The Notes On Histochemical Stains

H The notes on
ISTOCHEMICAL STAINS
Compiled By
Dr S Suban Mohammed Gouse
Assistant Professor,
Department of Pathology, SBMCH,
Bharath University, Chennai, India.
Dr S Sarojini
Professor,
Department of Pathology, SBMCH,
Bharath University, Chennai, India.
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The Human Path Education

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S Suban Mohammed Gouse & S Sarojini 1
The notes on
Histochemical Stains
© The Human Path Education, India
First Edition @ 2010

Editor : S Suban Mohammed Gouse
Compiled by : S Suban Mohammed Gouse
S Sarojini
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complies with latest legislation and standards of practice.
2 The notes on Histochemical Stains

Dedicated to …..
The Lord who created us…

The Parent who nurtured us…
The Teachers who taught us…
The Students who encourage us…

Contents
Pages
Acknowledgement 7
Preface 8
Introduction to the book 9
1. ROUTINE STAINING
Haematoxylin and Eosin 11
I Basics of staining mechanisms 11
II Staining methods 20
III Problems and solutions 23
IV Frequently asked exam questions 26
2. SPECIAL STAINS
A. Carbohydrate staining 35
I Basics of carbohydrate staining 35
II Periodic Acid Schiff Stain 37
III Periodic Acid Schiff - Diastase Stain 41
IV Mayer's Mucicarmine Stain 44
V Alcian Blue Stain (pH2.5/pH1.0/VEC) 47
VI Alcian Blue / PAS / Haematoxylin Stain 51
VII Applicability of Carbohydrate Stains 54
VIII Frequently Asked Exam Questions 60
IX Periodic Acid Methenamine Silver Stain 66
B. Connective Tissue Staining
I Basics of Collagen Staining 68
I.A. van Gieson's Picric Acid -
Acid Fuchsin Stain 69

I.B. Masson's Trichrome Stain 72
I.C.Frequently Asked Exam Questions 75
II Basics of Elastic Staining 78
II.A. Verhoeff's Elastic Stain 79
II.B. Frequently Asked Exam Questions 82
III Basics of Reticulin Staining 84
III.A. Gomori's Stain For Reticular Fibers 87
III.B. Gordon and Sweets Stain For
Reticular Fibers 90
III.C. Frequently Asked Exam Questions 94
C. Pigment Staining
I Basics of Pigments 97
II Basics of Haematogenous Pigments 98
III Prussian Blue Stain For Ferric Iron 99
IV Frequently Asked Exam Questions 102
D. Micro Organism Staining
I Basics of Microbes Staining 105
II Brown and Brenn Technique for Gram Stain 106
III Ziehl Neelsen Stain (AFB) 109
IV Fite Acid Fast Stain for M. Leprae 112
V Grocott's Methenamine Silver
Nitrate Fungus Stain 115
VI Frequently Asked Exam Questions 120
For Further Reading 122

ACKNOWLEDGEMENT
“True Gratitude is difficult to express”
We thank The Lord Almighty for providing us

health and wellness for writing this manuscript.
Before writing began, we had a panel of
professors, Prof B O Parijatham, Prof Shanthi
Vijaya lakshmi, Prof Hemalatha and Prof
G Bheema Rao helped us to determine the
appropriate content for this book, we thank
them all for helping us. Those who assisted in
other tasks are invaluable members of the
book’s team. Thank you to the post graduates
for assisting in photographic section.
We express our thanks to Dr. Sri Nisha,
Chairperson; Dr. Kamal Sheriff, Director;
Dr. Bala Krishnan, Dean; and Dr. P. Saikumar,
Vice Principal for giving support and
encouraging us for all the academic works.
We thank Br. Mohammed Mansoor, M. F. Ali
and Mr. Ramesh who involved in the work up
of this book.
We thank our family members who directly or
indirectly supported us. We thank Dr. Helen
who devoted her time in many days and nights
at the computer making, editorial changes and
proofreading.
We thank all the professors from whom we
learnt and still learning….

PREFACE
This book represents an interaction of three

elements,
i) Hard scientific background which contains
principle and procedures,
ii) Application of the stains with its problems
and solutions,
iii) Frequently asked questions for the post-
graduates at the exam bench. We hope that
this book will benefit the readers.
S Suban Mohammed Gouse

[[email protected]]
S Sarojini

INTRODUCTION TO THE BOOK
AND ITS USE
For whom this book is written for?

This is a book, in a nutshell for the commonly
used histochemical stains which not only covers
the principle and staining. But it also discuss the
problems and solutions and in detail about its
applications. More over it can serve as guide
for the postgraduates in regarding the commonly
used histochemical stains.
What is the scope of this book?
The authors maintained the synchronicity in
explanation of all the stains which are commonly
used in various histopathological tissues. The
methodology used in the book emphasis more
on applicability which makes the reader to
understand the concept. The book in detail
discusses about the Q & A section in a separate
subtitle FAEQ (Frequently Asked Exam
Questions), which was compiled after detailed
discussion with many eminent professors.
Where did the information in this book come from?
The authors specifically used the word
‘compilation’, to express the fact involved in this
work. This book is a compilation of knowledge
gathered from the various histotechnique books
written by the eminent scholars such as Culling,
Lynch, Carlton, Carson, Horobin, Bancroft,
Newman, Sumida, Lilly etc and from our senior
teachers in our field. We thank them all.

1. ROUTINE STAINING
HEMATOXYLIN AND EOSIN
I. BASICS OF STAINING MECHANISM
I n the past, there was a great deal of concern about whether staining
reactions were physical or chemical. Today it is recognized that most
reaction involves both physical and chemical factors. The fat stain is
an example of a purely physical stain, with the dye absorbed (soaked
up) by and dissolved in the lipid. However, most stains depend upon
adsorption of the dye, or the attraction for minute particles from the
surrounding solution by the surface of certain tissue components; the
dye is then bound to the tissue primarily by ionic, covalent, or hydrogen
bonds.
Ionic or electrostatic bonding occurs when the dye and the
substance to be dyed develop different charges, and thus become
attracted to each other. For example, we can stain the cytoplasm by
developing a positive charge on the cytoplasmic proteins and a
negative charge on the dye. This type of binding is also referred to as
salt linkage.
Hydrogen bonding occurs when hydrogen is attracted to atoms
that have a strong electronegative charge. Frequently this type of
bonding occurs between hydrogen and oxygen. Hydrogen bonds are
weak and occur naturally in water; they may form between the dye
and the water in which it is dissolved. Water also competes for
hydrogen bonding sites in tissue, so this type of bonding is probably
not important in most staining reactions.

Covalent bonding occurs when atoms share electrons. This type
of bond is typical of organic chemicals, because carbon, hydrogen,
and oxygen commonly form covalent bonds. In the water molecule,
one oxygen atoms shares two electrons, one with each of two
hydrogen atoms. Each hydrogen atom also shares an electron with
the oxygen atom. Vander waals force are due to attraction of the
molecules for the electrons of its neighbouring molecules. These are
weak physical forces that are effective over only very short
distance.
Nuclear Staining
It hypothesized that nuclear staining occurs by two different
mechanisms; (1) staining done with the basic (cationic or positively
charged) dyes, and (2) staining done with dyes combined with,
or followed by, metal mordents. The first mechanism depends
on the negatively charged phosphate groups of the nucleic acids
attracting cationic dyes. The second type of staining occurs in tissues
from which the nucleic acid has been removed (eg, decalcified
tissue).
The term “basophilic” (base-loving) may be applied properly to
acidic (anionic or negatively charged) tissue substances that are readily
stained with basic dyes, the term is not proper for the metal-mordant
type of staining. The metal-mordant dyes stain many of the same
tissue elements as the basic dyes. However, under some circumstance;
the metal-mordant dyes stain substances lacking acid groups, such
as myelin and neutral mucopolysaccharides. Stainability with the
aluminium hematoxylin should not be indicated with the term
basophilia; instead, more appropriate term “metallophilia” should be
used to indicate tissue characteristics that induce staining with metal-
mordant complexes.
There are two possible binding sites for the hematoxlyin-aluminium
complex: the phosphate groups of DNA and the histones that are
cationic (+) nuclear proteins bound by the phosphate groups. It appears
that a variety of forces are involved in the birding of metal-hematein
by nuclei, and that a simple explanation is not possible. Ionic and
hydrogen bonding as well as Vander Waals and hydrophobic forces
probably all have some role in the staining of nuclei by aluminium-
hematein solutions.

Cytoplasmic Staining
Fortunately the routine staining of non - nuclear elements is
understood much better than nuclear staining; non - nuclear staining
is due primarily to proteins or to charged groups on the side chains of
amino acids constituting the proteins.
Proteins, or polymers (chains) of amino acids, contain a terminal
amino (-NH2) group on one end and terminal carboxyl (-COOH)
group on the other; in additition, amino acid side chains may have -
NH2 or –COOH groups. Because of these two groups, proteins may
be positively (+) or negatively (-) charged. This charge is pH-
dependent and because proteins can carry either positive or negative
charges, they are said to be amphoteric.
Based on its net charge, a substance will migrate in an electrical
field; a protein with a net positive charges (more positive than negative
charges) will migrate to the cathode (-), and a protein with a net
charge will migrate to the anode (+). At the point where the positive
and negative charges are equal, there will be no migration. This is
termed the iso - electric point (IEP). To understand the staining of
non - nuclear elements, one must understand how pH affects the
ionization of proteins and how pH relates to the IEP. The IEP of
proteins is approximately pH 6.0; below the IEP, or below pH 6.0,
the net charge on the nonnuclear proteins will be positive and the
attraction will be for an anionic dye; above the IEP, the net charge
will be negative and the attractions will be for a cationic dye.
Substances attracting basic dyes are said to be “basophilic” and
substances attracting acid dyes are “acidophilic”.
The Dyes
When we discuss dyes, we must answer two questions: why is
the substance a dye or colored, and why is there an affinity between
the dye and the element being dyed. All dyes are organic compounds
and most are coal tar or benzene derivatives. Replacing two hydrogen
atoms in the benzene ring with oxygen, or with another atom or group
having two valency bonds instead of one, results in a readjustment of
the double bonds and the formation of a colored compound. In the
past, formation of the quinine ring was thought to the responsible for
the development of color, but it was proved to be wrong. Benzene

would have color if our eyes were sensitive in the UV range, but
certain modifications to the benzene ring will push the absorption
band into the visible spectrum. A group that confers the property of
color is called a chromophore. Chromophores differ greatly from
one another; however they have one common property; they are
easily reduced because they all have an unsatisfied affinity for the
hydrogen. If reduction occurs, the chromagen is destroyed and color
is lost.
Although compounds containing chromophores are colored, they
may or may not be able to act as a dye, or to combine with the
substance to be colored. An ionizing group, called an auxochrome, is
required to enable the dye to link firmly to the tissue. The amino
(-NH2) groups is one of the most frequently occurring auxochromes;
aniline contains this group and many of the dyes are aniline derivatives.
The application of the terms “acid” and “basic” to dyes can be
confusing, because when referring to dyes, these terms have nothing
to do with pH. A basic dye is one in which the charge on the dye ion
is positive; these dyes are more properly called cationic dyes. Acid
dyes are those with a negative charge and are more properly referred
to as anionic dyes. Crystal violet and safranin are typical basic dyes;
orange G and picric acid are typical acid dyes, and hematein and
lithium carminate are amphoteric dyes, with isoelectric points of about
6.6 and 4.5 respectively.
Factors Affecting Dye Binding
1. Solutions pH determines whether or not a dye will be bound by
certain tissue elements by establishing the appropriate charges
on both the tissue element and the dye molecule.
2. An increase in temperature will increase the rate of staining by
increasing the diffusion rate of the dye molecules. Swelling of
tissue components, caused by the increase in temperature,
probably is an important factor in dye penetration.
3. An increase in dye binding usually occurs with an increase in
concentration of the dye molecules.
4. Salts, other than the dye, dissolved in the staining solutions can
decrease or increase the staining intensity of certain tissue
components; probably salt ions and dye ions compete for the
same binding sites.
5. By reacting with certain chemical groups in tissue and making
those groups unavailable for dye binding, the fixative alters the
staining characters of the tissue. In general, formaldehyde,
mercuric chloride, and osmium tetroxide increase tissue basophilia,
or the uptake of cationic or positively charged dyes. Picric acid
increases the binding of anionic or negatively charged dyes and
ethyl alcohol is intermediate between these two groups.
Differentiation
Many dyes, including most of those used as counterstains or
cytoplasmic stains, are used progressively that is, once the desired
intensity of color is achieved, the reaction is stopped. Mordant-dyes
also may be used progressively, but frequently regressive staining is
required. Mordants are substances, or metals, that act as a link
between dye and tissue. The mordant combines with the dye to form
a “lake” that is usually basic in action. In regressive staining the
tissue is overstained and the differentiated, or decolorized, until only
the desired element is left stained. Many of the mordant-dyes are, or
must be used regressively to achieve differential staining of the desired
structure. There are three basic methods of differentiating sections
in the histopathology laboratory when mordant-dyes are used
regressively.
1. Basic cationic dyes are differentiated by weak acid solutions,
and acidic or anionic dyes are differentiated by weak alkaline
solutions. For example, the aluminium hematoxylins can be
differentiated with a dilute solution of hydrochloric acid, and eosin
can be removed from overstained sections with a dilute solution
of ammonium hydroxide. If the differentiating solutions are
prepared in alcohol rather than water, better control of
differentiation is possible.
2. Excess mordant will break the tissue/mordant/dye complex. Since
the amount of mordant in the differentiating solutions is large
compared with that bound to tissue, the dye will dissociate from
the tissue. The structures that have bound the most dye will the
last to completely lose color; careful control of the differentiating
process will leave the desired structures well stained and the
background colorless. Sections stained with regressive iron
hematoxylin methods are differentiated with excess mordant. Iron,

hematoxylin is also the nuclear stain of choice in many special
stains that employ acidified solutions following nuclear staining.
Iron hematoxylin is fairly resistant to decolorization with acid;
aluminium hematoxylin is not.
3. Oxidizers presumably work by oxidizing the dye to a colorless
substance, although the effect is that of differentiation, because
those substances containing the most dye will still remain colored
if the oxidation process is stopped at the appropriate stage.
Potassium permanganate and chromium trioxide have been used
as oxidizing differentiators.
The Nuclear Dyes
Haematoxylin, the most widely used nuclear stain, is extracted
from logwood (also known as campeachy wood). The Haematoxylon
campechianum is a tree that has been scientifically cultivated in
Jamaica since 1715. The freshly cut wood is colorless but becomes
dark reddish-brown when exposed to atmospheric oxygen; the oxidized
dye is hematein. Haematoxylin is not dye, hematein, the oxidation
product of hematoxylin, is a weak anionic dye. Oxidation of
hematoxylin is necessary and may be achieved naturally by exposing
the solutions to atmospheric oxygen or by using oxidizing agents such
as sodium iodate, mercuric oxide, and potassium permanganate this
oxidation process is also called “ripening.” Solutions should always
contain some unoxidized hematoxylin, since complete or overoxidation
leads to a breakdown of the solution and the loss of good staining.
Oxidized hematoxylin (hematein) has little affinity for tissue but
becomes a strong dye with a particular affinity for nuclei when
combined with a metallic mordant.
In some solutions of hematoxylin, the oxidizer also serves as the
mordant; these solutions, most commonly the iron hematoxylins, are
not stable. To achieve stability, the mordant should not oxidize the
solution. Ammonium or potassium aluminum sulfate, phosphotungstic
acid, and phosphomolybdic acid are in this category of non oxidizing
mordants. The mordant-dye combination is called a lake, and the
most commonly used hematoxylin lakes are combinations of hematein
with either aluminum or iron. The terms alum is frequently misused
in histotechnology, alums are double sulfates such as potassium

aluminum sulfate, ammonium aluminium sulfate, ferric ammonium
sulfate, or chromium potassium sulfate. The use of terms such as
ferric alum or iron alum are to be deplored; students have no idea of
the true nature of this compound and most frequently have trouble
trying to find the dry chemical for solution preparation. The routine
nuclear stains should be called aluminum hematoxylins, or more
properly aluminum hemateins, since aluminium is a mordant.
More selective nuclear staining can be achieved by adding either
an excess of acid or an excess of aluminum. The H+ of the acid will
combine with weakly acidic groups in the tissue sections and prevent
them from taking up hematoxylin; excess aluminum added to aluminum
hematoxylins solutions will counteract overoxidation by chemical
oxidizers. However, too much aluminum or improperly dissolved
aluminum salts can precipitate on top of the tissue, giving a crystalline
artefact.
Formulas for some of the aluminum hematoxylins follow,
Harris’ Hematoxylin
Hematoxylin….. 5.0 g
Absolute ethyl alcohol….. 50.0 ml
Ammonium aluminium sulfate….. 100.0 g
Distilled water….. 1000.0 ml
Mercuric oxide….. 2.5 g
Dissolve the hematoxylin in the alcohol (it may be necessary to
add about 20 ml of the water to completely dissolve the hematoxylin).
Completely dissolve the ammonium aluminium sulfate in the water
with heat. Remove from heat and carefully mix the two solutions.
Bring to a boil as rapidly as possible. Remove from heat and slowly
add the mercuric oxide. Reheat to boiling, and boil for 2 to 3 minutes
or until the solution becomes dark purple. Remove from heat, and
immediately plunge the vessel into a basin of ice. The stain is ready
for use as soon as it cools, or as soon as a metallic sheen develops on
the surface of the solution. Filter just before use and add glacial acetic
acid to give a final concentration of 4% (4ml glacial acetic + 96 ml
hematoxylin).

