan Black-B Staining

The principle is based on two theories. One theory is of differential solubility and other is
adsorption. These have been discussed earlier.
8.19.1 Staining Solution

It consists of saturated Sudan Black ± B solution in 70% ethanol. When bromine step
(bromination) is included in the staining technique, the following are also required:
2.5% Aqueous Bromine

0.5% Sodium or K metabisulphite
8.19.2 Procedure
i. Frozen sections post fixed in formol calcium are taken (Prefixation can be done).
ii. Take sections in aqueous solution of bromine for 30min in a fume cupboard. The
step is called bromination.
iii. Teat section with 0.5% metabisulphite solution to remove excess bromine solution.
iv. Wash in tap water and perform Sudan Black ± B staining on brominated sections
along with an unbrominated section. For the staining use saturated solution of Sudan
Black ± B for 15 minutes.
v. Differentiate in 70% ethanol (fresh) till a delipidize section appears colourless.
vi. Mount all the sections in glycerin gel.
75
8.19.3 Results
Neutral fats, Esterified Cholesterol, Blue Black/ Triglycerides Deep Blue
Phospholipids Grey
Free cholesterol, fatty acids, lecithins are also stained in this technique and show somewhat
light colour.
8.19.4 Interpretations
Saturated Sudan Black ± B staining incorporated with bormination is wLGHO\XVHGIRUOLSLG¶V
histochemical demonstration because it is capable of staining almost all types of lipids.
Neutral fats and cholesterol esters are naturally Sudanophilic but Sudan Black ± B can also
stain excellently hydrophilic lipids, i.e. phospholipids.
Free cholesterol can be demonstrated by Sudan Black ± B when bromination is done. It is

found that free Cholesterol is done. It is found that free Cholesterol which is crystalline at
room temperature is converted to such derivatives which are oily and amorphous after
bromination. Similarly free fatty acids and Lecithins, which are normally readily dissolve
out in ethanol, are considerably retained after bromine treatment and so can be
demonstrated. About Sudan ± B, it is said that it has 02 parts; hydrophilic and hydrophobic,
so reacts with both types of lipids respectively.
76
9. FROZEN SECTIONING
9.1 Aim/Significance
1. For certain staining procedures, e.g. the demonstration of lipid by Oil red O method,
for certain impregnation methods, e.g. the silver impregnation, frozen sections are
essential.
2. Frozen sections are indispensable for rapid diagnosis of malignancies during
operations, e.g. metastatic tumors of breasts and lymphomas of different types. In
such cases time matters and quick reporting is required.
3. All enzymes are destroyed at temperature above 56°C and they all are best shown in
cryostat or frozen sections of fresh tissue. Although, phosphatases may be
demonstrated in paraffin section.
4. Frozen sections are thicker and easily be handled during staining.
5. Frozen sections are also helpful in immunohistochemistry where antigen antibody
complexes are studied in tissues as in lymphomas.
9.2 Disadvantages
1. Cost Effect:
To produce frozen sections, special devices like cryostat are required. These
instruments are very expensive and routine laboratories cannot afford it.
2. Poor Staining:
Staining on frozen sections is rarely satisfactory. The colours are usually faint.
3. Freezing Artifact:
Freezing artifact may be produced by inappropriate techniques, e.g.
x Presence of ice crystals in tissues;
x Nuclear ballooning and vaculation;
x Separation of a mucosal or other epithelial surface.
4. Poor Quality Sections:
It is almost impossible to obtain serial sections on frozen section.
5. Because of the lack of an embedding mass, structural details tend to be somewhat
distorted during cutting and handling.
6. For frozen sections a lot of skill, experience and supervision is required.
7. Different tissues have different optimal cutting temperature (O.C.T) and it is very
difficult to acquire temperature changes for different types of tissues.
77
O.C.T. for Different Tissues
I II II
-18 to -20 °C -15 to -25 °C -35 °C
Lymphnode, soft Uterus, cervix, non- Fatty skin, fatty
tumors, fatty skin, non-fatty breast, omentium
endometrial breast tissue
curvettings
9.3 Principle of Frozen Sections