The mordant in this solution is aluminum and the chemical ripening
agent is mercuric oxide. Sodium iodate (0.37 g) may be used to oxidize
the solution instead of mercuric oxide. Culling recommended acidifying
the solution with 1% hydrochloric acid to a pH of 1.0 to 1.2 to achieve
very selective nuclear staining.
Mayer’s Hematoxylin
Hematoxylin….. 1gm
Distilled water….. 1000ml
Sodium Iodide….. 0.2gm
Ammonium or Potassium
aluminium sulphate….. 50gm
Citric Acid….. 1gm
Chloral Hydrate….. 50gm
Dissolve the hematoxylin in water, boil the solution for 5 mints.
Remove from heat and as soon as the boiling stops add the sodium
iodide. Let ripen for 10 mints add the remainder of the reagents in
order, making sure each one is dissolved completely before adding
the next.
Aluminium is the mordant and sodium iodide is the oxidizer. Citric
acid is added to adjust the pH. Chloral hydrate helps to prevent the
scum and precipitates that tend to form in aluminum hematoxylin
solutions. This stain can be kept for long period of time (2 to 3 months)
without overripening. This stain can be used in regressive staining
and counterstain for Immunohistochemistry.
Weigert’s Hematoxylin
Solution A Solution B
Ferric chloride 29%….. 4ml Hematoxylin….. 1gm
Distilled water….. 95ml 95% Alcohol….. 100ml
Conc. Hcl….. 1ml
Mix equal part of solution A and B for use
This Solution can be used for 2 to 3 days. This stain is used
progressively for 5 to 30 mints.
*This is the only hematoxylin can be used as counterstain in Van
Gieson Stain
Ehrlich’s Hematoxylin
Hematoxylin….. 2gm
95% Alcohol….. 100ml
Glycerol….. 100ml
Ammonium or Potassium
Aluminium sulphate….. 3gm
Glacial acetic acid….. 10ml
This stain is suitable for decalcified (acid treated) tissue, long stored
tissue and Bouins fixed tissue
Plasma Stains
The plasma stains are most frequently anionic, or negatively
charged, dyes that combine with very cationic, or positively charged,
tissue groups. The basic amino acids such as Arginine, Histidine, and
Lysine are common sites for dye binding.
Eosin is the most widely used counterstain in the routine staining
of sections. Usually Eosin Y is used. Eosin is the sodium salt of a
color acid; the chromophore is in the anionic (-) part of the molecule.
Eosin is fully charged at a pH of 7, but because the IEP of proteins is
approximately 6.0, we must stain below pH 6.0 to develop a net
positive charge on the protein. Below pH 4.0, the amount of charged
dye will be greatly decreased because the eosin is converted to a
free acid at a lower pH. The best staining with eosin will occur at a
pH of approximately 4.6 to 5.0. Used properly, at least three shades
of pink can be obtained with eosin alone; erythrocytes, collagen, and
the cytoplasm of muscle or epithelial cells should stain with different
shades or intensities of pink.
Eosin solution
Eosin Y (1% aqueous solution)….. 200.0 ml
95% ethyl alcohol….. 600.0 ml
Acetic acid, glacial….. 004.0 ml
Even eosin-phloxine B solution, can be preferred as the pink shades
are more vivid. It is very easy to over stain with this solution and to
lose some of the fine differentiation possible with counterstains. This
solution also must be acid to develop the appropriate charge on
proteins.
Eosin-Phloxine B
Eosin Y (1% aqueous solution)…….. 100.0 ml
Phloxine B (1% aqueous solution)……. 010.0 ml
95% alcohol…………….. 780.0 ml
Acetic acid, glacial….. 005.0ml
II. STAINING METHODS

Progressive Method
The following procedure works very well in a surgical pathology
laboratory. The use of Mayer’s hematoxylin increases the time needed
for each basket of slides and can dramatically increase the time
required to stain the day’s routine surgical slides. For this reason, it is
preferred to use Harris’ hematoxylin with 4 mL of glacial acetic acid
added to every 96 ml of hematoxylin. Harris’ hematoxylin used
progressively stains rapidly with very reproducible results. It’s
preferable to use eosin alone, but if more intense red shades are
desired, eosin-phloxine can be used. The formulas for the solutions
are the ones given previously.
1. Xylene, three changes….. 2minutes each
2. Absolute alcohol….. 10 dips
3. 90% alcohol, 80% alcohol
and 70 % alcohol ….. 10 dips each
4. Tap water*….. rinse until water runs off evenly
5. Haematoxylin, Mayer’s….. 15 minutes
or Harris’ with acetic acid …. 1 to 3 minutes
6. Tap water, two changes….. 10 dips each*
7. Ammonia water, 0.25 or

lithium carbonate, 0.5%….. until blue
9. Eosin….. 10 to 20 dips
or eosin-phloxine….. 1 to 3 minutes
10. 70% alcohol, 80% alcohol and
90 % alcohol….. 10 dips each
11. Absolute alcohol, three changes….. 10 dips each
12. Xylene, three changes….. 10 dips each
13. Mount the slide in synthetic resin
Let slides remain in last container until a coverslip is applied.
*Change water frequently; where two changes are indicated,
one container should be changed after each basket. Rotate the
containers so that the clean water is in the second container.
With a large volume of slides, we find it is best to use a staining,
time of 1 minute with fresh Harris’ hematoxylin staining solution
and add 3 second is per day until 3 minutes of staining time is
reached, then change to fresh solution.
Do not agitate in the ammonia water as most section loss will
occur at this point. This step requires 10 to 30 seconds, and the
solution should be changed when it becomes discolored.
Results
Nuclei….. blue
Erythrocytes and Eosinophilic granules….. bright pink to red
Cytoplasm and other tissue elements….. various shades of
pink.
Regressive method
1. Xylene, three changes….. 2 minutes each
2. Absolute alcohol….. 10 dips
70 % alcohol ….. 10 dips each
4. Tap water…. rinse until water runs of evenly*
5. Hematoxylin- Delafield’s, Ehrlich’s, or
Harris’ without acetic acid….. 10 to 15 minutes
7. 1% Hydrochloric acid in 70% alcohol….5 to 10 dips
8. Running water… wash well*
9. Ammonia water, 0.25% or
lithium carbonate, 0.5%….. 30 seconds**
11. Eosin….. 10 to 20 dips
or eosin-phloxine….. 1 to 3 minutes
90 % alcohol….. 10 dips each
13. Absolute alcohol, three changes….. 10 dips each
14. Xylene, three changes….. 10 dips each
15. Mount the slide in synthetic resin
Let slides remain in last container until coverslips are applied.
*Change water frequently; where two changes are indicated, one
container should be changed after each basket. Rotate the container
so that the clean water is in the second container.
**Do not agitate in the bluing agent as most section loss will occur at
this point. The solution should be changed when it becomes discolored.
Slides should be checked microscopically at this point until sufficient
experience is gained with differentiation, if necessary return the slides
to the hydrochloric acid solution.
Results
Nuclei…. blue
Erythrocytes and Eosinophilic granules…. bright pink to red.
Cytoplasm and other tissue elements…… various shades of
pink.
When using hematoxylin regressively, great care must be taken in
the differentiation step so that the nuclei are not overdifferentiated or
underdifferentiated; improper nuclear staining may lead to the loss of

important diagnostic features. Marked overstaining of the cytoplasm
is also undesirable.
Rapid Staining Procedure [Frozen Section]
1. Cut the frozen section and fix in 37% to 40% formaldehyde for
20 seconds [We prefer put the specimen in the test tube containing
formalin and gently heat it in Bunsen burner but see to it not to
overheat which means not reaching the boiling point because it
will damage the protein]
2. Rinse the section very well in at least three changes of tap water.
3. Stain in Harris’ hematoxylin with acetic acid for 60 to 90 seconds.
4. Rinse in two changes of tap water.
5. Place slide in 0.25% ammonia water and leave until blue.
6. Rinse in two changes of tap water.
7. Stain in eosin (formula given previously) with 15 to 20 dips or
until the desired intensity is achieved.
8. Dehydrate with 95% alcohol - 10 dips in two changes
9. Dehydrate with absolute alcohol – 10 dips in two changes
10. Clear the sections with Xylene – 10 dips in three changes.
11. Mount with synthetic resin.
Results
Nuclei….. blue
Cytoplasm and other tissue elements….. various shades
of pink.
III. PROBLEMS AND SOLUTIONS

FOR ROUTINE STAINING
Check points in staining
1. Microscopically check a slide from each basket to be sure that
proper staining has occurred.

2. Do not allow section to dry at any point during staining.
3. Keep the solutions covered when not in use to prevent evaporation.
If any precipitate is noted at the top of the hematoxylin container,
filter the solution into a clean dry container.
Stain or staining solution not as expected
The formation of precipitates in chemically ripened hematoxylins
indicates deterioration. Filter before use and, if necessary, extend
the staining times. If large num-bers of slides are stained, prepare
a fresh batch of stain each month.
Tissue stains unexpectedly weakly
1. If the stain was naturally ripened, check that it has ripened for a
sufficient period. The weather conditions can affect the ripening
so it may be speeded up by placing an unstoppered bottle in a
warm place.
2. If stain was chemically ripened, was the solution pre-pared several
months ago? Or has a precipitate formed in the stock solution?
3. If stain is heavily used, it will deteriorate more rapidly. Try longer
staining times.
4. Specimens exposed to acidic solutions prior to staining (e.g.
decalcification media, or unbuffered formalin or picric acid
fixatives) may lose DNA and hence require longer staining times.
5. If hematoxylin-stained sections are exposed to acidic solutions,
such as Van Gieson’s stain, the stain decom-poses. In such cases
use an alternative stain, such as an iron hematoxylin, or an iron
Celestine Blue method.
6. Tissues embedded in hydrophilic resins may stain slowly due to
occlusion. Lengthen staining times, or add a little alcohol to the
stain.
7. After applying the ammonia water, wash the sections very well
with tap water; the pH of the eosin is critical and if too much
ammonia is carried over into the eosin, cytoplasmic staining will
be lacking.
8. Do not pass the slides through the dehydrating solutions too
quickly, as dehydrating solutions also serve to differentiate eosin.

9. Tissues that have been fixed for longer than normal will require
increased staining times. For autopsy tissue, we increase the
staining times in H&E by one third.
10. Staining times also may need to be adjusted according to the
fixative used; the time in hematoxylin may need to be increased
after fixation in Helly’s, Zenker’s, or B-5 fixative, and the time in
eosin will frequently need to be decreased.
Unexpected structures stain
1. Over staining with hematoxylin can occur under various
circumstances:
(a) If the standard protocols, typically designed for paraffin
sections, are applied unmodified to frozen sections or
cytological specimens. Reduce the staining times
appreciably; in the first instance, try halving them.
(b) If tissues are embedded in hydrophilic resin, high resin
background staining can occur. Try differentiating in
alcohol.
2. Over staining with Eosin can occur with routine proce---dures if
mercuric fixations have been used. Shorten the Eosin staining
time, or lengthen the post staining wash.
3. We advice each individual laboratory has to form their own
standard protocol according to their pH of water, because pH is
more important which determines the IEP which will have effect
on both the hematoxylin and eosin.
Staining is of an unexpected color
Yellowish or brownish tinges seen following hematoxylin staining
may be due to the stain being contaminated with oxyhematin. If
this is a prob-lem, prepare a fresh batch of stain.
FOR RAPID STAINING
Expectation on fixation used
Many laboratories use acetone or alcohol as the fixative for frozen
sections; however, concentrated formaldehyde yields morphologic
preservation more like that seen in permanent sections.

Tissue stains unexpectedly weekly
1. The hematoxylin may need to be changed twice weekly depending
on the number of slides stained, as any formaldehyde carried
over into the hematoxylin will act as a reducing agent.
2. This rapid H&E method is preferred by many pathologists to
other frozen section stains; it required about 2 minutes to complete
the procedure and the slide is permanent. Other frequently used
methods are those involving metachromatic dyes (toluidine blue
O) and polychrome solutions (polychromed methylene blue).
These methods are most often used on unfixed sections and the
slides are not permanent. If dehydrated, cleared, and mounted
with synthetic resins, metachromatically stained sections become
monochromatically stained.
3. Fix cut sections immediately; do not allow air-drying or
morphologic preservation will be poor.
IV. FREQUENTLYASKED EXAM QUESTIONS
1. What is the principle behind H & E staining?

Haematoxylin and Eosin are basic and acidic dyes respectively which
were attracted by the nuclear (Basophilic) and cytoplasmic protein
(Acidophilic) component of the cell which end in blue and pink staining
of the corresponding substances.
2. What is meant by Vander Waals forces?
It is the attractive or repulsive forces between molecules (or between
parts of the same molecule) other than those due to covalent bonds.
3. What is the source of Haematoxylin?
It’s from the bark of the logwood otherwise called as campeachy
wood [Botanical Name: Haematoxylon Campechianum]
4. What is haematin?
Haematin is the oxidative product of hematoxylin.
5. What is chromophore?
In a dye, the group which is responsible for the color is called
chromophore example benzene ring.

6. What is auxochrome?
The substance required to enable the dye to link firmly to the tissue.
The amino (-NH2) groups is one of the most frequently occurring
auxochromes; aniline contains this group and many of the dyes are
aniline derivatives.
7. What is Iso Electric Point (IEP)?
The point where the positive and negative charges are equal, there
will be no migration in the electrode. This is termed the iso - electric
point (IEP). In other words in an IEP, protein will have equal positive
and negative charges.
8. If the cytoplasm stains blue in color, what is the cause of
this artefact?
The artefact is mostly due to alteration in the pH of the water used
because if IEP of the Protein is maintained in pH 6.0, if the pH goes
down it becomes acidic and attract the basic dye and becomes blue
in color. Other causes may be of fixation delay or keeping more time
in the lithium tray i.e., increasing the blueing time.
9. What is pH?
pH is a measure of the acidity or basicity of a solution
10. What is ripening?
Ripening means oxidization of the haematoxylin (colourless) into a
colourful haematin by natural oxidation by exposure of the dye to
light and air. This is the long process requires 3 to 4 months. Examples
of naturally oxidised haematoxylin are Ehrlich’s and Delafield’s
Haematoxylin
11. What is other mode of oxidation?
Chemical oxidation is done by using sodium iodate or mercuric oxide.
It is done almost instantaneously. It has a shorter life than naturally
occurring oxidation. Example Meyer’s Haematoxylin.
12. What is mordant?
Mordants are substances, or metals, that act as a link between dye
and tissue. The mordant combines with the dye to form a “lake” that
is usually basic in action.

13. What is the use of Mayer’s Haematoxylin?
It is used as regressive as well as progressive stain and counterstain
in Immunohistochemistry.
14. What is the use of Weigert’s Haematoxylin?
It is used as counterstain in van Gieson. The reason when
aluminum mordent haematoxylin is used picric acid will react
with aluminium and the stain colour will be washed off, to avoid
that Weigert’s is used because it contain ferric chloride as
mordant.
15. What is the use of Delafield’s Haematoxylin?
This stain is suitable for decalcified (acid treated) tissue, long stored
tissue and Bouins fixed tissue [same as like that of Ehrlich’s
Haematoxylin]
16. What is differentiation?
The process of selective removal of excessive dye is called
differentiation, commonly done in regressive staining. Basic cationic
dyes are differentiated by weak acid solutions, and acidic or anionic
dyes are differentiated by weak alkaline solutions. For example, the
aluminium hematoxylins can be differentiated with a dilute solution
of hydrochloric acid, and eosin can be removed from overstained
sections with a dilute solution of ammonium hydroxide. If the
differentiating solutions are prepared in alcohol rather than water,
better control of differentiation is possible. Postgraduates can easily
remember differentiators are decolourisers where 1 to 2 % acid alcohol
is used. Which is the first step used in restaining process but be
careful in regular staining because if you keep it for longer duration it
will completely remove all the colour (stain).
17. What is blueing?
Alum (Alum means it is a mixture of salt of ammonium aluminium
sulfate and potassium aluminium sulfate) in watery solution tends to
dissociate: the aluminium combined with the –OH group of the water
to form insoluble Aluminium hydroxide [Al (OH) 3], the free hydrogen
from the water tends to form sulphuric acid by uniting with the sulfate

from the alum. However if excess of acid (sulphuric or other acid) is
present, the aluminium hydroxide cannot form. Under such
circumstances in an alum haematoxylin dye the insoluble dye lake
(‘Lake’ is formed due to the combination of mordant with the dye
that is usually basic in action) cannot form because of lack of –OH
ions. Hence acid solutions of alum haematoxylin are reddish in color,
where as the alum lake of haematin is blue. In the blueing of sections
which have been stained by an alum haematoxylin the alkaline solution
used for blueing neutralizes the free acid and makes –OH groups
available so that the insoluble blue aluminium-haematin-tissue lake is
formed. Accordingly, for blueing of alum haematoxylin stained sections
warm (40° to 50°C) tap water is commonly used since it is generally
sufficiently alkaline. However, in many areas that tape water is acid
and unsuitable in such regions lithium carbonate (0.5% to 1% in water),
bicarbonate (0.2 to 0.5% in water), sodium or potassium acetate may
be used. Alternatively Scott’s tape water substitute may be employed
sodium or potassium bicarbonate 2 to 3.5 gm and magnesium sulfate
20gm dissolved separately, then combined and made upto 1000ml
with distilled water. (A few crystals of thymol or 5 to 10 ml of 40%
formaldehyde solution are added to prevent the growth of molds.
For the postgraduates easy to remember blueing means bringing blue
colour to the section by dipping into the 0.5 to 1.0% of lithium
carbonate. The students are asked to agitate the slide in the lithium
carbonate basket because lithium tends to settle down and if there is
discolouration in the basket, the solution has to be changed
18. What is the best blueing agent ammonia or lithium
carbonate?
Lithium carbonate because ammonia causes hardening and loosens
the section so there is high chance to loss the section in the further
procedures.
19. What is the difference between the progressive and
regressive staining?
The step of the differentiation (i.e., dipping in the 1% acid alcohol) is
not there in the progressive staining because in the regressive staining

the section is overstained and it differentiated to remove the excess
stain until the desired color is reached.
20. What type of eosin is used in H & E?
Eosin Y
21. Enumerate the rapid staining method?
(i) Cut the frozen section and fix in 37% to 40%
formaldehyde for 20 seconds [We prefer put the
specimen in the test tube containing formalin and
gently heat it in Bunsen burner but see to it not to
overheat because it will damage the protein]
(ii) Rinse the section very well in at least three changes
of tap water.
(iii) Stain in Harris’ hematoxylin with acetic acid for 60
to 90 seconds.
(iv) Rinse in two changes of tap water.
(v) Place slide in 0.25% ammonia water and leave until
blue.
(vi) Rinse in two changes of tap water.
(vii) Stain in eosin (formula given previously) with 15 to
20 dips or until the desired intensity is achieved.
(viii) Dehydrate with 95% alcohol - 10 dips in two changes
(ix) Dehydrate with absolute alcohol – 10 dips in two
changes
(x) Clear the sections with Xylene – 10 dips in three
changes.
(xi) Mount with synthetic resin.
22. What type of alcohol used and percentage of it?
Isopropyl alcohol (propan-2-ol) - (CH3)2CHOH, In our lab we use
95%, 90%, 80% and 70% grade of alcohol but few labs follow 95%

of alcohol in all the grades. We recommend standardized has to be
achieved in individual laboratory.
23. What is the effect of pH on H&E staining?
It can effect on the stains by altering the iso electric point of the dye.
Example the hematoxylin (haematin) has less affinity towards
tissue when there is increase in pH it affinity becomes stronger to
the nucleus.
It can affect the protein content of the tissue by altering the iso electric
point, if the pH goes below protein becomes acidic and attract the
basic dye vice versa happens when pH goes up.
24. What is the reason for bubble artefact seen in the H & E
section?
The bubble artefacts may be of water or air bubble. The water bubble
is due to improper dehydration process because Xylene will not mix
with water so proper clearing will not occur so checking the percentage
of the alcohol is essential. The air bubble is due to improper mounting,
which can be corrected after remounting the slide.
25. What are the methods to check the water content of the
alcohol?
There are various methods a) Adding a small amount of dried
powdered white copper sulfate, if water presents the copper sulfate
will become tinged with blue. b) Mix 1 ml of alcohol in 1ml of Xylene,
if cloudiness appears there is more water content. c) Hydrometer
26. When usually the sections float during staining process?
If the sections are not well embedded, it floats after differentiation.
27. What alterations are required in the staining process
when the mercuric (zenker’s) fixatives are used?
During the hydration process, immerse in a bath of 95% alcohol
for 1 or 2 mints then immersing for 5 mints in a 0.5% solution of
iodine in 80 to 95% alcohol. Following this, the section should be
rinsed in water and the iodine removal by placing for 1 to 5 mints
in 3% sodium thiosulfate solution, after which the solution must

be washed well by placing in running water for 3 to 5 mints.
Alternatively, mercury deposits may be removed after the sections
are hydrated by immersing in Gram’s or Lugol’s iodine for 5 mints,
followed by sodium thiosulfate and washing. Sections of mercurial
fixed tissues are then ready for staining.
28. What is the alternative if there is haematoxylin
shortage?
Celestine blue B is an alternative. [Celestine Blue B…..1gm;
Ferric Ammonium sulfate…6gm; Conc. Hcl…2ml;
Glycerine…15ml; Distilled water…50ml]
Dissolve the ferric ammonium sulfate in the distilled water,
add Celestine blue B and gently boil for 3 mints to facilitate
solution. Cool, add glycerine and Hcl. The solution is then ready
to use.
Working solution of eosin is prepared by adding 1 part of alcoholic
solution of eosin 1% with 3 parts of 80% alcohol. Just before use,
add 0.5 ml glacial acetic acid to each 100ml of eosin solution stir
well.
i) Bring the sections to water
ii) Celestine blue B – 15 mints
iii) Dip in distilled water until excess Celestine blue B
is washed off
iv) Dehydrate in 2 changes of 95% alcohol and 3
changes of absolute alcohol each time for 2 mints
v) Clear in 3 changes of Xylene
vi) Mount
Results, Nuclei….blue; Cytoplasm….red.
29. What are the methods to save haematoxylin?
i) Over oxidation is avoided when closing the lid during the
staining process.
ii) 30 second wash in 0.5% of aqueous citric acid before
staining in haematoxylin will prolong the staining life of
the haematoxylin solution.

iii) After a solution of harris haematoxylin has reached the
end of its staining usefulness allow to evaporate to about
half its volume. Then add 10 ml saturated aqueous
ammonium aluminium for each 100 ml of haematoxylin
solution. Filter after 24 hrs and employ in staining of
tissues doubling the usual time.
iv) Using Mayer’s haematoxylin instead of Harris’s yields a
saving of 4/5 in the amount of dye used.
30. What are accentuators?
These are the substances which heighten the color intensity,
crispiness and selectivity of a stain. They differ from mordants
in that they do not bind or link the dye to the tissue. Examples
aniline used in gentian violet, barbiturates used in metallic
impregnation of nerve fibers
31. What is the alternative for Eosin?
Several substitutes are available examples phloxine, erythrosine,
azophloxin, Biebrich scarlet and orange G, all in concentrations
and modes similar to eosin.
32. How the section holds to the slide?
Due to the surface tension and capillary attraction created
between the slide surface and the section.
33. What is the difference between the Harris’ and Mayer’s
Haematoxylin?
Oxidising agent [Harris’ – Mercuric oxide; Mayer’s - sodium
iodate]. Mayer is used in immunohistochemistry and
iummunoperoxidase because it does not contain alcohol.
34. What is distilled water?
Natural water usually contains a number of microscopic
contaminants, along with dissolved minerals such as calcium and
iron. The water is to boil it until it changes to steam, a process
known as distillation. When this steam is allowed to cool down
and condense into liquid form again, the result is a purified form
called distilled water. Distilled water should ideally be nothing but

hydrogen and oxygen molecules, with a PH level of 7 and no
additional gases, minerals or contaminants.
The distilling process relies on the principle that most solid
materials found in water are heavier than the water molecules
themselves. When water is heated in a distiller, any dissolved
solids such as salt, bacteria, calcium or iron remain solid while
the pure water converts to a much lighter steam and is drawn out
for condensation. Distilled water has a noticeably bland taste
because all of the minerals which give water its flavour have
been removed.
35. What is feulgen reaction?
The feulgen reaction is to demonstrate DNA, which is based on
the mild hydrolysis of DNA by hydrochloric acid, which rapidly
remove the purine bases (adenine and Guanine) but leaves the
sugar and phosphates of DNA intact. This hydrolysis generates
an aldehyde group that can be demonstrated with Schiff’s
reagent. The molecular structure of RNA is different and
hydrolysis with hydrochloric acid does not occur so RNA is not
demonstrated by this method.

2. SPECIAL STAINS
A. CARBOHYDRATE STAINING
I. BASICS OF STAINING MECHANISM

CULLING’S HISTOCHEMICAL CLASSFICATION
They are four groups;
Group I: Neutral Polysaccharides (non-ionic homoglycans)
1. Glucose containing: Glycogen, Starch, Cellulose.
2. N-Acetyl-Glucosamine containing: Chitin.
This group gives a very positive PAS reaction and a negative
reaction with the other frequently used carbohydrate stains (Alcian
blue, Colloidal iron, Mucicaramine)
Group II: Acid Mucopolysaccharides (anionic heteroglycans)
1. Carboxylated (COOH): Hyaluronic acid [Found in the
connective tissues and umblical cord]
2. Sulfated (OSO3H) and Carboxylated (COOH)
a) Chondroitin Sulfate A (Chondroitin-4-sulfate).
b) Chondroitin Sulfate C (Chondroitin-6-sulfate) [Found in
cartilage, chondrosarcoma, cornea and blood vessels]
c) Chondroitin Sulfate B (Dermatan sulfate) [Found
principally in skin, also in connective tissue, aorta and
lung]
d) Heparin [Found in mast cells and the intima of
arteries]

3. Sulfated only (COOH – free) [human aorta and bovine
cornea]
This group are acidic (anionic) and are thought to be attached to
protein, even though the word protein does not appear in the name.
They are negative for PAS reaction and Alcian blue positive (both
pH2.5 and 1)
Group III: Glycoproteins (mucins, mucoids, mucoproteins,
mucosubstances)
1. Neutral: Ovimucoid (egg white), mucin in stomach, Paneth
cell granules.
2. Carboxylated (COOH): sialoglycoproteins that contain sialic
acid but no sulfate.
a. Sialomucins [Found in submaxillary gland mucins, small
intestine mucins, fetal mucins, the upper part of colonic
crypts and human sublingual gland].
b. Serum glycoproteins
c. Blood group substances.
3. Sulfated (OSO 3 H) and Carboxylated (COOH):
sialoglycoproteins that contain both sialic acid and sulfate
[Found in colonic mucins of sheep and humans]
These are mostly “epithelial mucins” but some may occur in
connective tissue. These glycoproteins are potentially but not
necessarily, PAS positive.
Group IV: Glycolipids
1. Cerebrosides: Fatty residue bound to a carbohydrate
structure
2. Phosphatides: PAS-positive, non carbohydrate containing
lipids, including lecithin, cephalin and sphingomyelin. This
compound is included because of PAS positivity.
Almost invariably, polysaccharides occur in the body as a mixture.
The histochemical differentiation between those components with a
series of long chain carbohydrate polymers attached to a small protein
core and those with short chain carbohydrate polymer attached to a

large protein core is not possible, but frequently the histological
localization permits an educated guess (Culling uses the word
‘informed guess’). The information that can be obtained is based on
the groups present in carbohydrates that can be histochemically
demonstrated. Specifically, these groups are 1:2 glycols, carboxyl
(COOH) and ester sulfate (OSO3H). Addition information can be
obtained with enzyme digestion procedures involving the use of
diastase, hyaluronidase and sialidase. Blocking procedures may also
add information and aid in identification of carbohydrates, but blocking
techniques are less frequently used in routine histopathology.
With these basics, we will move to the stains which is useful in the
demonstration of neutral polysaccharides, acid mucopolysaccharides
and glycoproteins (mucins).The stains that deal with glycolipids are
beyond the scope of this book.
II. PERIODIC ACID SCHIFF STAIN

PRINCIPLE AND PROCEDURES
Purpose
The demonstration of polysaccharides (glycogen, chitin), neutral
mucosubstances (mucins in stomach) and basement membrane.
Principle
The reaction is based on oxidation of certain tissue elements to
aldehydes by periodic acid. The most common group is the 1:2 glycol
group, but other groups are also selectively oxidized by periodate.
1:2 Glycol + Periodic acid Aldehyde
Schiff reagent is prepared by treating pararosaniline with sulphurous
acid. Reduction causes the loss of the quinoid structure and a
colourless compound, referred to as leucofuchsin, is formed. Following
the Schiff reaction, washing in running water causes the loss of the
bound sulphurous acid group attached to the central carbon atom, the
restoration of the quinoid structure in the dye bound by the aldehyde
and the visualisation of the typical Schiff color. Metabisulfite rinses
are used to remove excess Schiff reagent and prevent false colorization
of the tissue elements due to oxidation of any adsorbed reagent.
Fixative
10% neutral buffered formalin or Bouin’s solution. Blood smears
should be fixed with methyl alcohol for 10 to 15 minutes.
Quality control
A section of kidney is most sensitive control. If the procedure is
used to demonstrate glycogen, use a section of liver containing
glycogen. Among the gastrointestinal specimens, appendix can be
used as control.
Reagents
0.5% Periodic Acid
Periodic Acid…… 2.5gm
Distilled water…… 500ml
1N Hydrochloric Acid
Hydrochloric Acid,
concentrated (specific
gravity, 1.19)…… 83.5ml
Distilled water…… 916.5ml
Add acid to the water and mix well.
Schiff Reagent
Basic Fuchsin…… 4.0gm
Sodium Metabisulfite…… 4.0gm
1N Hydrochloric Acid…… 80.0ml
Heat water to the boiling point. Remove from flame, add basic
fuchsin, and again heat solution to the boiling point. Cool the solution
to 50°C and then filter. Add 80.0 ml of 1N HCl, cool completely, and
add 4.0 gm of sodium meta bisulfite. Let the solution stand in the
dark overnight; it should turn light amber. Add 2.0 g of activated
charcoal and shake for 1 minute. Filter the solution and store in the
refrigerator.

Test for the quality of Schiff reagent
Place 10 ml of 37% to 40% of formaldehyde in a beaker. Add a few
drops of Schiff reagent. If the solution rapidly turns reddish purple, it
is good. If the reaction is delayed and the resultant color is a deep
blue – purple, the solution is breaking down.
0.55% of Potassium Metabisulfite
Potassium Metabisulfite…… 2.75gm
Procedure
1. Cut paraffin sections ate 4 to 5 microns thick
2. Deparaffinize and hydrate slides to distilled water.
3. Wash slides in three changes of distilled water.
4. Place sections in Schiff reagent for 15 minutes.
5. Wash for 1 minute in each of two jars of 0.55% potassium
Metabisulfite to remove excess stain.
6. Wash in running tap water for 10 minutes to develop full
color.
7. Counterstain ½ minute in Harris’ hematoxylin with acetic
acid (2ml acetic acid/48 ml haematoxylin)
8. Wash sections well to blue the haematoxylin.
9. Dehydrate with 95% and absolute alcohol, clear with Xylene
and mount the section
Results
Glycogen, neutral mucosubstances (Mucins of stomach), certain
epithelial sialomucin (Mucins of the sub maxillary glands, small
intestine, upper part of the colonic crypts) and sulfomucin (Mucins of
the colon), colloidal material of the thyroid and pars intermedia of the
pituitary, basement membranes and fungal walls show a positive PAS
(BRIGHT ROSE) reaction.
PROBLEMS & SOLUTIONS
If the Schiff’s reagent looks yellow brown, this suggests
contamination by acridines. Use another batch of reagent; or try
de colorizing with activated charcoal as in the initial preparation
of the reagent.
If the Schiff reagent is pink tinged and smells only weakly of
sulphur dioxide it may be overaged, as Basic Fuchsin is slowly
formed on standing. Use fresh reagent or reconstitute with
bisulfite.
Extraction of the polysaccharides by the fixatives, try picric –
formaldehyde or alcoholic fixative. Tissue containing the anionic
polysaccharides (connective tissue substances) and
glycosaminoglycans (connective tissue ground substance) tend
to stain weak, so give more oxidation time or adding magnesium
chloride to the periodate solution.
A) Pink background staining may be due to decomposition of
Schiff reagent. This can occur for several reasons:
i) Aging
ii) Carryover of periodate. Try more extended washing
following oxidation.
iii) Thermal decomposition during microwave accelerated
staining. If the stain becomes transiently pink during staining,
reduce the time or temperature setting on the oven.
B) Pink background staining also occurs due to artefactual tissue
aldehyde groups. These arise in several ways:
i) Glutaraldehyde fixation
ii) Insufficient washing of tissue following formaldehyde
iii) Lipid rich material such as myelin can be oxidised by
periodate, becoming Schiff positive
C) Localised purple or red staining of ‘non target’ structures
can occur for various reasons:
i) Depositions of carbonates and other salts can decompose
the Schiff reagent. This can be detected by the routine control.
ii) Cysteine rich sites, such as hair shafts, may be oxidized by
periodate, becoming Schiff positive.

D) Intra cellular location of PAS staining can vary with the
fixation method used. Try varying the fixative agent or fixative
method.
Yellow brown background staining may be due to contamination
of the Schiff reagent with acridines.
III. PERIODIC ACID SCHIFF STAIN WITH DIASTASE

DIGESTION
Purpose
The demonstration of glycogen in tissue sections.
Principle
This is a very sensitive histochemical method for glycogen.
Diastase and á – amylase act on glycogen to depolymerise it into
smaller sugar units that are washed out of the section. The Schiff
reaction has been described in the PAS procedure.
Fixative
10% neutral buffered formalin or formalin alcohol or absolute
alcohol
Quality Control
Two controlled sections of liver containing glycogen must be used,
one labelled ‘with’ and one labelled ‘without’.
Reagents
0.5% Periodic Acid
Periodic Acid…… 2.5gm
1N Hydrochloric Acid
Hydrochloric Acid,
concentrated (specific gravity,
1.19)…… 83.5ml
Add acid to the water and mix well.

Schiff Reagent
Basic Fuchsin…… 4.0gm
Sodium Metabisulfite…… 4.0gm
1N Hydrochloric Acid…… 80.0ml
Heat water to the boiling point. Remove from flame, add basic fuchsin,
and again heat solution to the boiling point. Cool the solution to 50°C
and then filter. Add 80.0 ml of 1N HCl, cool completely, and add 4.0
gm of sodium meta bisulfite. Let the solution stand in the dark
overnight; it should turn light amber. Add 2.0 g of activated charcoal
and shake for 1 minute. Filter the solution and store in the refrigerator.
Test for the quality of Schiff reagent
Place 10 ml of 37% to 40% of formaldehyde in a beaker. Add a few
drops of Schiff reagent. If the solution rapidly turns reddish purple, it
is good. If the reaction is delayed and the resultant color is a deep
0.55% of Potassium Metabisulfite
Potassium Metabisulfite…… 2.75gm
Malt diastase solution
Diastase of malt…… 0.1gm
Phosphate buffer, pH 6.0…… 100ml
Phosphate Buffer, pH 6.0
Sodium chloride…… 8.0gm
Sodium phosphate, monobasic…… 1.97gm
Adjust pH to 6.0 of necessary
Procedure
1. Cut two paraffin sections at 4 to 5microns thickness. Label
one section ‘with’ and one section ‘without’.

2. Deparaffinize and hydrate slides to distilled water
3. Place the sections labelled ‘with’ in preheated diastase
solution at 37°C for 1 hour. Hold the section labelled ’without’
in distilled water.
4. Wash in running water for 5 mints.
5. Place all the sections (‘with’ and ‘without’) in 0.5% periodic
acid solution for 5 minutes.
6. Wash in three changes of distilled water.
7. Place in Schiff reagent for 15 minutes.
8. Wash for one minute in each of two jars of 0.55% potassium
Metabisulfite to remove the excess stain.
9. Wash in running tap water for 10 minutes to develop full
color.
10. Counter stain ½ minute with Harris’ haematoxylin with acetic
acid (2ml acetic acid/48ml haematoxylin)
11. Wash well in running tap water to blue the haematoxylin.
12. Dehydrate with two changes each of 95% and absolute
alcohol, clear with Xylene, and mount the slide.
Results
Glycogen will stain bright rose red on the section labelled ‘without’
and will be absent from the section labelled ‘with’.
PROBLEMS AND SOLUTIONS
Loss of section…
Malt diastase, containing both á and â amylase is commonly used
for digestion but tends to loosen the sections. For this reason as
well as decrease the digestion time, many laboratories prefer to
use human saliva, which contains only á – amylase. If preferred,
digest with saliva for 20 minutes at room temperature.
Both ‘with’ and ‘without’ section shows positive for
PAS reaction…
Check the fixatives, if the fixatives are of picric acid formalin,
then glycogen is resistant for digestion. So wash the section in
the water for longer duration. Another reason the material in the

cell may not be of glycogen it could be of neutral mucin or other
epithelial mucins or glycolipids.
IV. MAYER’S MUCICARMINE STAIN

Purpose
Demonstration of “epithelial” mucin (i.e., neutral mucins of stomach,
sialomucins of small intestine, submaxillary gland & upper part of
colonic crypts and sulfamucins of colon) in tissue sections.
Principle
This is primarily an empirical stain. Aluminum is believed to form a
chelation complex with the carmine; the resulting compound has a
net positive charge and attaches to the acid groups of mucin.
Fixative
10% neutral buffered formalin.
Quality Control
A section of colon, small intestine or appendix
Reagents
Mucicarmine Stock Solution
Carmine, Alum Lake…. 4.25gm
Aluminium hydroxide…. 4.45gm
Ethyl alcohol 50%…. 375ml
Ethyl alcohol 25%…. 25.0ml
Ammonium chloride, anhydrous…. 2.05gm
Use hood for the preparation of this reagent. Thoroughly mix the dry
carmine and aluminium hydroxide in the 50 ml test tube. Add the 25
ml of 25% ethyl alcohol to the test tube, and stir thoroughly with a
glass rod until as much of the dry mixture as possible is in solution.
Using the test tube holder, warm the solution by lowering the tube
intermittently into the water bath, stirring continuously with the glass
rod. Warming should last no longer than one minute. Do not allow the

solution to boil or contaminated with the boiling water. Using the
premeasured 375ml of 50% of alcohol, rinse the entire contents of
the test tube into the 1000ml flask, stirring with the glass rod each
time so that the mucicarmine mixture is removed from both the rod
and the inside of the test tube. Use the dry stainless steel spatula;
slowly and gradually add the aluminium chloride to the solution in the
flask, swirling after each addition. Do not breathe the hydrochloric
acid vapours. After adding all the aluminium chlorides immediately
place the flask into boiling water bath and watch closely for signs of
boiling inside the flask. Boil for exactly 2 ½ minutes. Promptly remove
from the water bath and allow to cool. Seal the cooled flask of solution
with parafilm and refrigerate for 24 hours. Remove from the
refrigerator and allow to reach room temperature, agitating
periodically. Filter once with standard laboratory filter paper to obtain
stock solution. Store in the refrigerator.
Mucicarmine working solution
Mucicarmine stock solution…… 10ml
Prepare just before use and use one time only.
Weigert’s Iron Haematoxylin
Solution A
Haematoxylin…… 10gm
Alcohol, 95%.... 1000ml
Solution B
Distilled water…. 475ml
Hydrochloric acid, concentrated…. 5ml
Ferric chloride, 29% solution…. 20ml
Working solution
Mix equal parts of solution A and B.
This solution may be used for 2 or 3 days.
0.25% Metanil Yellow solution
Metanil yellow… 0.25gm
Glacial acetic acid…. 0.25ml
Procedure
1. Cut paraffin sections at 4 to 5 micron thickness.
2. Deparaffinize sections and hydrate to distilled water.
3. Stain in working Weigert’s hematoxylin solution for 7
minutes.
4. Wash in running water for 10 minutes.
5. Stain in working mucicarmine solution for 60 minutes.
6. Rinse quickly and remove excess water before the next
step.
7. Stain in Metanil yellow solution for 30 seconds to 1 minute.
8. Dehydrate with three changes of 95% alcohol and three
changes of absolute alcohol.
9. Clear in Xylene and mount with synthetic resin.
Results
Mucin…… deep rose to red
Capsule of Cryptococcus…… deep rose to red
Nuclei…… black
Other tissue elements…… blue or yellow
Stain not as expected…
Carminophillic properties will be obscured if sections are
overstained with either hematoxylin or metanil yellow.
All mucins are stained…why?
Mucin is a term used to describe the intracellular secretions of a
variety of cells and although these secretions appear to be
microscopically similar they differ slightly in composition. Culling
et al list the following properties of mucin :(a)staining with basic
dyes, (b) metachromatic,(c) precipitated by acetic acid (except
gastric mucin) and (d) soluble in alkaline solutions.

Best stain for determining the presence or absence of
mucin…
According to Bancroft and Stevens, the combined alcian blue
and Pas technique is best to establish the presence or absence of
mucins with more certainty and also provide more information.
V. ALCIAN BLUE STAIN

A. ALCIAN BLUE STAIN PH 2.5
Purpose
The demonstration of acid mucopolysaccharides.
Principle
Alcian blue is a copper phthalocyanin basic dye that is water soluble
and colored blue because of its copper content. When used in a 3%
acetic acid solution (pH 2.5), alcian blue stains both Sulfated and
Carboxylated acid mucopolysaccharides and sulfated and
Carboxylated sialomucins (glycoproteins). It is believed to form salt
linkages with the acid groups of acid mucopolysaccharides.
Fixative
10% neutral buffered formalin or Bouin’s solution
Quality control
A section of small intestine, appendix or colon should be used as a
positive control.
Reagents
3% Acetic Acid solution
1% Alcian Blue Solution
Alcian blue 8GX….. 5g
Acetic acid, 3% solution….. 500ml
Adjust the pH to 2.5. Filter and add a few crystals of thymol.