The principle of frozen sections is very simple. As a tissue is frozen, water present in it is
converted to ice which then gives an internal support to the tissue and its sectioning is
facilitated. The ice acts as an embedding medium. Frozen sections can be produced from
fresh as well as fixed biopsy. But the former is preferred than fixed material but it is
hazardous so UV light is used for disinfection.
When quick reporting is required frozen sections are taken from fresh material because no
time for fixation is available. The use of fresh material is associated with problems like risk
of infection and streaming artifact. On the other hand, fixed tissues can be used when the
aim is histochemical study. Fresh biopsy material can be easily sectioned in a cryostat
because they require O.C.T in the range of 0 to -15 °C to -30 °C which is easy to obtain in a
cryostat. On the other hand, fixed biopsy material cannot be excellently sectioned in a
cryostat because they require an O.C.T which is difficult to attain on a cryostat.
Freezing microtome is commonly used for fixed frozen sections because O.C.T for fixed
biopsies can easily be produced which is not possible for fresh biopsy material. The fact
that fixed biopsies require a higher O.C.T than that for fresh biopsy which requires a lower
O.C.T is due to difference in their water content. Fixed biopsy contains more water and so
are firmly solidified, even at a higher temperature -15 °C to -10 °C whereas fresh biopsies
are firmly solidified at a much lower temperature like -15 °C to downwards.
The working principle, merits and demerits of freezing Microtome are described in chapter
6.
78
10. CRYOSTAT
The best method of preparing sections from unfixed tissues is by use of a cryostat. The
cryostat consists of a microtone housed in a deep freeze cabinet, maintained at a
temperature of -15°C to -30°C. A thermostat controls the temperature of cabinet. The
optimum working temperature of cryostat is -18°C to -20°C. The mounting media used in
cryostat is O.C.T which stands for optimal cutting temperature. The microtome is handled
by an operating handle outside the cabinet. There is a variety of cryostats manufactured, the
fundamental difference between them is the type of microtone employed. The earliest
models manufactured in UK incorporated the Cambridge Racking microtone. However,
now a days rotary microtome is commonly used with wedge profile knife because this type
of knife is very rigid. Several models of cryostat fitted with Base sledge microtone, suitable
for cutting larger and tougher tissue blocks are also available.
Figure 10.1 Cryostat

Adapted from http://www.colorado.edu
10.1 Anti Roll Plate

In cryostats a special device called Anti Roll Plate is incorporated which is meant for
prevention of curling or rolling of frozen sections. This device is adjusted to knife edge in
such a way that a small gape or slot is present between knife edge and anti-roll plate. As
frozen sections are produced these smoothly go beneath the anti-roll plate on the surface of
the knife. Anti roll plate is necessary because frozen sections show a natural tendency of
curling.
79
When sections are produced, anti-roll plate is set apart and sections are directly picked on
slides, properly smeared with an adhesive and kept at room temperature. As there is
temperature difference between sections (-20°C) and slides (room temp) so due to this
difference in temperature sections firmly stick with the slide. Frozen sections of fresh
material rarely detach of the slide so adhesive is not required. But sections of fixed material
may detach of the slide, some adhesive is required.
10.2 Free Floating Sections

Sometimes frozen sections are picked on cover glass or these are directly smeared as free
floating sections. The idea of free floating sections is favoured by the fact that in certain
techniques like enzyme histochemistry, staining reagents are very costly and definitely free
floating sections save the staining reagents which is not possible when sections are taken on
slides or cover glass.
10.3 Merits of Cryostat

With the expansion of knowledge of histochemical studies and requirements of quick
reporting of emergency biopsies, cryostat has gained wide acceptance in most of the routine
laboratories. Cryostat is actually a Hospital device. Fresh emergency biopsies can easily and
promptly be sectioned in a cryostat. The results can be given by the pathologist while
surgery is still continuing and so it is possible for the surgeon to operate accordingly.
10.4 Pre-Requisites for Cryostat Sectioning

The following two pre-requisites must be kept in mind before sectioning on cryostat:
10.4.1 Preparation of fresh biopsy material for cryostat sectioning:

In the preparation of fresh biopsy material quick freezing is required because the material is
fresh and must be safe against post-mortem changes, and also to avoid large ice crystals
formation in case of slow freezing. For quick freezing different freezing agents are used
which include liquid gases like liquid N2 at a temperature of -190°C, liquid CO2 at a
temperature of -70°C, dry ice or cardice at a temperature of -70°C, aerosols at a temp of -
50°C and isopropanol at a temperature of -150°C. In case when cardice is used as a freezing
agent, gloves should be worn before touching the cardice.
Comparison of Freezing Agents:

Of the above freezing agents, merits of liquid nitrogen (N2) is that it causes rapid freezing
but on the other hand it is not suitable for delicate biopsies due to its over cooling effect.
Secondly, it has been observed that serious damage occur to tissue block as well as to knife
whenever it is attempted to take sections at temperature below -70°C. Usually cardice is
80
recommended for freezing of biopsy material. Two blocks of cardice are taken with gloved
hands, the tissue block holder is held in between cardice blocks and a biopsy material is
frozen by means of a water bath on the upper surface of the block holder.
10.4.2 Accurate Operation of a Cryostat:
This is the second prerequisite and includes:

x Optimal cutting temperature
x Adjustment of cryostat chamber and knife
x Correct operation of cryostat
x Correct adjustment of anti-roll plate
Optimum Cutting Temperature

An optimum cutting temperature is required depending upon the type when biopsy material
is prepared. Prior to sectioning, the cryostat chamber and microtome knife must be at
correct temperature. As a general rule, it is described that most of the fresh unfixed biopsies
are sectioned in a range of temperature, i.e. -15 to -23°C. Normally biopsies containing
more water are sectioned at relatively warmer temperature with respect to above described
range. Similarly harder biopsies are sectioned towards cooler temperature. Biopsies
containing a lot of fat are sectioned on temperatures as lower as possible. For fixed biopsies
the temperature range in cryostat is -7 to -12°C.
Cryostat Knife
The sharpening of cryostat knife is essential because an inaccurate facet creates problems in
two ways:
1. In the adjustment of antiroll plate
2. In picking up sections on slides or cover slip from facet of the knife.
Adjustment of Anti-Roll Plate:

The main points in the adjustment of antiroll plate are as follows:
i. Correct angle anti-roll plate is lifted upwards from behind.
ii. Correct height of anti-roll plate.
iii. Upper edge of the anti-roll plate must be intact (not damaged).
iv. Another point must be considered during adjustment of anti-roll plate, i.e. the anti-
roll plate must be at the cabinet temperature because sections will stick to it due to
difference in temperature.
81
10.5 Cryostat Sectioning
If the pre-requisites already mentioned are properly fulfilled, then it is easier to produce
sections in a cryostat. Similarly cryostat sectioning requires good practice of the worker.
Speed of cutting the section is also important. As a rule softer tissue requires somewhat
slower speed of cutting whereas harder material requires faster speed of cutting.
For cryostat sectioning, fresh biopsy material is received in gauze piece soaked in isotonic
saline. It inhibits the attacks of bacteria and prevents the biopsy from deterioration. An idea
is given that two blocks should be prepared from the material. One block is sectioned and
the other is kept ready in case when the 1st one is detached from the holder and so by this
way there is no wastage of time. Sections in cryostat rest on knife surface underneath the
anti-roll plate which are then picked up on albumin slide. Sometimes, in place of albumin
some other adhesive is used, i.e. gelatin formaldehyde mixture. The slides are then kept at
37°C for 1 hour.
Slides/Cover Slips for Picking Up Sections:

In case of cryostat sectioning, sections can be picked on slides or cover slips. Usually it is
RQSHUVRQ¶VRZQSHUIRUPDQFH+RZHYHULQFDVHRIHQ]\PHKLVWRFKHPLVWU\LW is advisable to
take sections on cover slip because in this case reagents used are very costly and so the idea
is to minimize the load on laboratory.
Rapid H/E Staining Technique:

i. Fix sections in formol calcium for 20 seconds.
ii. Rinse in water
iii. Stain with Harris Hx regressively.
iv. Differentiate and blue the sections
v. Stain with eosin for 10 seconds
vi. Rinse in water
vii. Mount the sections in aqueous solution.
viii. Dehydrate, clear and mount the sections.
O.C.T Compound:
Now a days, in cryostat sectioning, OCT compound is used which is optimal cutting
temperature compound. This keeps the tissue at optimum temperature properly for a longer
time and more over this compound also acts as an embedding media.
It can be used for fresh biopsy material.