Nuclear- Fast Red Solution
Nuclear- fast red ….. 0.5 g
Aluminum sulfate….. 25.0g
Dissolve the aluminum sulfate in the distilled water and then add the
nuclear fast red. Heat the solution until the nuclear fast red has
dissolved. Cool, filter; add a few grains of thymol as a preservative.
Procedure
1. Cut paraffin sections at 4 to 5 micron thickness
2. Deparaffinize and hydrate sections to distilled water.
3. Place slides in 3%acetic acid solution for 3 minutes.
4. Place slides in alcian blue solution for 30 minutes.
5. Wash slides in running tap water for 10 minutes.
6. Rinse in distilled water.
7. Counterstain in nuclear fast red solution for 5 minutes
8. Wash in running tap water for at least 1 minute.
9. Dehydrate in two changes each of 95% alcohol and
absolute alcohol and clear in Xylene.
10. Mounting.
Results
Weakly acidic sulfated mucosubstances,
hyaluronic acid and sialomucins….. dark blue
Background….. pink to red
B. ALCIAN BLUE STAIN PH 1.0

Purpose
The demonstration of sulfated mucosubstances
Principle
When used in a 0.1 N hydrochloric acid solution (pH1.0), alcian blue
stains only sulfated acid mucopolysaccharides [skin, cartilage, blood
vessels, cornea and lung] and sulfated sialomucins [mucins of the
colon]. Acid mucopolysaccharides and sialomucins that are only
Carboxylated will not be stained.
Fixative
10% neutral buffered formalin or Bouin’s solution.
Quality control
A section of appendix or colon should be used as a positive control
Reagents
0.1 N Hydrochloric acid solution
Hydrochloric acid , concentrated…. 8.2 ml
Distilled water ….. 991.8ml
1% Alcian Blue solution pH 1.0
Alcian blue, 8GX….. 3.0g
Hydrochloric acid 0.1 N….. 300 ml
Nuclear –fast Red Solution
Nuclear- fast red ….. 0.5 g
Aluminum sulfate….. 25.0g
Dissolve the aluminum sulfate in the distilled water and then add the
nuclear fast red. Heat the solution until the nuclear fast red has
dissolved. Cool, filter; add a few grains of thymol as a preservative.
Procedure
2. Deparaffinize and hydrate sections to distilled water.
3. Stain in 1% alcian blue in 0.1 N hydrochloric acid for 30
minutes. Filter solution back into stock bottle.
4. Rinse sections briefly in 0.1 N hydrochloric acid.
5. Blot sections dry with fine filter paper. Do not wash in water
since this can change the pH and cause non specific staining
to occur.
6. Counterstain with nuclear fast red solution for 5 minutes.

7. Wash in distilled water.
8. Dehydrate in two changes each of 95 % and absolute
alcohol and clear in xylene.
9. Mounting.
Results
Sulfated mucosubstances….. pale blue
Background….. pink to red.
C. VARYING ELECTROLYTE CONCENTRATIONS
To detect the various fractions of acid mucosubstances Add
magnesium chloride in the Alcian blue solution and make the desired
molarities.
Molarity Magnesium Substances tested
Chloride
0.06M 1.20G Carboxylated and Weakly Sulfated
Mucosubstances
0.3M 6.10G Weakly & Strongly Sulfated
Mucosubstances
0.5M 10.15G Strongly Sulfated Mucosubstances
0.7M 14.20G Strongly Sulfated Mucosubstances
0.9M 18.30G Strongly Sulfated Mucosubstances,
Keratan Sulfate
The sections should be stained for atleast 4 hours; overnight
staining for about 12 hours give better results. Background staining
may occur.
Stain or staining solution not as expected….
If the dye is not readily soluble, or if it rapidly precipitates from the
salty solution, check the dye label. Do not use samples labelled Alcian
Blue 5G or 7G, as they precipitate easily from salty solutions; and
discard unstable dye batches, however they are labelled.

Tissue stains unexpectedly weakly….
This problem occurs when resin embedding used because it tends to
enclose the targeted material so occlusion occurs, section fail to stain.
Try cutting thinner resin sections or use paraffin sections.
Unexpected structures stain….
Check the alkalinity of the solution, and if the pH is altered, chance
the entire stock solution and prepare new solution.
VI. ALCIAN BLUE / PAS / HEMATOXYLIN STAIN

Purpose
To differentiate between neutral and acid mucosubstances
Principle
Acidic mucosubstances are stained by the Alcian blue technique and
neutral mucosubstances are stained by PAS reaction.
Fixative
10% neutral buffered formalin or Zenker’s solution
Quality control
Use a kidney or a mucin control, depending on the diagnostic tissue
to be stained.
Reagents
3% Acetic Acid Solution
Glacial acetic Acid ….. 3.0ml
Distilled water….. 100 ml
Alcian Blue, pH 2.5
Alcian Blue….. 5.0 g
Acetic acid, 3 %….. 500ml

Stock reducing Rinse
Sodium Metabisulfite….. 10g
Distilled water….. 100 ml
Working reducing rinse
Stock reducing rinse….. 2.5 ml
Prepare just before use.
Schiff Reagent
Distilled water….. 800.0ml
Basic Fuchsin….. 4.0gm
Sodium Metabisulfite….. 4.0gm
1N Hydrochloric Acid….. 80.0ml
Procedure
1. Cut the paraffin sections at 4 to 5 micron thickness
2. Deparaffinize sections and bring to water a s usual
3. Place sections in 3 % acetic acid for 1 minute
4. Stain sections in alcian blue for 30 minutes.
5. Wash sections in running tap water then rinse in distilled water.
6. Place sections in 0.5 % periodic acid for 10 minutes.
7. Wash slides in running tap water for 5 minutes then rinse in
distilled water.
8. Place sections in Schiff reagent for 10 minutes.
9. Place slides in reducing rinse for 5 minutes
10. Wash in running tap water for 10 minutes.
11. Stain sections with Harris’ hematoxylin containing acetic acid
(46 ml hematoxylin/ 2ml acetic acid) for 1minute

13. Dehydrate in two changes each of 95% and absolute alcohols,
clear in Xylene and mount the slide.
Results
Exclusively acid mucosubstances….. blue
Neutral polysaccharides….. magenta
Certain substances will be colored by both PAS
and alcian blue….. purple.
If Alcian Blue is not readily soluble or if it rapidly precipitates
from solution, discard dye batch.
If in paraffin sections structures expected to be alcianophilic do
not stain, try extending the hydration step during the de-waxing
process as some alcianophilic structures hydrate rather slowly.
After embedding in water miscible resins such as
glycolmethacylate, alcianophilic target materials enclosed within
the resin section may fail to stain due to occlusion. Try cutting
thinner resin sections or using paraffin sections.
1. High Alcian Blue background staining may due to
presence of either salt or dextrin impurities in the dye.
Try another batch of dye.
2. Routinely nonalcianophilic basophilic structures such as
cell nuclei, sometimes stain blue. The causes of this
include the following:
a) Over lengthy staining, check the Alcian Blue staining
time.
b) Pre-treatment with reagents that increase staining
rate, eg enzyme extractions or extremes of pH.
Check if such were used and if so be skeptical.

3. If structures expected to stain with PAS instead stain
with Alcian Blue check that the staining sequence used
was indeed Alcian Blue- PAS and not PAS- Alcian Blue
as periodate oxidation can generate alcianophilic
materials.
VII. APPLICABILITY OF CARBOHYDRATE STAIN
IDENTIFICATION OF CARBOHYDRATES BY USING
HISTOCHEMICAL STAINS*
AB - pH2.5
AB - pH1.0
AB - PAS
Substance
Reactive
Location
PAS - D
group
Type
PAS
I 1: 2 Glycol Glycogen Liver etc., + - - - -
COOH Hyaluronic CT’s & - - + - +AB
Acid Umblical cord
CS - A & C Cartilage, - - + + + AB
Cornea, BVs
II COOH & CS - B Skin, CT’s, - - + + + AB
OSO3H Aorta, Lung
Heparin Mast cells, - - + + + AB
Intima - aorta
OSO3H Keratosulfate Connective - - + + + AB
Tissues
1:2 Glycol Neutral Stomach, + + - - + PAS
Mucin Paneath cell
1:2 Glycol Sialomucin Sub Maxillary/ (+) (+) + - + AB
& Lingual Glands,
COOH Small intestine,
III
Upper - colonic crypts
1:2 Glycol Sulfated Colon (+) (+) + + + AB
& COOH Sialomucin
& OSO3H
IV 1:2 Glycol Glycolipid Cerebrosides + + - - + PAS

& Double
Bond

CS – Chondroitin Sulfate; COOH – Carboxylated; OSO3H – Sulfated;
CT’s – Connective Tissues’; BVs – Blood Vessels
(+) = Inconsistent positivity; + AB = Positive for Alcian Blue (targeted
area);
+PAS = Positive for PAS (targeted area)
Notes: Alcian Blue/PAS/ Haematoxylin stain is applied only to
differentiate neutral and acid mucosubstances.
*The Table was modified from a lecture by C.F.A. Culling to the
annual convention of the National Society of Histotechnology,
Washington, D.C., 1975. This was modified by the authors of this
book to give more applicability and stress on the Epithelial Mucins.
IDENTIFICATION OF MUCOSUBSTANCES BY USING
HISTOCHEMICAL STAINS
In routine histopathological practice to identify precisely the type of
mucosubstances being secreted by a particular type of epithelium or
even a neoplasm is important. As we discussed earlier in the book,
mucosubstances are chiefly sulphated and sometimes non sulfated in
types, whereas adenocarcinoma are known to secrete neutral
mucosubstances. The types of intestinal metaplasia in the stomach
which is associated with the secretion of sulfa-mucosubstances
predispose to malignancy. In the cervical epithelium, the presence of
sialic acid has been thought to increase the viscosity of mucus.
Sulfated acid mucosubstances can be identified from non – sulfated
acid mucosubstances by the following methods:
i) Gomori’s Aldehyde Fuchsin method
ii) Aluminium Sulphate method
iii) Alcian Blue Methylation method at 37°C
iv) High Iron Diamine method
Gomori’s Aldehyde Fuchsin Stain
The aldehyde fuchsin stain has a great affinity for sulfated
mucosubstances. The traditional method employs an initial oxidation
step followed by a variable time in a solution consisting of Basic
Fuchsin, concentrated HCl and Paraldehyde. The value of Aldehyde
Fuchsin in identifying mucins is greatly increased when Aldehyde
Fuchsin and Alcian blue are combined. This combined technique will
give colour separation of sulfated and non sulfated acid
mucosubstances. The differentiation depends on the greater
affinity of Aldehyde Fuchsins for sulfate groups than for
carboxyl groups as prior staining with aldehyde groups
blocks the sulfated mucosubstances and subsequently staining with
Alcian Blue demonstrates the carboxylated acid mucosubstances.
Reagents
Gomori’s Aldehyde Fuchsin Solution
Dissolve 1gm of Basic Fuchsin in 100ml of 60% alcohol; add
1ml of Conc. HCl and then 2ml of Paraldehyde (Use Fresh).
Allow to ripen by standing for atleast 2 days at room
temperature (Solution develop a blue color) and store at 4°C
Alcian Blue Solution
1% Alcian Blue in 3% glacial acetic acid.
Procedure
2. De-paraffin the sections and take down to 70% Alcohol.
3. Stain with Aldehyde Fuchsin for 20 minutes.
4. Rinse well in 70% Alcohol, then in water.
5. Stain with Alcian Blue solution for 5 minutes.
6. Wash the section,
7. Dehydrate [dipping into the high grades of alcohol], clear
[dipping in xylene] and mount [mounting the section using
natural like Canada Balsam or synthetic resin like DPX with
coverslip]
Results
Strongly sulfated mucins….. deep purple
Weakly sulfated mucins….. purple
Non - sulfated acid mucins….. blue
Phenylhydrazine – PAS Stain
Generally acid mucosubstances are Alcian blue positive, and neutral
mucosubstances are PAS positive and the combined AB – PAS reaction
detects both these substances. However, there are few acid
mucosubstances are also PAS positive. To identify these,
phenylhydrazine – PAS method is used. The Phenylhydrazine
condenses periodate treated aldehyde groups of neutral mucosubstances
thus blocking the subsequent reaction with Schiff reagent, the PAS
reactivity of the acid mucosubstances are not changed.
Reagents
1% Aqueous Periodic Acid
5% Aqueous Phenylhydrazine Hydrochloride [Phenylhydrazine
Hydrochloride – 5gm, Distilled Water – 95%]
Procedure
2. De – Paraffinize the sections and take down to distilled water
3. Treat all sections (test and controls) with periodic acid
solutions for 2 minutes
4. Wash well in distilled water
5. Treat the test and positive control sections with the
Phenylhydrazine solution for 1 hour (at room temperature)
and the negative control sections with distilled water for the
same period of time.
6. Wash well in distilled water, and then treat all sections with
Schiff’s reagent for 8 minutes.
7. Wash in running tap water for approximately 10 minutes
followed by nuclear staining with haematoxylin in the
conventional manner.
8. Dehydrate [dipping into the high grades of alcohol], clear
[dipping in xylene] and mount [mounting the section using
natural like Canada Balsam or synthetic resin like DPX with
coverslip]
Results
Neutral Mucins….. negative
Acid Mucins….. rose red or magenta
Enzyme Digestion Methods
There are chiefly three types of enzymes which are in common
use for mucosubstances identification they are i) Diastase, ii) Sialidase
or Neuraminidase, and iii) Hyaluronidase. The potential drawback to
the use of certain enzymes like sialidase and hyaluronidase they are
high cost, which prevents their routine use in the laboratory. Sialidase
or Neuraminidase extracted from vibrio cholerae. Hyaluronidase
extracted from the testes or from the cultures of bacteria like
staphylococci, streptococci, clostridium etc. Diastase extracted from
the Malt but best diastases are of human saliva. The digestion is
followed by staining with Alcian Blue to know whether or not digestion
has occurred. Should digestion be followed with the combined AB –
PAS reaction, it will be seen that the sialidase labile mucosubstances
lose their alcianophilic character and give a positive PAS reaction.
(i.e., they change their staining from blue to magenta.

A few sialic acid containing mucosubstances are not digested by
sialidase. In such substances, if sialidase digestion is preceded by
deacetylation the sialidase resistant sialo-mucosubstances are
rendered liable to the enzyme. Alternatively, sulphuric Acid hydrolysis
may be employed to remove both sialidase – sensitive and sialidase –
resistant forms of sialic acid mucosubstances.
The commonly used Hyaluronidase is bovine testicular in
origin which digests in addition to hyaluronic acid, Chondroitin Sulphates
A and C. the digestion procedure is followed by Alcian Blue staining.
The diastase digestion is useful both to the Alcian Blue – PAS
and Phenylhydrazine – PAS as digestion by diastase will exclude the
presence of non mucosubstances such as glycogen
Simplified Classification of Mucosubstances [Applicability based]
TYPES OF MUCOSUBSTANCES LOCATION
Neutral Mucosubstances Lining epithelium of Stomach,
Brunner’s gland of Duodenum
Acid Mucosubstances
[Sulfated & Carboxylated]
A) Strongly Sulfated Chondroitin sulfate, Keratan
sulfate, Heparan sulfate, Bronchial
serous glands, Minor fraction of
intestinal goblet cells.
B) Weakly Sulfated Usually Epithelial in orgin,
Colonic goblet cells.
C) Sulfated Sialo Prostatic Carcinoma,
Mucosbustances Malignant Synovioma
Acid Mucosubstances
[Carboxylated/Sialo] [Non-Sulfated]
A) Enzyme Labile Goblet cells of peripheral airways of
lungs, Intestine, Submandibular
Glands,
B) Enzyme Resistant Mucous glands of major bronchi,
Gastric Epithelium
Hyaluronic Acid
[Non - Sulfated]
A) Enzyme Labile Connective tissue, Synovial fluids of
joints, Pleural Malignant
Synovioma Mesotheliomas.
B) Enzyme Resistant Gastric pyloric glands.

Flow chart for the elucidation of the type of Mucin
MUCICARMINE

Pink
Neutral or Acid Mucins

AB - PAS

Blue GAF -Pink Neutral Mucin, Magenta

Acid AB - AS -Blue Glycogen,

PAS - D

Mucin AB - M -Blue Some Acid Mucin

HID/AB -Brown Blue
Sulfated Acid Mucin MagentaNeutral No color

Neutral Mucin Glycogen
AB - Molarity Acid Mucin

0.06M 0.3M 0.5 M 0.7 M 0.9 M
Blue Blue Blue Blue Blue No color

PAS - PH

Neutral
Magenta Mucin
Strongly Acid Mucin
Sulfated Mucin
Carboxylated Acid Mucin

Weakly & Strongly
Sialidase Digestion Hyaluronidase
Sulfated Mucin with AB Digestion with AB

Carboxylated & No color Blue No color
Sulfated Mucin Sialidase Sialidase Resistant Hyalu-
labile Sialomucin or Weakly ronic
sulfated mucin
Acid

Blue Acid Hydrolysis - AB No color

Weakly Sialidase Labile
Sulfated or Resistant
Mucin Sialomucin
AB: Alcian Blue / GAF: Gomori’s Aldehyde Fuchsin / AB – AS: Aluminium Sulphate /
AB – M: Alcian Blue Methylation / HID: High Iron Diamine.

VIII. FREQUENTLYASKED EXAM QUESTIONS
1. What are mucins?
Mucins are glycoproteins present or secreted by the epithelial cells
(Epithelial Mucins) If it contains 1:2 Glycol group is called neutral
mucins, 1:2 Glycol plus COOH is called Sialomucins and 1:2 Glycol
plus COOH & OSO3H is called Sulfa - Sialomucins. Neutral Mucins
are present in stomach, Paneth cell granules. Sialomucins
[carboxylated] are present in sub maxillary/ lingual gland, Small
intestines, upper part of the colonic crypts. Sulfa – Sialomucins
[sulfated and carboxylated] are present in colon.
2. What is the fixative for Mucins and Mucosubstances?
Generally carbohydrates are aqueous soluble, Formal – calcium,
acidic fixatives, Formaldehyde – Alcohol, Carnoy’s fixatives can give
better results. Over fixation in formalin tends to reduce the strength
of the PAS reaction in mucins owing to polymerisation, but this can
be reversed by treating hydrated sections with 0.2 NaOH for 10 to
15 minutes prior to staining- a procedure that almost invariably causes
detachment of the sections.
3. What is Schiff Reagent?
Basic Fuchsin is a mixture of three dyes of tri amino tri phenyl
methane type, viz., rosanilin, pararosaniline and magenta II. Over a
one and half centuries ago H. Schiff (1866) showed that aldehydes
restore the magenta color to fuchsin which has been decolorized
(Leucofuchsin) by sulfur dioxide. Leucofuchsin is colorless because
there is loss of quinoid structure i.e., the chromophore is absent.
Reoxidation (slowly by exposure to air and light will restore the quinoid
structure and color. This also occurs by aldehyde groups, if aldehyde
is added to the ‘Fuchsin – sulfurous acid compound’, reddish purple
color will be produced.
Basic Fuchsin + Sulfurous Acid Loss of quinoid structure

+
Aldehyde COLORLESS

Reddish Purple Color

4. What is PAS reaction?
Periodic acid oxidises compounds having free hydroxyl group: when
the OH groups are next to each other, e.g., 1:2 Glycol, the bond
between the neighbouring carbon atoms that carry the OH groups is
broken and a dialdehyde structure is produced, which will react
strongly with Schiff reagent and produces the reddish purple colour.
5. Which step is more important to produce the full color in
PAS staining?
After the Schiff reagent, washing in the running tap water is
important for the development of the full color. It causes the loss of
the bound sulfurous acid group which was attached to the basic
fuchsin, then aldehyde group reacts and the restoration of the quinoid
structure occurs which leads to visualisation of the full color.
6. What is the color of the Schiff reagent and how to store it?
Its colorless however it looks light amber in color, it should be
stored in the dark colored bottle, closed air tight in the refrigerator.
The stored should be free of formalin vapours.
7. How to check the quality of the Schiff reagent?
Place 10 ml of 37% to 40% of formaldehyde in a beaker. Add a
few drops of Schiff reagent. If the solution rapidly turns reddish purple,
it is good. If the reaction is delayed and the resultant color is a deep
8. What are the criteria to give a positive PAS reaction?
It depends on the i) substance must contain 1:2 Glycol groups, ii)
it must not diffuse away during fixation, iii) it must give an oxidised
product which is not diffusible and iv) sufficient concentration must
be present to give a detectable final color.
9. What is the best counterstain to demonstrate the fungus in
PAS staining?
Fast green can be used instead of Haematoxylin, which provides
good contrast background.
10. What happens when the tap water is chlorinated?
Chlorinated water causes oxidation of any non adsorbed Schiff
reagent to become basic fuchsin, so Metabisulfite rinse is more
important to remove any non adsorbed Schiff reagent during the
staining process.
11. Which is fixative is not recommended for PAS staining?
Glutaraldehyde is a dialdehyde and one aldehyde group may not
be involved in protein cross linking during fixation, but may be left
free to react with the Schiff reagent.
12. Why acid mucopolysaccharides are negative for PAS?
There are three reasons i) 1:2 Glycol group may be strongly
attached so it requires more time for oxidation with periodic acid. ii)
There is need of two aldehyde group after oxidation of periodic acid
to react with Schiff reagent. iii) In case of hyaluronic acid, 1-3 linkage
between glucuronic acid and N- acetyl – glucosamine prevents
oxidation so it is negative.
13. What are the other oxidising agents?
Oxidising agents other than periodic acid have also been used,
preceded by Schiff reagent (e.g., chromic acid in the Bauer – Feulgen
reaction and KMnO4 in the Casella reaction), but these other reagents
are stronger oxidizers than periodic acid and will oxidize many groups
beyond the reactive aldehyde stage.
14. What are the methods in demonstration of glycogen?
Enzyme digestion technique is commonly used by using the diastase
enzyme (PAS – D reaction). Other methods are iodine method
(Mahogany brown for glycogen and dark blue for starch) but it is not
specific, it stains amyloid & ceroids. The Best’s Carmine method,
which demonstrate glycogen in brilliant red again it is not specific, it
stains mucin and fibrin but in very much lighter shade.
15. What is blockade procedure?
Blocking of the positive reaction is called blockade procedure. In
blocking the PAS reaction; the color caused by the presence of 1:2
Glycol group in the original molecule. In order to prove that it is correct
assumption, it is necessary to change chemically the 1:2 Glycol groups
present (by blocking them) so they do not react with PAS reaction.
This can be done by acetylation, benazoylation, boration and
bromation. Most popular is the acetylation which blocks the 1:2 Glycol
and hydroxyamino groups preventing the oxidation by periodic acid.
The process can be restored back by deacetylation (saponification)
by treating with dilute alkali. Through this method it can be proved
that the PAS reaction is due to neutral polysaccharides or due to
other compounds such as sphingomyelin.
16. What is aldehyde blocking technique?
These are used to block the preformed aldehyde in tissue sections
and are used along with acetylation, to determine the PAS reaction is
caused by reactive group other than aldehydes or 1:2 Glycol groups
17. What is enzyme digestion technique?
By using the specific enzymes which digest the specific molecule
are used to confirm their presence of the molecule when the
corresponding stains become negative. Examples are diastase
digestion of glycogen (PAS), hyaluronidase digestion of hyaluronic
acid and Chondroitin sulfate A and C (Alcian Blue pH2.5), and sialidase
[Neuroaminidase] digestion of sialic acid (Alcian Blue pH2.5). Note:
the source of hyaluronidase from the testes and bacteria – gram
positive; the source of sialidase [Neuroaminidase] from vibrio cholerae.
18. What is the principle of PAS – D reaction and source of
Diastase?
Diastase the enzyme which digests the glycogen and the followed
stain by PAS must show negative reaction, thus it confirms the
presence of glycogen in the section. The source is commercially
available malt diastase and another source is human saliva. Best is
saliva because it has only á – amylase which has effect on
the glycogen not on the embedding media so the section doesn’t
float.
19. What is the fixative for PAS staining in blood smear?
The fixative for blood smear is methyl alcohol for 10 to 15 minutes.
20. What is metachromasia?
Most of the dyestuffs stain the tissue orthochromatically i.e., in
shades of their own dyestuffs color. Certain dyes, however, also stains
certain tissues in a color or hue that is quite different from that of the
stain itself. Examples, Thionine, New methylene blue, Azure, Toluidine
blue, Crystal violet, Methyl violet etc

21. What is the simple method for mucin staining?
Metachromatic staining, they are Feytrter’s enclosure Technique,
Southgate’s Mucicarmine, Mayer’s Mucihaematein, Lison’s Alcian
Blue – Chloranline Fast Red Stain.
22. What are the stains for amyloid?
The staining of amyloid includes Iodine staining, Metachromatic
staining, Congo red staining, Fluorescence (thioflavin T) staining and
Silver reduction method for amyloid.
23. What are the stains for fibrin?
Fibrin stains includes i) with H & E which has a high content of
basic aminoacids, appears as a homogeneous Eosinophilic substance
ii) it is stained blue to purple by Mallory’s PTAH iii) PAS – Positive
iv) Gram – Weigert stain, fibrin is strongly positive, staining blue black
to deep violet v) Picro – Mallory vi) Martius – scarlet – Blue , stains
fibrin red in color.
24. What are the stains for Acid mucopolysaccharides?
Alcian Blue pH 2.5, Alcian Blue – PAS, Colloidal iron, Iron diamine
methods
25. What are the stains for Mast cells?
Best results are attained by alcoholic fixatives; Stains are Giemsa,
Toluidine Blue, Acridine orange, Bismarck brown stain and
Haematoxylin – Neutral red.
26. What is the principle of Mucicarmine Staining?
This is primarily an empirical stain. Aluminum is believed to form
a chelation complex with the carmine; the resulting compound has a
net positive charge and attaches to the acid groups of mucin.
27. What is the principle of Alcian Blue pH 2.5?
Alcian blue is a copper phthalocyanin basic dye that is water soluble
and colored blue because of its copper content. When used in a 3%
acetic acid solution (pH 2.5), alcian blue stains both Sulfated and
Carboxylated acid mucopolysaccharides and sulfated and
Carboxylated sialomucins (glycoproteins). It is believed to form salt
linkages with the acid groups of acid mucopolysaccharides.

28. What is the principle of Alcian Blue pH1.0?
When used in a 0.1 N hydrochloric acid solution (pH1.0), alcian
blue stains only sulfated acid mucopolysaccharides [skin, cartilage,
blood vessels, cornea and lung] and sulfated sialomucins [mucins of
the colon]. Acid mucopolysaccharides and sialomucins that are only
Carboxylated will not be stained.
29. What is the principle of Alcian Blue pH2.5 – PAS –
Haematoxylin Staining?
Acidic mucosubstances are stained by the Alcian blue technique
and neutral mucosubstances are stained by PAS reaction.
30. How many types of Alcian Blue are there and what type
is used in the Alcian Blue staining?
Alcian Blue 5G, 7G, 8GS and 8GX. The 8G is used in histopathology
staining. These variations are due to presence of cationic substituents.
The 8GS (S – Standard) and 8GX (X – Extra) they contain the same
dye so anything can be used so remember it as 8G [contains four
cationic substituents at eight possible position]. The other dyes such
as 5G or 7G have lesser cationic substituents. [J E Scott (1972)
Histochemistry of Alcian Blue, the structure of the Alcian Blue 8GX.
Histochemie 30; 215 – 234.]
31. Why the combined stain is Alcian Blue – PAS but not PAS
– Alcian Blue?
Acid mucin and neutral mucin are clearly separated by this
technique, which is also useful as a routine demonstration technique
for the presence of any mucins. The rationale is that by first staining
all acid mucins with alcian blue, those acid mucins which are also
PAS positive will not react in the subsequent PAS reaction; only the
neutral mucins will. In this way, a good color distinction can be made
between acid and neutral moieties.
32. What is streaming artefact?
In the process of fixation and gradual penetration of fixative,
aggregates of glycogen are seen in certain areas of the cell giving a
stream like appearance called the “the streaming artefact”. This
usually cannot be avoided. The formol saline fixation proves adequate
although much has been written about ethyl alcohol as a fixative.
The latter usually causes severe artefactual cell shrinkage.

IX. PERIODIC ACID –
SILVER METHENAMINE STAIN (PASM)
Purpose
To demonstrate basement membrane in the kidney.
Principle
The polysaccharides present in the basement membrane and fungal
wall are oxidised to aldehyde by periodic acid, when reduces the
metallic silver to give black color.
Gomori utilized a silver solution to demonstrate aldehydes exposed
by periodate treatment, the method cannot however be used in a
Feulgen technique. While it is not generally recommended as a
substitute for Schiff reagent, it does not give very good results with
basement membranes (particularly in kidney) and fungi in tissue
sections. This modification in which the stock silver Methenamine
solution is diluted in equal parts with distilled water avoids the grossly
overstained sections which sometimes resulted with the original
method.
Reagents
1% Periodic Acid
Methenamine silver solution
Take 100ml of 3% Methenamine solution to it add 5ml of 5%
silver nitrate which turns to a white precipitate and clear
5% (Trisodium tetraborate) Borax
Borax …. 5gm
Incubating Solutions
To 25ml of Methenamine silver solution add 25 ml of distilled water
and 5 ml of borax solution. The solution is filtered in a clean coupling
jar and rinsed in distilled water and kept in the oven at 50 - 60°C.
Procedure
1) Dewax the sections with two changes of xylene and hydrate
the section with descending grades of alcohol
2) Flood the section in 1% Periodic acid for 15 minutes.
3) Wash thoroughly in distilled water.
4) Pre heated incubated solution for 1 to 3 hours.
5) Check microscopically at 5 minutes interval to see the
adequate staining of basement membrane
6) Remove the sections from silver solution and wash in distilled
7) Place in 3% sodium thiosulphate for 5 minutes, and then wash
in running water for 2 -3 minutes.
8) Sections may be counterstained if desired with hematoxylin
or 0.2% light green in 0.2% acetic acid.
9) Dehydrate, clear and mount
Results
P.A.S. positive structures
Basement membranes, Fungi, mucin, etc….. black- brown
Background….. according to
counterstain
Stain not as expected
Check the process of oxidation and the strength of the periodic
acid. It can be solved by giving more time for the oxidation
process.
Incubation is critical, in our laboratory we standardized it for 1 ½
hours but this has to done by careful checking of the sections
at 5 minutes interval.
Check the fixatives used, if mercuric fixatives are used remove
the mercury before starting the process.
If structures expected to stain with PASM, then do the routine
stain of PAS to know the constituents of the targeted area.

B. CONNECTIVE TISSUE STAINING
I. BASICS OF COLLAGEN STAINING

The connective tissue consists of collagen fibres which may be
either mature or immature, may be in the form of Reticulin, muscle,
elastic tissue, adipose tissue, bone and cartilage as also basement
membrane, fibrin and fibrinoid material which may form the basis of
newly formed connective tissue.
Collagen of both the mature and immature varieties stains varying
shades of pink with routine H & E methods. In certain instances
such as neoplasms admixed with an inflammatory exudates,
granulation tissue or collagen, it would be necessary to define the
collagenous nature of a pink substance that is found interlaces with
other cellular material. Most commonly used stains are van Gieson,
Masson’s Trichrome and Mallory Trichrome stains.
The most insensitive collagen stain is the van Gieson which stains
only the mature extruded collagen (does not stain Reticulin) and cannot
differentiate with great ease from such mature extruded collagen
and maturing collagen. This is particularly needed in the spindle cell
neoplasm like fibrous histiocytomas, fibrosarcomas and smooth muscle
neoplasms where the collagen is constantly maturing. In such
instances Mallory’s and Masson’s Trichrome stain is used. They stain
both the collagen and muscle especially smooth muscle.
Aniline dyes have strong affinity to adhere to collagen in acid
environment. This affinity is taken advantage of by using these dyes
in the various collagen stains. It should be remembered that when
such an acid requirement is necessary the nuclear counter stain,
Haematoxylin should be of the Iron i.e., Weigert’s Haematoxylin which
will not be affected by the acid environment. Common aniline dyes
are Aniline blue, Acid fuchsin, Methyl blue etc.
The acid most commonly used in a stain such as the van Gieson,
is picric acid which also produce a contrast colour for non collagenous
stained areas, these combination in the van Gieson is called as the
Picro Fuchsin. The other acids used are phosphomolybdic and
phosphotungstic acid for the Masson’s and Mallory’s Trichrome
respectively.
With this above introduction we will discuss about the collagen
staining in detail.
I.A. VAN GIESON’S PICRIC ACID – ACID FUCHSIN STAIN

Purpose
Although the van Gieson technique may be considered a primary
connective tissue stain, it is rarely used as such: however, it serves as
an excellent counterstain for other methods such as the Verhoeff
elastic technique ,referred to as in many institutions as the Verhoeff
– van Gieson stain.
Principle
In a strongly acidic solution, collagen is selectively stained by acid
fuchsin, an acid aniline dye. Picric acid provides the acidic pH
necessary and also acts as a stain for muscle and cytoplasm. The
low pH is very important, as selective staining of collagen will not
occur at higher pH levels. The addition of 0.25 ml of hydrochloric
acid to 100ml of van Gieson’s solution will sharpen the differentiation
between collagen and muscle. Saturated picric acid solutions are
important in the preparation of the stain, again for the selective staining
of collagen. If the picric acid solution is not saturated, collagen may
stain pale pink to pale orange, and collagen, cytoplasm and muscle
may all stain the same colour.
Fixation
Any well fixed tissue can be used.
Quality Control
Practically every tissue has an internal control, so no other control
sections are needed; however, if a control is desired, uterus, small
intestine, appendix or fallopian tube will provide good material.
REAGENTS
Weigert’s iron hematoxylin
Hematoxylin...... 10g Distilled water......475ml
95% alcohol......1000ml Conc. hydrochloric acid.......5ml
Ferric chloride,29% solution........20ml
Working solution: Mix equal parts of solutions A and B.

Acid fuchsin, 1%solution
Acid fuchsin….. 1g
Picric acid, saturated solution
Picric acid….. 13g
Stir the solution on the mechanical stirrer for several hours. Some
picric acid should remain undissolved in the bottom of the flask. The
solubility of picric acid is 1.23g/100ml water at 20p C. The amount
used may have to be adjusted, depending on whether or not water
has been added to the stock powder to insure that the water content
does not drop below at least 10%.
van Gieson’s solution
Acid fuchsin, 1%solution….. 5ml
Picric acid, saturated solution…. 95ml
Procedure
1) Cut paraffin sections at 4 to 5 µm.
2) Deparaffinise sections and hydrate to distilled water.
3) Stain sections with Weigert’s iron hematoxylin for 10 to 20
minutes. Sections should be over stained, as they will be
slightly decolourised by the picric acid.
4) Wash in running tap for 10 minutes
5) Stain sections in van Gieson’s stain for 5 minutes. Discard
solution.
6) Place slides in 95% alcohol
7) Dehydrate as usual, clear with xylene, and mount with a
synthetic mounting medium.
Results
Nuclei….. black
Collagen….. brilliant red
Muscle and cytoplasm….. yellow

Uneven staining of collagen fibers, with some areas of the sections
being satisfactory, may be due to stain not being sufficiently acidic.
Check the pH of the working solution.
If nuclei fail to stain, or are weakly colored, check that an iron
hematoxylin or Celestine Blue was used. If an alu-minum hematoxylin
was used, replace with one of the aforementioned. In any event,
ensure that nuclear stain-ing is intense before application of the picro-
red acid dye solution, since the picric acid acts as a differentiating
agent.
Non collagenous structures are stained in unusual manner, check if a
change in fixative has occurred, as such surprises may be fixative
effects.
If sections are redder or yellower than you anticipate, check the
fixative used. A coagulant fixative tends to pro-duce redder tones,
and a cross-linking fixative produces yellower colors.
If thick sections cut for neuroanatomical work are yellow-er than
anticipated, this may be a section-thickness artefact. Try longer
staining times, or staining in a heated dye-bath. ‘
If sections become redder during removal of excess stain and
dehydration, the small somewhat lipophilic picric acid may have been
selectively extracted by the rinse (if used) or the dehydration alcohols,
leaving an excess of the large hydrophilic red acid dye. Replace any
aqueous rinse step by blotting or rinsing in alcohol and/or short-en
dehydration times.
Specimens embedded in hydrophilic resins may show no red staining
in regions expected to be collagen-rich. This may be due to resin
excluding the larger dye. Try longer staining times, adding a little
ethanol to the staining bath as plasticizer, or staining in a heated dye-
bath.
If sections become yellower on storage, the red acid dye may have
faded. Acid Fuchsin is especially prone to do this. Keep out of direct
sunlight.

I. B. MASSON’S TRICHROME STAIN
[Modified Mallory: Masson 1929]
Purpose: they are frequently used to differentiate between collagen
and smooth muscle in tumours and to identify increases in collagenous
tissue in diseases such as cirrhosis of liver.
Principle: Trichrome stains are so named because of 3 dyes which
may or may not include the nuclear stain, are used. Sections are first
stained with acid dye such as Biebrich’s scarlet. All acidophilic tissue
elements such as cytoplasm, muscle and collagen will bind the acid
dyes. The sections are then treated with phosphotungstic and/or
phosphomolybdic acid. Because cytoplasm is much less permeable
than collagen, phosphotungstic and phosphomolybdic acids cause the
Biebrich’s scarlet to diffuse out of the collagen but not out of cytoplasm.
Phosphotungstic and phosphomolybdic acids have numerous acidic
groups that most likely act as a link between the decolourised collagen
and aniline blue, the collagen dye. Probably the pH of phosphotungstic
and phosphomolybdic acid solution also increases selective collagen
staining and aids in the diffusion or removal of Biebrich’s scarlet.
Fixative
Bouin’s solution is preferred but 10%neutral buffered formalin may
be used.
Quality control
Every tissue has an internal control so no other control is required. If
required uterus, small intestine, appendix or fallopian tube will provide
good material.
Reagents
Weigert’s iron hematoxylin
Hematoxylin........10g Distilled water ....... 475ml
95% alcohol......1000ml Conc. hydrochloric acid......... 5ml
Ferric chloride,29% solution........ 20ml
Working solution: Mix equal parts of solutions A and B.

Bouins solution
Picric acid, Saturated aqueous solution….. 75ml
Formaldehyde 37 to 40%….. 25 ml
Biebrich’s scarlet - acid fuchsin solution
Biebrich’s scarlet, 1%aqueous solution….. 360ml
Acid fuchsin solution, 1%aqueous solution….. 40ml
Phosphotungstic and Phosphomolybdic acid solution
Phosphotungstic Acid ….. 25g
Phosphomolybdic Acid….. 25g
Aniline blue solution
Aniline blue….. 25g
Distilled water….. 1,000ml
1% Acetic acid solution
Procedure
1. Cut the paraffin section at 4 to 5 micron thickness
2. Deparaffinize section and hydrate to distilled water.
3. Rinse well in distilled water.
4. Mordant the section in bouin’s solution for 1 hour at
56 °C.
5. Remove slides from oven, allow to cool, and wash in
running water until yellow colour disappears.