Disadvantage includes high cost.
82
10.5.1 Merits of Cryostat Sectioning
1. Cryostat can primarily be used for emergency biopsies where quick reporting is
required, e.g. lymphomas and malignancies.
2. Cryostat sectioning is useful for histochemical demonstration.
3. Thin sections can be obtained as compared to those produced by freezing microtome
and similarly serial sections are possible.
4. Cryostat sectioning is also useful for immunohistochemistry.
10.5.2 Demerits
1. The use of cryostat is not applicable for a routine busy laboratory especially when it
is situated for away from a hospital.
2. Fixed biopsy creates a difficulty in sectioning by cryostat.
3. When this technique is compared with standard histological technique, thin sections
obtained by wax sectioning cannot be obtained in cryostat.
4. Cryostat is costly instrument and its proper maintenance is also necessary to keep it
working for longer time.
5. Ice crystals artifact causes severe morphological damage to tissues by rapid freezing.
10.6 Freeze Drying

Freeze Drying Technique is used to overcome number of problems such as:
In routine histological techniques, there are number of problems for a histotechnologist
like loss of diffusible substance.
Streaming artifact of diffusible molecules of chemical substances is observed in routine.
The alteration of chemical reactivity of molecules during routine fixation and processing
is also observed.
Denaturing of protein and inactivation of enzymes is also common problem.
To overcome above mentioned problems, freeze drying technique is employed by a

histotechnologist. Use of this technique is almost restricted to histochemical studies for
research purpose and this technique is not being used in routine histopathological
laboratories.
In this technique, fresh material is rapidly frozen by a freezing agent at a temperature, say -
160°C and this phenomena is called Quenching and this is the first step in freeze drying
technique.
Next, the frozen water (in the form of ice) is removed by the process of sublimation, i.e. in
vacuum at a higher temperature, i.e. 40°C. The material is then raised to room temperature,
83
fixed, embedded and finally subsequent treatment is given. Four major steps are involved in
the procedure above mentioned which are:
Quenching
Drying
Freezing and Drying
Subsequent Sectioning
Box 10
Quenching is the rapid freezing of fresh material by means of freezing agent. The effect of
quenching is of paramount importance. Due to quenching, chemical reactions are slowed
down. Similarly, diffusion of molecules is retarted and rapid freezing results in small ice
crystals formation which saves the tissue against morphological changes by large ice
crystals due to slow freezing.
84
11. REFERENCES
Baker FJ, Silverton RE. Introduction to Medical Laboratory Technology. Butterworth & Co
(Publishers) Ltd. 6th Edition.
Gerabek W. , Bernhard D. Haage, Keil G. , Wegner W. (2005): Enzyklopädie
Medizingeschichte (Encyclopaedia of medical history), Walter de Gruyter, ISBN
3110157144
Hill, John . The Construction of Timer, from its early growth; Explained by Microscope,
and proven from Experiments, in a great Variety of Kinds.. London. 1770.
Mallory FB, Wright JH. Pathological Technique: a Practical Manual for Workers in
Pathological Histology and Bacteriology, W.B. Saunders, Philadelphia 1918.
Quekett, John. A Practical Treatise on the use of the Microscope. London: Hippolyte
Bailliere. 1848.
5DSKDHO 66 /\QFK¶V 0HGLFDO /DERUDWRU\ 7HFKQRORJ\ :% 6DXQGHUV &RPSDQ\ th
Edition 1983.
Sood R. Medical Laboratory Technology; Methods and Interpretations. Jaypee Brothers
(India) 1st Edition 1985.
http://ia360611.us.archive.org/1/items/journalofroyalmi1910roya/
http://www.radicalscientific.com/microtomes-44.html
http://websites.labx.com/rankin/detail.cfm?p=0&autonumber=80707
http://www.colorado.edu/che/ansethgroup/facilities_album/pages/Cryostat_jpg.htm
85
ABOUT THE AUTHORS
Usman Waheed is a Medical Laboratory graduate with a Masters in Biochemistry