7. Stain section in Weigert’s haematoxylin for 10 minutes
10. Stain sections in Biebrich’s scarlet - acid fuchsin solution
for 2 minutes, if desired the solution may be saved for
one more run only.
12. Place the slides in phosphotungstic and phosphomolybdic
acid solution for 10 to 15 minutes. Discard this solution.
13. Stain sections in aniline blue solution for 5 minutes. If
desired the solution may be saved for one more run only.
15. Place slide in1% acetic acid solution for 3 to 5 minutes.
Discard this solution
16. Dehydrate with 95% and absolute alcohol, two changes
each.
17. Clear with two or three changes of xylene and mount
with synthetic resin.
Results
Nuclei….. black
Cytoplasm, keratin, muscle fibers red
Collagen and Mucus….. blue
Weak nuclear staining occurs for many reasons.
Understanding by ‘cytoplasmic’ dyes can arise for a vari-ety
of reasons:
i) Many cytoplasmic dyes are removed by overextend-ed
water washes, leaving the collagen dye in place, as typical
cytoplasmic dyes are smaller, and thus faster diffusing,

than typical collagen dyes. Check wash times and try
shortening them.
ii) Many cytoplasmic dyes can also be selectively washed
out, leaving the collagen dye behind, by overextended
alcoholic dehydration, as typical cyto-plasmic dyes are
more alcohol soluble than typical collagen dyes. Check
the dehydration time and try-shortening it.
iii) Uneven staining (e.g. where adjacent muscle fibers have
markedly different color intensities) can arise if
specimens were formalin fixed. If so, check that a Bouin’s
or picric acid or mercuric chloride mordant was applied
prior to staining.
Background staining of the embedding medium can occur when
staining tissues embedded in hydrophilic resins. Acid dyes of
moderate size (e.g. Aniline Blue, Fast Green, Light Green and
Methyl Blue) stain such resins, and are then difficult to
remove.
The color balance is fixative dependent. Fixatives increas-ing
rate of staining (e.g. Carnoy’s fluid) tend to produce overstaining
by the connective tissue stain, while fixatives decreasing rates
of staining (e.g. Zenker’s fluid) tend to limit staining by the
collagen stain to the connec-tive tissue elements.
Changes in color balance can also occur in microwave-
accelerated procedures, with a tendency for the collagen dye
to over stain in heated dye-baths. Reduce the temper-ature or
staining time of the second acid dyeing step.
I. C. FREQUENTLY ASKED EXAM QUESTIONS

1. Why iron haematoxylin is used in collagen staining?
An iron hematoxylin solution is used for nuclear staining in the
trichrome procedures because iron hematoxylin is more resistant
than aluminium hematoxylin to decolourisation in subsequent acidic
dye solutions

2. What is the reason for the weak staining of the collagen?
The sharp colour differentiation is not obtained between collagen
and muscle, check the preparation of the saturated picric acid
solution, as the acidic pH provided by this solution is very important,
because the binding of aniline dye i.e., acid fuchsin occurs with
collagen in the acidic medium
3. What is the principle of Masson’s Trichrome Stain?
In a strongly acidic solution, collagen is selectively stained by
acid fuchsin, an acid aniline dye. Picric acid provides the acidic
pH necessary and also acts as a stain for muscle and cytoplasm.
The low pH is very important, as selective staining of collagen
will not occur at higher pH levels. The addition of 0.25 ml of
hydrochloric acid to 100ml of van Gieson’s solution will sharpen
the differentiation between collagen and muscle. Saturated picric
acid solutions are important in the preparation of the stain, again
for the selective staining of collagen. If the picric acid solution is
not saturated, collagen may stain pale pink to pale orange, and
collagen, cytoplasm and muscle may all stain the same colour.
4. What are the uses of trichrome stain?
To differentiate collagen and smooth muscle in the tumour, to
see the extent of collagen in cirrhosis liver, used as routine staining
in muscular biopsy.\
5. What are the best fixatives for trichrome stain?
Zenker’s, Formol – Mercury, Bouin’s or Picro – Mercuric alcohol
are the most satisfactory fixatives. If formaldehyde fixatives are
used, treat the tissue with picric acid or mercuric chloride solution.
6. Which counterstain is best when collagen is abundant?
Light green and if the collagen is less, Aniline blue is best.
7. What is one step trichrome stain?
In this procedure a plasma stain and a connective tissue stain are
combined in a solution of phosphotungstic acid (PTA) to which
glacial acetic acid is added. PTA favours the red staining of the
muscle and cytoplasm, the tungsten ion taken up by collagen and
the connective tissue stain is subsequently bound to this complex

colouring the collagen green or blue depending on the counter
stain used. Result: Nuclei – Black; Muscle – Red; Collagen –
Blue or Green.
8. What is the difference between the van Gieson and the
Trichrome stain?
The van Gieson is a relatively simple single unit stain utilising
picro fuchsin as the single unit, staining collagen pink and all other
non collagenous substances yellow. This stain gives sharper
differentiation if small amounts of acid are added to stronger
fuchsin solutions
As opposed to the van Gieson stain the Masson’s stain uses a
combination of Biebrich’s Scarlet Acid Fuchsin. This combination
when used, stains both the collagen and muscle red. With the use
of acid solutions such as Phosphotungstic Acid and
Phosphomolybdic Acid either alone or in combination the collagen
is altered. With the further use of the counterstain which may be
light green or aniline blue, the collagen stains on intense green
(Light Green) or a shade of blue (Aniline Blue)
The preferred fixative for the van Gieson is formalin where as
for the Masson’s Trichrome a mercurial fixative (Bouin’s or
Zenker’s) is preferred although equally good results may be
obtained with formalin fixed tissues. (The section may or may
not be treated with mercury chloride)
9. What is the reason for decrease in red & blue staining of
Trichrome?
Decreased red staining indicates staining solution has been aged
or overused and should be discarded, if the blue staining of
connective tissue appears faded the sec has been
overdifferentiated in acetic acid solution. Pathologically, altered
collagen (burns)may lose its affinity for aniline blue and bind to
acid dye instead.
10. How to store the picric acid?
Picric acid containing less than 10% water become as explosive
so it is important to keep the solution without getting dehydrated
while handling the solutions should not get spilled in the oven and

then allowed to evaporate. For this reason the staining jar should
be placed in another container while in oven.
11. Why Iron haematoxylin (Weigert’s Haematoxylin) used
in collagen staining?
An iron hematoxylin is used for nuclear staining because it is
more resistant than aluminium hematoxylin to decolourization in
acidic dye solutions. And many authors say the Weigert’s
Haematoxylin should be prepared fresh except Carson, where
they found to be good for several days.
12. What are the other stains for collagen?
Lillie’s Biebrich Scarlet Picroaniline Blue, Mallory’s Aniline Blue
and Hemalum – Phloxine – Saffron Trichrome stain.
13. What is the other histochemical benefit of van Gieson
stain?
It is mostly used as counter stain in Verhoeff elastin stain.
II. BASICS OF ELASTIC STAINING

Elastic fibers are strongly eosinophilic. They are easily identifiable
when they are compactly arranges due to their refractivity e.g., arterial
elastic lamina. They are insoluble in organic and inorganic solvents
where as collagen are easily soluble. (Collagen is soluble in 2% acetic
acid). In Verhoeff elastin stain most preferred counterstain is van
Gieson.
The elastic tissue stains are more useful in vascular disease where
such abnormalities of elastic laminae such as splitting, reduplication
(hypertensive vascular disease) and breaks (active or old) occurs.
Their importance in renal biopsies is of value in the diagnosis of benign
and malignant nephrosclerosis, old episodes of transplant rejection
and renal polyarteritis. It plays a major role in the study of heart valves
and skin diseases. Among its usage in neoplasm are very minimal.
Many methods are used to demonstrate elastin; they are Verhoeff,
Orcein, Weigert’s Resorcin Fuchsin and Gomori’s Aldehyde
Fuchsin.

The Verhoeff’s procedure is overstaining with a combination of
iodine – ferric chloride – haematoxylin combination followed by ferric
chloride differentiation. The technique works after any fixation, which
is most consistent stain giving an intense black staining of the coarse
elastic fibers. This stain is not good for demonstrating the very thin
delicate fibers. The stained sections show little fading even after
many years.
The Weigert’s Resorcin Fuchsin method, the principle of staining
involved is that the presence of ferric salts, which act as oxidizers,
the elastic fibers stain with basic fuchsin to give a brown to purple
color. The results obtained with this method are good but the
preparation of the stain is not easy and time consuming and at times
may give variable results or even fail to act. The ferrous salts contained
in the ferric chloride may also interfere with the staining.
The Gomori’s Aldehyde Fuchsin stain has been used to
demonstrate elastic fibres a deep purple because the stain is difficult
to prepare and deteriorates rapidly with time, the method has following
out of favour.
Orcein is the naturally occurring vegetable dye which is now
synthesized and stains elastic fibers in an acidic solution. In the
Resorcin – Fuchsin, Aldehyde - Fuchsin and Orcein methods are
hydrogen ion bonding between the staining molecule and the substrate
may be responsible for the staining of elastic tissue.
II.A. VERHOEFF’S ELASTIC STAIN
Purpose
Elastic fibre techniques are used for the demonstration of pathologic
changes in elastic fibres. These include atrophy of the elastic tissue,
thinning or loss that may result from arteriosclerotic changes, and
reduplication, breaks, or splitting that may result from other vascular
diseases. The techniques also may be used to demonstrate normal
elastic tissue, as in the identification of veins and arteries, and to
determine whether or not the blood vessel have been invaded by
tumour.
Principle
The tissue is over stained with a soluble lake of hematoxylin -
ferric chloride iodine. Both ferric chloride and iodine serve as mordants,

but they also have an oxidising function that assist in converting
hematoxylin to hematin. The mechanism of dye binding is probably
by formation of hydrogen bonds, but the exact chemical groups
reacting with the hematoxylin have not been identified. This method
requires that the sections be over stained and then differentiated, so
it is regressive. Differentiation is achieved by using excess mordant,
or ferric chloride to break the tissue - mordant - dye complex. The
dye will be attracted to the larger amount of mordant in the
differentiating solution and will be removed from the tissue. The elastic
tissue has the strongest affinity for the iron – haematoxylin complex
and will retain the dye longer than the other tissue elements. This
allows other elements to be decolourised and the elastic fibres to
remain stained. Sodium thiosulphate is used to remove excess iodine.
Van Gieson’s solution is the most commonly used counter stain, but
others may be used.
Fixative
Any well fixed tissue may be used.
Quality control
Use a section of aorta embedded on edge, or a cross section of a
large artery
Reagents
Lugol’s iodine
Iodine….. 10g
Potassium Iodide….. 20g
Put the iodine and potassium iodide in a flask with 200ml
of water. Stir on a mechanical stirrer until the iodine
dissolves and then add the remaining water.
10% ferric chloride
Ferric chloride….. 50g
store in the refrigerator
Verhoeff’s elastic stain
Prepare fresh each time and mix in order:
Hematoxylin , 5% in 95% alcohol

(may be kept as a stock solution)….. 30ml
Ferric chloride, 10% solution….. 12ml
Lugol’s iodine….. 12ml
van Gieson’s solution
Acid fuchsin, 1% aqueous….. 20 ml
Picric acid, saturated solution….. 380ml
5% Sodium Thiosulfate
Sodium thiosulfate….. 50g
Procedure
1) Cut paraffin sections at 4 to 5µm
2) Deparaffinise sections and hydrate to distilled water.
3) Place sections in Verhoeff’s elastic tissue stain for 1 hour.
4) Wash in two changes of distilled water.
5) Differentiate sections microscopically in 2% Ferric chloride
until the elastic fibres are distinct and the background is
colourless to light grey. If the sections are differentiated too
far, restain.
6) Rinse sections in distilled water.
7) Place sections in sodium thiosulfate for one minute.
8) Wash in running tap water for 5 minutes.
9) Counter stain sections in van Gieson’s stain for 1 minute.
10) Differentiate in 95% alcohol.
11) Dehydrate in absolute alcohol, clear in xylene and mount with
synthetic resin.
Results
Elastic fibres….. blue black to black
Nuclei….. blue to black
Collagen….. red
Other tissue elements….. yellow

Over differentiation may lead to stain the tissue weakly
because this is regression procedure, so again restain it and
repeat the process. But over overdifferentiated sections can
be restained at any step if they have not treated with alcohol
Do not prolong staining with Van Gieson’s solution as picric
acid also will differentiate the stain further.
The time of differentiation is somewhat dependent on the
amount of elastic tissue present
The preparation of Van Gieson’s solution is critical for proper
differentiation of muscle and collagen. If the picric acid is not
saturated, collagen will not stain red, and cytoplasm, muscle, and
collagen may all stain the same colour.
To prepare the Verhoeff’s elastic staining solution the reagents
must be added in the order given, with mixing after each addition,
or poor staining may result.
II.B. FREQUENTLY ASKED EXAM QUESTIONS
1. What is the principle of Verhoeff’s Stain?
The tissue is over stained with a soluble lake of hematoxylin -
ferric chloride iodine. Both ferric chloride and iodine serve as
mordants, but they also have an oxidising function that assist in
converting hematoxylin to hematin. This method requires that
the sections be over stained and then differentiated, so it is
regressive. Differentiation is achieved by using excess mordant,
or ferric chloride to break the tissue - mordant - dye complex.
The dye will be attracted to the larger amount of mordant in the
differentiating solution and will be removed from the tissue. The
elastic tissue has the strongest affinity for the iron – haematoxylin
complex and will retain the dye longer than the other tissue
elements. This allows other elements to be decolourised and the
elastic fibres to remain stained.

2. What are the uses of elastin stain in neoplasm?
The use of elastin stain in neoplasm is minimal however it can be
useful in demonstrating the blood vessel invasion by tumour and
sometimes useful in breast neoplasm.
3. Is elastin emitting fluorescence?
Elastin is isoelectric and gives yellowish (blue if unstained)
fluorescence in UV light.
4. What is the control tissue can be taken?
The control tissue of aorta, lung and old aged person skin (wrinkled
skin) from the autopsy.
5. What are the other elastin stains?
Orcein, Weigert’s Resorcin Fuchsin and Gomori’s Aldehyde
Fuchsin.
6. Which stain is useful for demonstrating fine elastin
fibers?
Gomori’s Aldehyde Fuchsin, the sharpness and intensity can be
increased by prior oxidation with periodic acid or peracetic acid
or potassium permanganate. [Verhoeff’s elastin stain is not good
for fine elastin fibers]
7. How to improve the sharpness and intensity of Verhoeff’s
staining?
Pre treatment with 1% KMnO4 for 5 minutes, followed by oxalic
acid, improves sharpness and intensity of staining.
8. What is the best counter stain for Verhoeff’s stain?
Van Gieson’s Stain because it also demonstrates the collagen
and gives a good contrast staining.
9. What is the best counter stain for Aldehyde Fuchsin
Elastic stain?
Definitely van Gieson’s stain is not recommended because the
collagen staining of the van Gieson’s stain makes it difficult to

identify the fine elastic fibers, so any nuclear stain is
recommended.
III. BASICS OF RETICULIN STAINING

Reticulin is procollagen. It is finer than collagen, stains black with
Reticulin stain and is unstained with collagen stain. Collagen fibers
on the other hand are coarse, doubly retractile, stain red with a collagen
stain like van Gieson and yellow or brown on silver impregnation.
Reticulum and collagen are basically similar and though there may
be chemical differences in the amino acid content of collagen and
Reticulin, most of the observed differences between the two
substances may be the result of differences in the physical
arrangements of molecules and the presence of additional binding or
cementing substances in collagen such as a mucopolysaccharides
resembling hyaluronic acid.
Three methods are available for the demonstration of the reticulin,
i) silver impregnation ii) gold method and iii) PAS technique.
The last method depends on the carbohydrate content of reticulin
and it is widely held, the silver methods depend on the same factor
i.e., the local reduction and selective precipitation of silver by the
aldehydic groups of the carbohydrate of reticulin.
Many methods of silver impregnation of reticulin exists of these,
one method of Foot and the methods of Bielschowsky – Maresch,
Perdrau da fano, Wilder, Gomori and Lillie all depend on silver oxide
or hydroxide in ammonical solution. The del Rio – Hortega, Foot and
Laidlaw variants use ammonical solution of silver carbonate. Most
are produced by precipitate from silver nitrate with sodium, potassium
or ammonium hydroxide or with lithium or sodium carbonate. The
type of reaction involved in the preparation of the silver solution may
be exemplified by Laidlaw’s method:
2AgNO3 + Li2CO3 = Ag2CO3 + 2LiNO3
The lithium nitrate is then removed by washing and the precipitated
silver carbonate is dissolved with ammonia water:
Ag2CO3 + 4NH3 = [Ag (NH3)2]2CO3
[Ammonium silver carbonate]
The essential reactions in the impregnation are believed to be:
1. The aldehydic groups of reticulum reduce the colorless silver
complex (mixed solution and colloid) to a dark brown lower
oxide, which is precipitated in particulate form on the reticulin
fibers.
2. This silver oxide is reduced to black metallic silver by formalin.
Sodium sulfite and hydroquinone will also accomplish this
but formalin is used in the majority of methods.
3. The unreduced silver is removed by solution in sodium
thiosulfate. Various refinements are employed in different
methods:
a) Many methods recommend some form of pre-silvering.
All of these pre treatments involve oxidation which
includes KMnO4, 10% Phosphomolybdic acid, acidified
KMnO4 [Gordon & Sweet’s], 4% chromic acid, and 0.5%
periodic acid for 15 minutes. Many of the ammonia silver
methods employ ‘sensitizers’ example uranium or silver
nitrate, ferric chloride or iron alum. These substances
act as oxidants and many others recommended other
substances like H2O2, NaIO3 and K2Cr2O7
b) Toning with gold chloride. This is a very valuable step. If
the sections are untonned after silver impregnation, the
background will be yellowish because of colloidal metallic
silver. Toning removes the silver and replaces it with gold
chloride. This in turn reduced to metallic gold by
Metabisulfite thus giving a very pale grey background
which is better for photography and also improves
subsequent counterstaining.
As regards fixation of tissues for silver impregnation, most methods
specify formalin, but other fixatives may be employed. The high
alkalinity of the silver solution tends to detach the section from the
slides this may be overcome by 1) Fixing the section well to an
albuminized slide by thorough drying, 2) Celluloidinizing the mounted
sections, this is done after removal of the wax by transferring the
slides from absolute alcohol to 1% celluloidin where it is allowed to
soak for 5 minutes. The excess celluloidin is then drained of and the

celluloidin film is hardened by immersing for 5 minutes in 80% alcohol
followed by water. 3) carrying out the steps of the procedure by
floating the paraffin section on the solution, the section being mounted
on the completion of the staining procedure, drained dry, ‘Baked’ by
placing in the paraffin oven for 10 minutes, cleared in xylene and
mounted.
Silver impregnations are notoriously capricious, painstaking
technique will ensure consistent results. In particular all glassware
use especially before and for the silver bath and for the preparation
of the silver solution must be thoroughly washed with 10% nitric acid
solution and then rinsed with several changes of distilled water. Some
workers including Culling prefer to use silvered flask which has been
well rinsed in distilled water. Over a period of years such a flask will
become completely coated with metallic silver and look filthy, however
it will found to give good results with even the most capricious silver
technique similarly whenever indicated the particular method, washing
of the sections with distilled water must be thorough especially prior
to impregnation. In particular, dust must be avoided since it is the
greatest single factor in the deterioration and precipitation of silver
solution. Precipitate of silver is also more likely to occur with the
more concentrated silver solution example Laidlaw’s. Silver precipitate
is less likely to form in the Gordon & Sweet’s and Gomori’s method,
but improper procedure will lead to precipitate formation in almost
any method. Splashing, or the use of glass rods without careful washing
between solutions, must be avoided. Glass distilled water should be
used.
Explosive hazard: In the preparation of the commonly used silver
impregnation solution various chemical reactions occur. With aging
or exposure of ammonical silver solution to air or light, shiny black
crystals of explosive silver compounds e.g., ‘fulminating silver’, silver
nitride and silver azide are formed. Violent explosion may occur while
removing the stopper, throwing the solution down a sink or even holding
it up to light. In order to avoid this; a) All ammonical silver solutions
should be prepared fresh just before use. b) Any used solutions should
be inactivated by adding excess of sodium chloride solution or dilute
hydrochloric acid.

III.A. GOMORI’S STAIN FOR RETICULAR FIBERS
Purpose
The demonstration of reticular fibers in tissue sections can be
important in the differential diagnosis of certain types of tumors. A
change from the normal reticular fiber pattern, as is seen in some
liver diseases, is also an important diagnostic finding.
Principle
The tissue is first oxidized by potassium permanganate to enhance
subsequent staining of the reticular fibers, and the excess potassium
permanganate is removed by potassium Metabisulfite. Ferric
ammonium sulfate acts as the sensitizer and is subsequently replaced
by silver from the diamino silver solution. Following impregnation,
formalin is used to reduce the silver to its visible metallic form. Toning
with gold chloride and followed by removal of unreacted silver with
sodium thiosulfate. The final step is to counterstain if desired.
Fixative
10% neutral buffered formalin is preferred.
[Nonmetallic forceps, chemically clean glassware (Coplin jars,
graduated cylinders, Erlenmeyer flasks, and pipettes), filter paper
should be used]
Quality Control
Liver is a very good control tissue.
Reagents
10% Silver Nitrate Solution
Silver nitrate ….. 10.0 g
Distilled water ….. 100.0 ml -
10% Potassium Hydroxide Solution
Potassium hydroxide ….. 10.0 g
Distilled water ….. 100.0 ml

Ammonical Silver Solution
Combine 20 ml of 10% silver nitrate solution and 4.0 to 5.0
ml of 10% aqueous solution of potassium hydroxide. Add
concentrated ammonium hydroxide, drop by drop, while
shaking the container continuously, until the precipitate is
completely dissolved. Cautiously add 10% silver nitrate
solution, drop by drop, until one drop causes the solution to
become permanently cloudy. Only a faint cloudiness is
desirable. Measure the resulting solution, dilute with an equal
amount of distilled water, and filter into a chemically clean
Coplin jar.
0.5% Potassium Permanganate Solution
Potassium permanganate….. 2.5 g
2% Potassium Metabisulfite Solution
Potassium metabisulfite ….. 10.0 g
2% Ferric Ammonium Sulfate Solution
Ferric ammonium sulfate ..... 10.0 g
Distilled water ..... 500.0 ml
Formalin Solution
Formaldehyde, 37% to 40% ..... 10.0 ml
0.2% Gold Chloride Solution
Stock gold chloride solution (1%) …….. 10.0 ml
2% Sodium Thiosulfate Solution
Sodium thiosulfate . ... 10.0 g

Nuclear-Fast Red Solution
Nuclear-fast red ….. 0.5 g
Aluminum sulfate ….. 25.0 g
Dissolve the aluminum sulfate in the distilled water, and then
dissolve the nuclear fast red in this solution, using heat. Cool,
filter, and add a few grains of thymol as a preservative.
Procedure
1. Cut paraffin sections at 4 to 5 microns thickness
2. Deparaffinize sections and hydrate to distilled water.
3. Oxidize sections in 0.5% potassium permanganate solution
for 1 minute.
4. Rinse in tap water for 2 minutes.
5. Differentiate in 2% potassium metabisulfite for 1 minute.
6. Wash in tap water for 2 minutes.
7. Sensitize sections in 2% ferric ammonium sulfate for 1 minute.
8. Wash slides in tap water for 2 minutes followed with two
changes of distilled water for 30 seconds each.
9. Impregnate sections with the silver solution for 1 minute.
10. Rinse in distilled water for 20 seconds.
11. Reduce for 3 minutes in the formalin solution.
13. Tone in 0.2% gold chloride solution for 10 minutes.
15. Place sections in 2%, potassium metabisulfite for 1 minute.
16. Fix in 2% sodium thiosulfate for 1 minute.
18. Counterstained if desired with nuclear-last red for 5 minutes.
Generally, the counter stain is net applied to liver sections
but is applied to all other sections. Wash well in tap water.

19. Dehydrate in 95% and absolute alcohols. Clear in xylene
and mount with synthetic resin.
Results
Reticulin …… black
III.B. GORDON AND SWEETS STAIN FOR RETICULAR

FIBERS
Purpose
The demonstration of reticular fibers in tissue sections can be
important in the differential diagnosis of certain types of tumor. A
change from the normal reticular fiber pattern, as is seen in some
liver diseases, is also an important diagnostic finding.
Principle
The tissue is first oxidized by potassium permanganate to enhance
subsequent staining of the reticular fibers, and excess permanganate
is removed by oxalic acid. Ferric ammonium sulfate acts as the
sensitizer and is subsequently replaced by silver from the diamino
silver solution. After impregnation, formalin is used to reduce the
silver to its visible metallic from. Before toning with gold chloride,
unreacted silver is removed with sodium thiosulfate. The final step is
to counter stain if desired.
Fixative
10% neutral buffered formalin is preferred,
[Nonmetallic forceps, chemically clean glassware (Coplin jars,
graduated cylinders, Erlenmeyer flasks, and pipettes), filter paper]
Quality Control
Liver is a very good control tissue.
Reagents
10% Silver Nitrate
Silver nitrate ….. 10.0 g
Distilled water….. 100.0 ml

3% Sodium Hydroxide
Sodium hydroxide ….. 3g
Ammonical Silver Solution
Place 5ml of 10% silver nitrate solution in an Erlenmeyer
flask and add concentrated ammonium hydroxide, drop by
drop, while shaking the container continuously, until the
precipitate that forms is completely dissolved. Do not add
any excess ammonium hydroxide. Add 5 ml of 3% sodium
hydroxide solution and cautiously redissolve the precipitate
until only a faint cloudiness remains. If this step is carried
too far and no cloudiness remains, add 10% silver nitrate
solution, drop by drop, until one drop causes the solution to
become permanently cloudy. Only a faint cloudiness is
desirable. Dilute the resulting solution to 50 ml with distilled
water, and filter into a chemically clean coplin jar.
1 % Potassium Permanganate Solution
Potassium permanganate ….. 1.0 g
1 % Oxalic Acid Solution
Oxalic acid ..... 1.0 g
2.5% Ferric Ammonium Sulfate Solution
Ferric ammonium sulfate ..... 12.5 g
Formalin Solution
Formaldehyde, 37% to 40% ..... 10.0 ml
0.2% Gold Chloride Solution
Stock gold chloride solution (1 %) ..... 10.0 ml
Sodium thiosulfate ….. 25.0 g
Nuclear-Fast Red Solution
Nuclear-fast red ….. 0.5 g
Aluminum sulfate ….. 25.0 g
Distilled water. ... 500.0 ml
Dissolve the aluminum sulfate in the distilled water, and then
dissolve the nuclear-fast red in this solution, using heat. Cool,
filter, and add a few grains of thymol as a preservative.
Procedure
1. Cut paraffin-sections at 4 to 5 microns thickness
2. Deparaffinize sections and hydrate to distilled later.
3. Oxidize sections in 1.0% potassium permanganate
solution for 5 minutes.
4. Rinse in tap water for 2 minutes.
5. Bleach in 1 % oxalic acid for 2 minutes, or until sections
arc colorless.
7. Sensitize sections in 2.5% ferric ammonium sulfate for
at least 15 minutes.
8. Wash in several changes of distilled water.
9. Impregnate sections with the silver solution for 2 minutes.
10. Rinse well with distilled water.
11. Reduce sections for 2 minutes in the formalin solution.
13. Tone in 0.2% gold chloride solution for 10 minutes.
15. Place slides in 5% sodium thiosulfate for 1 minute.

17. Counterstain, if desired, with nuclear-fast red for 5
minutes. Generally liver sections are not counterstained
and all other sections are. Wash well in tap water.
18. Dehydrate in two changes each of 95% and absolute
alcohols, clear in xylene, and mount with synthetic resin.
Results
Reticulin..... black
Collagen……….. taupe [dusky brownish grey color]
The silver impregnation solution may contain either excess or
insufficient ammonia. Carry out solution prepa-ration with care
and, if possible, ask the advice of an experienced worker.
Ammonia is constantly lost from the impregnation solu-tion by
evaporation, eventually giving unstable solutions with poor staining
properties. Avoid methods that do not use ammonia in excess.
Stain in a Coplin jar to reduce evaporation.
The polysaccharide, which gives rise to the staining, is water
soluble and can be lost prior to silver reduction. If unfixed
cryosections were used, try fixing them prior to staining; if an
aqueous fixative was used, try a non aqueous; if a microwave-
accelerated variant was used, try reducing pre impregnation
temperatures.
To increase staining intensities, try increasing the silver
concentration or the temperature of the impregnation step.
(Beware: High background staining may result)
Non selective staining of other tissue elements can occur if the
alkalinity of the silver impregnation solution falls below pH 11.
Check that ammonia is not lost.

If cell nuclei stain over intensely, try reducing the time in the
alum mordant solution.
When hydrophilic resin embedding media are used, silver can be
retained in the embedding medium, resulting in strong background
staining. This is sometimes pronounced when using microwave-
accelerated variants. Try extending the pre incubation rinse: but
beware that too much silver is not lost, so reducing sensitivity.
III. C. FREQUENTLY ASKED EXAM QUESTIONS
1. What is the principle of silver impregnation?
The aldehyde groups of the carbohydrate of reticulin fibers reduce
the colourless silver complex to a dark brown lower oxide of
silver which is precipitated in particulate form on reticulin fibers
and subsequently reduced to black silver by formalin.
2. What are the uses of reticulin stain as an aid in diagnosis?
Reticulin stains are often useful in diagnosing
hemangiopericytomas and in differentiating these from
hemangioendotheliomas. In the hemangiopericytomas, the
pericytes are seen external to the basement membrane of the
vascular channels. Both glomangioma and hemangiosarcoma cells
tend to be separated from each other by fine reticulin, but former
are external to, and the latter within the reticular sheaths of the
vessels.
In paragangliomas, reticulin stains reveals the cell nests to
advantage.
Some tumours of Lymphnode produce more reticulin e.g.,
reticulum cell sarcoma, Hodgkin’s disease.
A negative finding such as absence of reticulin is seen in the
Ewing’s sarcoma of the bone help in arriving at diagnosis.
The thymus whish has very little amount of reticulin, in myasthenia
gravis, the germinal centres shows reticulin fibers arising from
the cortex.
A few tumours of the CNS [According to the Dr. Lynch’s opinion,
exclusively arising from the mesodermal origin] show abundant

reticulin e.g., microgliomas, certain meningeal tumours,
hemangiopericytomas, CNS sarcomas. Reticulin stain readily
differentiates the latter from the medulloblastoma which has only
strands of reticulin associated with vasculature.
In liver it is very helpful in early cirrhosis.
In kidney, the lesions of diabetic glomerulosclerosis display
laminated argyrophilia, those of chronic lobular glomerulonephritis
show tangles skein of reticulum, and amyloid glomerular lesions
are colored a diffuse pale gray with silver reticulum stains.
In bone marrow, it is very helpful in diagnosing the myelofibrosis,
and fibrosis due to cancer therapies.
3. What stains are beneficial in Small round cell tumours?
Especially PAS, Reticulin and GMS for small round cell tumours.
But still it is not conclusive because PAS is positive in Ewing’s,
Certain lymphomas, Rhabdomyosarcoma, sometimes non specific
reaction of PAS occurs in Neuroblastomas. GMS gives positive
reaction in argentaffin cells it help in the neuroendocrine tumours
and carcinoids, even non appendicular carcinoids may not express
argentaffin reaction.
[Note: The applicability of histochemical stains in tumour diagnosis
was more helpful during 19th century, but the revolutionary
development of Immunohistochemical profiles made the
applicability of histochemical stains for tumour diagnosis
questionable. For example the newer definition for Heman-
giopericytoma - the tumour should have stag horn pattern,
with reticulin surrounding the individual cells everywhere
and that is negative for muscle, nerve sheath and epithelial
markers but positive for CD34 and CD99. However still, a
good reproducibility can be provided through the mucin stains.
But still, the histochemical stains remain gold standard for many
non tumour diseases. For example renal disorders]
4. What is toning?
Toning removes the silver and replaces it with gold chloride. This
in turn reduced to metallic gold by Metabisulfite thus giving a
very pale grey background which is better for photography and
also improves subsequent counterstaining. Otherwise the
background will be yellowish because of colloidal metallic silver.
5. Which is the best method for reticulin?
The best method is Gordon and Sweet’s method because, rapid
impregnation, reversibility of the reaction, relatively slight tendency
to form precipitation, gives much less background and nuclear
staining.
6. When the section floats in reticulin stain?
When the silver solution is added, The high alkalinity of the silver
solution tends to detach the section from the slides this may be
overcome by 1) Fixing the section well to an albuminized slide by
thorough drying, 2) Celluloidinizing the mounted sections, this is
done after removal of the wax by transferring the slides from
absolute alcohol to 1% celluloidin where it is allowed to soak for
5 minutes. The excess celluloidin is then drained of and the
celluloidin film is hardened by immersing for 5 minutes in 80%
alcohol followed by water. 3) carrying out the steps of the
procedure by floating the paraffin section on the solution, the
section being mounted on the completion of the staining procedure,
drained dry, ‘Baked’ by placing in the paraffin oven for 10 minutes,
cleared in xylene and mounted.
7. How to overcome the hazardous effect of silver?
All ammonical silver solutions should be prepared fresh just before
use. Any used solutions should be inactivated by adding excess
of sodium chloride solution or dilute hydrochloric acid.
8. How to store the silver solution?
Store the impregnation reagent in a dark bottle, since there is
high chance for formation of silver azide and silver nitride which
are highly explosive in nature. so preferably store it in plastic
bottle rather glass bottle.
9. How to clean the glassware before and after the
procedure?
All glassware, especially that used in the preparation of silver
solutions, should be washed in 10% Nitric Acid and then washed
in several changes of distilled water

C. PIGMENT STAINING
I. BASICS OF PIGMENTS
Pigments could be of artefacts, endogeneous and exogeneous.
The common artefactual pigments are formalin, mercuric and chrome
pigments. The formalin pigments are dark brown, double refractile
pigments (acid formaldehyde hematein) produced by the interaction
of acidic formaldehyde solution and blood. There are various methods
to remove those pigments; i) treating the sections with the mixture of
70% ethyl alcohol and ammonia water for 5 minutes to 3 hours
depends on the amount of pigment [Kardasewitsch’s Method] ii)
after bringing down to water place the sections for 1 to 5 minutes in
a mixture of acetone, 3 vol hydrogen peroxide and 28% ammonia
water; this should be followed by washing in 70% alcohol and then in
running water [Lille’s Method] iii) place sections after bringing to
water in a saturated alcoholic solution of picric acid for 5minutes to 2
hours. Then wash for 10 to 15 minutes in running tap water.
The mercuric deposits are gray black granular deposits resulting
from the use of mercuric fixatives. Remove by treating sections with
alcoholic iodine followed by sodium thiosulfate. The chrome deposits
are brownish black granules which are the result of alcohol treatment
following chrome fixation; such pigment cannot be removed. Chrome
fixed tissues must be washed in running water for 12 to 18 hours
immediately following fixation. This washing removes excess
chromate from the tissue which can then safely be dehydrated in
alcohol.
Endogeneous pigments derived from haemoglobin includes
hemosiderin, hematoidin, bile pigments and some porphyrins; malarial
and schistosomal pigments. Pigments not derived from haemoglobin
include melanin, enterochromaffin, lipofuscins, hemofuscins, ceroids,
and lipochromes. Endogeneous deposits include calcium, urates,
oxalates, cystines alcoholic hyaline in liver etc. Exogeneous pigments
include carbon dust, silica, asbestos, therapeutic agents include gold,
silver etc.
In this book we will be dealing only with the hemosiderin related
pigments and the details of other pigments are beyond the scope of

this book. However we try to cover certain aspects in the Frequently
Asked Questions section but it only the tip of the iceberg.
II. BASICS OF HEMATOGENEOUS PIGMENTS

Haemoglobin is a conjugated protein that is found normally in red
blood cells and that is responsible for transporting oxygen from the
lungs to other parts of the body. Haemoglobin may be found
pathologically in areas of recent haemorrhage or in renal tubules after
excessive haemolysis. Haemoglobin stains vividly with acid (anionic)
dyes such as eosin.
Erythrocytes have a normal life span of about 120 days; after
circulating for that period, they are destroyed by splitting open
(Haemolysis) or by phagocytic cells (macrophages) in the spleen.
Haemoglobin breaks down into two parts: globin (protein that is
returned to amino acid pool) and heme (iron – containing). The
heme portion splits again into iron and a greenish bile pigment
(biliverdin)
Iron is conserved by the body for use in the production of new
haemoglobin. If the iron is not needed immediately, it is stored primarily
in the bone marrow and spleen as hemosiderin, a yellow to brown
pigment. Since much of the iron is needed for production of new
RBC’s, large deposits of hemosiderin are found only in pathologic
conditions. If the production and destruction of red cells are not
balanced (e.g., increased destruction in haemolytic anemia), there
may be increases deposition of hemosiderin in tissues.
Hemochromatosis, a disease caused by excessive absorption of dietary
iron, is also characterized by excessive hemosiderin deposits.
Hemosiderin is differentiated from other yellowish brown pigments
by Prussian blue reaction.
The bile pigment biliverdin also results from destruction of RBC’s
and further breakdown of the heme portion of haemoglobin. Biliverdin
is transported to the liver, where it undergoes reduction to bilirubin.
Bilirubin is not normally deposited in tissue but is removed by circulation
by the liver and then secreted as one of the constituents of bile. An
obstruction of the normal bile flow may cause abnormal accumulation
of the pigments in blood and may impart a yellowish coloration of the

skin, a condition known as jaundice. In obstructive jaundice the bile
pigment may be seen in the liver in bile canaliculi and also deposited
in the cytoplasm of both kupffer’s cells and hepatocytes. Bile is
demonstrated with techniques relying on the oxidation of bilirubin
(yellow brown) to biliverdin (green). Hematoidin is a pigment similar
to bilirubin and also oxidised to biliverdin by bile demonstrating
techniques. Hematoidin is formed in tissues as a result of haemorrhage
and reduced oxygen tension.
III. PRUSSIAN BLUE STAIN FOR FERRIC IRON

Purpose
The detection of ferric (Fe3+) iron in tissues. Ferric iron is normally
found in small amounts in the bone marrow & the spleen. Abnormally
large deposits may be seen in Hemochromatosis & hemosiderosis.
Principle
This method detects the ferric ion in loosely bound protein complexes
(as in hemosiderin). Iron that is strongly bound, as in haemoglobin,
will not react. In the Prussian blue reaction, sections are treated with
an acidic solution of potassium ferrocyanide & any ferric iron present
reacts to form an insoluble bright blue pigment called Prussian blue
HCl

3k4Fe(CN)6 + 4 Fe3+ Fe4[Fe(CN)6]3 + 12 k
[Prussian Blue]
Fixative
Alcohol or 10% neutral buffered formalin
Quality control
A section containing ferric iron must be used. Excessive amounts of
iron are not desirable in the control, as the reaction product is slightly
soluble & may contaminate the incubating solution, giving a
background stain in all sections. Coplin jars that have been used for
iron haematoxylin solutions & not adequately cleaned may also
contaminate the staining solution.