and MPhil in Molecular Biology. He has received post graduate diplomas in Public
Health (SDC) and Epidemiology (LSHTM). He has also recently been trained in
Transfusion Medicine, Quality Management, Immunohaematology, Nucleic Acid
Testing and Plasma Fractionation from Germany and Netherlands.
UW has been involved with the teaching and training of MLT students at CMT,
PIMS and CMLT, NIH. He has published several research papers in national and
international Journals besides authoring two handbooks titled µHIV/AIDS Testing
6WUDWHJ\¶ DQG µHaematology - %ORRG %DQNLQJ IRU 0HGLFDO 7HFKQRORJLVWV¶. He is a
member of several professional bodies including International Society of Blood
Transfusion, International Federation of Infection Control, e-Health Association of Pakistan, Infectious
Diseases Society of Pakistan and Pakistan Society of Blood Transfusion. He is the Associate Editor of
Medical Review; A Medical Current Affairs Magazine. Currently serving as a Technical Advisor in National
Blood Transfusion Project GIZ.
Asim Ansari is a Medical Laboratory Technology graduate with a Masters in

Biochemistry & Molecular Biology. He has also received a few professional
diplomas/certificates during his carrier along with the immense and diverse
experience starting from a bench worker to a Manager. His core competencies
include Laboratory/Healthcare Management, ISO Quality Management, Infection
Control, and Personnel Capacity Building in Laboratory Sciences. He is a certified
ISO-15189 Assessor by Pakistan National Accreditation Council (PNAC). His
interest in Infection Control took him to the International Federation of Infection
Control (IFIC) as associate member. He remained affiliated with known quality
diagnostic facilities in Islamabad and currently serving as a Manager, Pathology
Laboratory & Blood Bank at a private hospital.
86
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Chapter No. Title Page No.

ABOUT THE AUTHORS 86

Figure No. Title Page No.

6.1 18th Century Microtome by Alexander Cummings 37

6.2 Cambridge Rocking Microtome 38

6.3 Rotary Microtome 39

6.4 Base Sledge Microtome 40

6.5 (a) Microtome Knives 43

6.5 (b) Microtome Knives 44

6.6 Direction of the movements in Honing 45

6.7 Direction of the movements in Stropping 46

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Although, a lot of educational matter is available on various disciplines of medical

Dr. Haroon Khan

These techniques, employed in a histopathology laboratory, help a histo-pathologist to

2.1 Rationale of Fixation:

2.2 Mechanism of Fixation:

2.4 Properties of an Idea Fixative:

A good fixative should be capable of fulfilling the following requirements:

2.4.1 It must penetrate the tissue rapidly and evenly.

2.5 Classification of Fixatives

There are three classifications of fixatives as below:

2.5.2 Classification II:

2.5.3 Classification III:

2.5.3.3Fixatives of Unknown Mechanism:

2.5.3.5Physical Methods of Fixation:

2.6 Fixation Artifacts:

 Formaldehyde fixatives give brown pigmentation to tissues.

2.7 SIMPLE FIXATIVES

Commercially available formaldehyde is a saturated solution of formaldehyde (HCHO) gas

i. Formalin is cheaper, easy to prepare and stable.

*formal saline + handful calcium carbonate + well shaken

2.7.2 Mercuric Chloride

It belongs to the class of fixatives of unknown mechanism. Mercuric chloride HgCl2 is a

2.7.3 Osmium Tetraoxide

It belongs to the class of fixatives of unknown mechanism. Its solubility is about 1% in

2.7.5 Potassium Dichromate

2.7.5.1 Advantages and Disadvantages:

2.7.6.1 Advantages and Disadvantages:

2.7.7 Acetic Acid

Trichloroacetic acid [CCl3COOH] is sometimes incorporated into compound fixatives. It is

2.8 Osmotic Considerations in Fixation

2.9.3 Penetration of Fixatives:

Where K is constant of proportionality and is known as Coefficient of Diffusibility. By re-

01 % Picric acid C6H3N3O7 0.5 mm/hour

2.9.4 Concentration of Fixatives:

Sometimes a fixative can be effective even at a low concentration, e.g. 3% glutaraldehyde is

2.9.5 Time of Fixation:

2.9.6 Osmolarity of Fixatives:

2.10 COMPOUND FIXATIVES

2.10.1.1 Formol-Saline 10%:

2.10.1.2 Neutral Buffered Formalin 10%:

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Formaldehyde fixatives give brown pigmentation to tissues.

Acidic and Basic Reaction