Reagents
2% Potassium Ferrocyanide
Pottassium Ferrocyanide ….. 10g
Distilled Water ….. 500ml
2% Hydrochloric Acid Solution
Hydrochloric Acid , Concentrated ….. 10ml
Nuclear – Fast Red Solution
Nuclear- Fast Red ….. 0.5g
Aluminium Sulfate ….. 25g
Dissolve the aluminium sulfate in the distilled water, & then dissolve
the nuclear – fast red in this solution, using heat. Cool, filter, & add a
few grams of thymol as a preservative
Procedures
1. Cut paraffin sections at 4-5 micron thickness
2. Deparaffinize & hydrate the section to distilled water.
Handle slides in the following steps with non metallic
forceps.
3. Place slides in a freshly prepared mixture of equal parts
of 2% Potassium Ferrocyanide & 2% Hydrochloric Acid
& heat for 20 minutes at 60 c
4. Wash sections thoroughly in several changes of distilled
water
5. Counterstain sections in Nuclear-Fast Red for 5 minutes
6. Rinse in running tap water for atleast 1 minute
7. Dehydrate sections in 95 % alcohol & two changes of
absolute alcohol
8. Clear in three changes of xylol & mount with synthetic
resin
Results
Nuclei & Hemofuchsin… bright red

Hemosiderin … blue
Background … pink
1. Whenever you are surprised by weak staining, or a fail-ure to
stain, look at your positive control as a check that there is nothing
wrong with the reagents or the procedure.
2. Low iron concentrations give pale staining. Try extending the
staining time. If, however, you extend staining times above 30
min, replace the working solution after that time.
3. Acidic fixation in acidic media may cause loss of iron. Try other
media in future work.
4. Failure to demonstrate the ferric iron of myoglobin or
haemoglobin, or in ferric oxide, can be due to the loss of the
highly water-soluble hydrated ferric iron from the specimen during
the pre treatments necessary to release the cation from its
complexed state. Explore shorter pre-treatment times.
5. It has been suggested that, since ferrocyanide ions diffuse slower
than hydrogen ions, acid-induced losses of iron can be reduced
by pretreating the section with potassium ferrocyanide solution
prior to staining with the acidified ferrocyanide reagent.
6. If the section was stained more than a year or two ago, the
staining may have faded. If permanent preparations are called
for, carry out a diaminobenzidine (DAB) stain (as in DAB
peroxidase procedures) after the ferrocyanide staining and before
any counterstain. This produces a brown, non fading DAB
polymer around the Prussian Blue deposits.
1. Iron contamination is one possible source of false-positives. This
can arise from the reagents, glassware or water. Check glass
washing procedures and the water source. When apply-ing a
group of stains to a liver biopsy always do the Perls’ stain first, in
order to avoid contamination with iron alum used in the reticulin
methods.

2. A finely granular blue deposit, throughout the section, can arise
with variants involving staining at elevated temperatures. Use a
low temperature method, if neces-sary with an extended staining
time.
3. Do not use a strongly staining control section, as some leaching
of the blue reaction product may occur and con-taminate the
test section, and false-positives may be seen.
Over Staining
If iron is abundant, the procedures suggested in staining manuals
may result in overstaining, obscuring surround-ing tissue elements.
Should this occur, reduce staining times.
IV. FREQUENTLYASKED EXAM QUESTIONS

1. How to differentiate various pigments?
PIGMENTS APPEA- NORMAL PATHO- BIREFRIN-
-RANCE SITES - LOGICAL -GENCE
IN SITES
SECTION
Formalin Dark Brown Blood containing tissues Positive
[acid formalde Black e.g., spleen
hyde Granules
haematin]
Mercury Brown All tissues Negative
Black
Haemoglobin Yellow RBC Renal Casts Negative
Brown
Droplets
Hemosiderin Yellow - Liver, Bone Negative
Brown Marrow Etc
Granules
Or Clumps
Hematoidin Yellow - Old Negative
Brown Haemorr-
Red -hages
Granules
Or Needles

PIGMENTS APPEA- NORMAL PATHO- BIREFRIN-
-RANCE SITES -LOGICAL -GENCE
IN SITES
SECTION
Haemazoin Dark Brown - Vascular & Positive
[Malaria To Black Reticuloendo
Pigment] Granules thelial
Argentaffin Pale Yellow Stomach, Si, Carcinoid Negative
Appendix Tumours
Melanin Yellow Eyes, Tumours, Negative
Brown Skin, Hair Freckles,
Black Addison’s
Granules Disease
Chromaffin Brown Adrenal Pheochro Negative
If Cortex mocytoma
Mordanted
Lipofuscins Yellow & Adrenals Heart, Liver, Negative
Brown Ganglion
Droplets Cells, Testes
2. What is Prussian blue reaction?

In the Prussian blue reaction, sections are treated with
an acidic solution of potassium ferrocyanide & any ferric iron
present reacts to form an insoluble bright blue pigment called
Prussian blue
3. What is the fixative used to study bone marrow biopsy?
Many methods are followed by different laboratories; the most
important thing is rapid acid decalcifying agents should not be
used because it will extract the iron. The best decalcifying agent
are EDTA chelating method or 1000 ml formic acid, 3250 ml
distilled water, and 250 ml formaldehyde. Some laboratories use
this combination; stain for iron on bone biopsy specimens that
are fixed overnight in Zenker’s solution containing 3% acetic
acid. Both fixation & decalcification are accomplished, no further
decalcification is needed & the iron is preserved.

4. What are the controls used?
Bone marrow biopsies and spleen.
5. What is the stain for the ferrous iron?
Turnbull’s blue stain for ferrous iron
6. How will you grade iron in bone marrow biopsies?
0 - No stainable iron
1+ - Small intracellular iron stores using oil objective
2+ - Small, sparse intracellular iron particles at low power
3+ - Numerous small intracellular iron particles
4+ - Larger particles with a tendency to aggregate into clumps
5+ - Dense, large clumps
6+ - Very large clumps and extracellular iron
7. What is Hemochromatosis?
The term haemosiderosis is generally used to indicate the
pathological effect of iron accumulation in any given organ, which
mainly occurs in the form of haemosiderin.
8. What is hemosiderosis?
Iron accumulation in the form of hemosiderin, hemosiderin is an
iron-storage complex. It is always found within cells (as opposed
to circulating in blood) and appears to be a complex of ferritin,
denatured ferritin and other material. The iron within deposits of
hemosiderin is very poorly available to supply iron when needed.
Iron is ferric in nature.
9. What is malarial pigment?
This is also granular and dark brown pigment and is hematin. It is
iron negative, differs from formalin pigment in that it is not doubly
refractile. It is found in the parasites, in red cells and macrophages
(liver)
10. What is formalin pigment?
This is acid formaldehyde hematin formed by the action of acid
formalin on haemoglobin. It is a dark brown granular pigment
found especially where blood is abundant. It is double refractile
and iron negative.

11. What is melanin?
These are granular, yellow, brown or black pigments which are
formed from tyrosine related compounds by the action of
tyrosinase. Melanins are slowly bleached by strong oxidising
agents such as hydrogen peroxide, peracetic acid, and potassium
permanganate. Melanins also reduce solutions of ammonical
silver nitrate to black metallic silver. [Masson – Fontana
ammonical silver reaction].
12. What are lipofuscins?
These are made up of a heterogeneous group of yellowish brown
basophilic granular pigments. Variously called as ‘brown atrophy
pigments’, ‘wear and tear pigments’. They were frequently seen
in the elderly people.
13. What are ceroids?
Ceroids are not a single substance but is a mixture of lipofuscins
like pigments but probably represents early stage of lipofuscins.
Ceroids exhibit auto fluorescence and appear greenish yellow in
frozen and brownish yellow in paraffin section, when these are
de - waxed and unstained.
14. What is the stain for calcium?
Von kossa’s stain – insoluble calcium such as carbonates and
phosphates
15. What are the other stains for iron?
Mallory’s method and The Quincke – Tirmann – Schmetzer
method for ferric iron.
D. MICROORGANISM STAINING
I. BASICS OF MICROBES STAINING

Gram-positive bacteria are those that are stained dark blue or
violet by Gram staining. This is in contrast to Gram-negative bacteria,
which cannot retain the crystal violet stain, instead taking up the
counterstain (safranin or fuchsine) and appearing red or pink. Gram-

positive organisms are able to retain the crystal violet stain because
of the high amount of peptidoglycan in the cell wall. Gram-positive
cell walls typically lack the outer membrane found in Gram-negative
bacteria. In the histopathological sections the stain is applied to detect
both the gram positive and negative organisms by using the counterstain
for gram negative bacteria so, both the organisms are stained.
Acid Fast techniques are valuable in detection of mycobacteria,
rod shaped organism sometimes exhibit filamentous growth. More
significant disease producing mycobacteria are mycobacterium
tuberculosis and mycobacterium leprae. Acid fast organism contains
large amounts of lipid in the cell wall; once the cell is stained it resists
decolorization with dilute mineral acids. Resistance to acid
decolorization is responsible for the application of the term ’Acid
Fast Bacteria’ to these organism. Some bacteria such as spirochetes,
are not well stained whether grams or acid fast techniques, so silver
stains are used for the demonstration of this organism.
Fungi are unicellular or multi cellular primitive plants that have a
distinct membrane bound nucleus containing the genetic material.
The fungal wall is made up of chitin. There are four types of fungus
which are medically important. I) The filamentous fungus, hyphae
which are divided transversely called septae e.g., Aspergillus. II)
Single round or oval cells that reproduce by budding, yeasts e.g.,
Cryptococcus. III) Yeast like fungus, reproduced by budding but tend
to elongate like filamentous e.g., Candida. IV) Dimorphic fungus
which grown in vivo (37°C) as yeast but in vitro (25°C) as hyphae
e.g., Histoplasma capsulatum.
II. BROWN AND BRENN TECHNIQUE FOR GRAM
STAIN
Purpose
The demonstration of gram- negative & gram- positive bacteria
in tissue.
Principle
Crystal violet is applied first and then followed by an iodine
mordant, forming a dry lake. At this point both gram negative and
gram positive organisms are stained. Although both types of bacteria

have a cell wall composed of peptidoglycan, the wall of gram positive
bacteria is thicker than that of gram negative organisms and gram
negative also contain a layer of lipopolysaccharide external to the
cell wall. These differences in the cell wall account for differences
in the way that bacteria are decolorized in the next procedural step.
The large crystal violet iodine molecular complex cannot be easily
washed out of the intact peptidoglycan layer of gram positive cells
although it is easily removed from gram negative bacteria because
alcohol or acetone disrupt the outer lipopolysaccharide layer and the
remaining thin peptidoglycan cell wall cannot retain the complex. The
decolorization step is a relative one and sections can be over
decolorized removing stain from both gram negative & gram positive
organisms. After decolorization, a counterstain is applied to color the
gram negative organisms.
Fixative
10% neutral buffered formalin
Quality Control
Known Positive cases serve as control
Reagents
1% Crystal Violet
Crystal violet .... 1.0 gm
Distilled water .... 100.0 ml
Filter into a dropper bottle. Stable for 1 year.
Lugol’s Iodine
Acetone
0.25% Basic Fuchsin
Basic fuchsin .... 0.25 gm
Filter into a dropper bottle. Solution is stable for 6 months.
Picric Acid-Acetone
Picric acid, saturated .... 100.0 ml
Acetone .... 900.0 ml
Discard after use.

Procedure
1. Cut paraffin sections at 4-5 micron thickness
2. Deparaffinize and hydrate to distilled water.
3. Place slides on staining rack, drop crystal violet stain onto
tissue section, stain for 1 minute.
4. Wash in tap water.
5. Lugol’s iodine, 1 minute.
7. Blot sections dry, breath on section then quickly pour
acetone over section until no color runs off.
9. Place slides on staining rack, drop Basic fuchsin on tissue
sections, stain 3 minutes.
10. Wash in tap water, blot gently but not completely dry.
11. Dip quickly into acetone, 2 dips.
12. Dip directly into picric acid-acetone mixture until a
‘salmon’ color.
13. Dip quickly into two changes of acetone.
14. Air dry, dip into xylene, and coverslip.
Results
Gram-Positive Bacteria …… blue
Gram-Negative Bacteria …… red
Nuclei …… red
Background …… yellow
Sections should not be allowed to be dried at any stage of
the procedure as drying leads to the formation of insoluble
compounds that are difficult or impossible to decolorize with
picric acid- acetone

Bacteria stain unexpectedly weakly
Check the crystal violet and iodide solution, and give more
time for the formation crystal violet iodide complex to be
formed. Check the control section if it stains good, ask
treatment history of the case, it may interfere the staining
and load of organism.
III. ZIEHL-NEELSEN STAIN (AFB)

Purpose
To detect the presence of acid-fast mycobacteria in tissue sections.
Principle
The lipoid capsule of the acid fast organism takes up carbol fuchsin
and resists decolorization with dilute mineral acid. Carbol- fuchsin is
more soluble in the lipids of the cell than in acid- alcohol but is readily
removed from bacteria that lack the waxy capsule. Staining is
enhanced by the phenol and the alcohol and both of these chemicals
also aid in dissolving the basic fuchsin. Alcoholic, rather than aqueous
solutions of acid are used because of uniform decolorization is obtained
with alcoholic solutions. The lipoid capsule of mycobacteria is of such
high molecular weight that it is waxy at room temperature and
successful penetration by the aqueous based solutions used in Gram’s
staining procedures is prevented.
Fixative
10% neutral buffered formalin is preferred others with the exception
of Carnoy’s solution may be used.
Quality Control
Known positive cases serve as control
Reagents
Ziehl Neelsen Carbol-Fuchsin Solution
Basic fuchsin .... 2.5 gm
100% alcohol .... 25.0 ml
Phenol crystals, melted .... 12.5 ml
Mix well, filter into brown bottle. Label bottle with date and initials,
solution is stable for 1 year.
1% Acid Alcohol
Hydrochloric acid .... 10.0 ml
70% Alcohol .... 990.0 ml
Mix well, label with date and initials, stable for 1 year.
Methylene Blue Stock Solution
Methylene blue .... 0.7 gm
Mix well, filter into bottle. Label with date and initials, stable for 1
year.
Methylene Blue Working Solution
Methylene Blue Stock .... 5.0 ml
Pour into coplin jar, stable for 2 months.
Procedure
1. Cut paraffin sections at 4-5 micron thickness.
2. Deparaffinize and hydrate to distilled water.
3. *Carbol-fuchsin solution, microwave 80 power, 45
seconds, allow slides to stand in hot solution for 5
minutes. Filter solution once a week.
4. Wash in running tap water.
5. 1% Acid alcohol until light pink and color stops
running.
6. Wash in running tap water for 5 minutes.
8. Working methylene blue for 30 seconds.
9. Rinse in water.
10. Dehydrate, clear, and coverslip.
*Conventional Method: 60°C oven for 1 hour.
Results
Acid-Fast Bacilli……. bright Red
Background…… blue
When using a ZN method based on Carbol Fuchsin, has this
stain deposited substantial amounts of red precipi-tate? If
so, discard and prepare fresh stain.
Bacteria stain unexpectedly weakly
Compare with the positive control and if this is also weak
check if the stain has been freshly pre-pared.
Exposure of the specimen to acids can reduce or elimi-nate
acid fastness. So;
(a) Check if an acidic fixative (e.g. Carnoy’s fluid) has
been used. If so, avoid this in future.
(b) Check if the specimen has been decalcified in strong
acid media. If so, use an alternative decalcification
system in future.
If a ‘cold’ method was used, the specimen might be
understained. Extend the staining period (try over-night).
Are there few or no stainable organisms, even though the
histological characteristics are indicative of tuberculosis? If
so, check if the patient had been treated with routine anti-
tuberculous drugs, since these can result in nonstaining
organisms.
If counterstaining is too intense, it can obscure the
mycobacteria. If this appears likely, try counterstaining for a
shorter time, or from an acidified solution.
Does differentiation fail to remove background staining by
the primary dye? Differentiation is slower with thicker
specimens, so check the thickness. In any event, with existing
specimens extend the time in the acid dif-ferentiator; be
relaxed about this, it is difficult to over differentiate.
Alternatively, try a method using a surfactant as co solute,
rather than phenol; or try a ‘cold’ method, which avoids
heating the Fuchsin solu-tion.
Does the counterstain give excessive coloration of the
background? If so, counterstain for a shorter time, or
counterstain from an acidified dye-bath.
Nature of staining is unusual
Check that stained bacteria are in the focal plane of the
section. If not, these acid-fast bacteria may be contami-nants
from the water supply.
If you stain by heating individual slides, and there are darkly
stained ‘odd-shaped’ objects present, try staining in a Coplin
jar in a heated water bath. Dye can precipitate if the solution
is overheated, causing excessive evapora-tion.
IV. FITE ACID FAST STAIN FOR M. LEPRAE

PRINCIPLE AND PROEDURES
Purpose
To detect the presence of mycobacterium leprae in tissue sections
Principle
Mycobacterial cell walls contain a waxy substance composed of
mycolic acids. These are ß-hydroxy carboxylic acids with chain
lengths of up to 90 carbon atoms. The property of acid fastness is
related to the carbon chain length of the mycolic acid found in any
particular species. The leprosy bacillus is much less acid and alcohol
fast than the tubercle bacillus, therefore alcohol is removed from the
hydrating and dehydrating steps and 10% sulphuric acid is used as a
decolouriser in place of acid / alcohol solution. The sections are also
deparaffinised using peanut oil/xylene mixture, this helps to protect
the more delicate waxy coat of the organisms.
Fixative
10% neutral buffered formalin is preferred others with the exception
of Carnoy’s solution may be used.

Quality control
Tissue containing leprosy organisms must be used as a control.
Millipore filtered water should be used in the floatation bath and a
negative control from the same day’s workload should be run. Do
not use any tap water or distilled water before applying carbol- fuchsin,
but use only Millipore filtered water.
Reagents
Xylene - Peanut oil
Peanut oil ….. 1 part
Xylene….. 2 parts
1% Acid Alcohol
Hydrochloric Acid, concentrated….. 10ml
Alcohol, 70%….. 990ml
Ziehl- Neelsen Carbol- fuchsin Solution
Phenol crystals, melted….. 5ml
Alcohol, absolute….. 10ml
Basic Fuchsin….. 1g
Stir on a mechanical stirrer. Filter before use. The solution keeps
well at room temperature.
Working Methylene Blue Solution
Methylene blue….. 0.5g
Glacial Acetic acid….. 0.5ml
Tap water….. 100ml
Procedure
1. Cut 4 -5 micrometers paraffin sections.
2. De - Paraffinize sections with two 12 – minute changes
of xylene - peanut oil mixture
3. Drain sections, wipe off excess oil and blot to opacity.
The residual oil helps to prevent shrinkage and injury of
the section.
4. Stain sections in freshly filtered Ziehl- Neelsen Carbol
– Fuchsin solution for 20-30 minutes at room
temperature. This solution may be saved for future reuse.
5. Wash sections in running tap water.
6. Differentiate slides individually with 1% acid alcohol until
the sections are faint pink.
8. Counterstain sections lightly with working methylene
blue solution. Do not overstain, the sections should look
sky-blue.
9. Rinse off excess methylene blue in tap water.
10. Blot sections and let stand for a few minutes to air- dry
completely
11. Mount air dried sections with synthetic resin. Do not
use alcohol and xylene.
Results
Acid Fast Bacteria….. bright red
Background….. light blue
Exposure of the specimen to acids can reduce or elimi-nate
acid fastness. So:
(a) Check if an acidic fixative (e.g. Carnoy’s fluid) has
been used. If so, avoid this in future.
(b) Check if the specimen has been decalcified in strong
acid media. If so, use an alternative decalcification
system in future.
Does differentiation fail to remove background staining by
the primary dye? Differentiation is slower with thicker
specimens, so check the thickness.

This method is not specific for leprosy bacteria. For example,
other acid-fast bacteria, and also tissue con-stituents such
as hair shafts and even erythrocytes, stain.
Does the counterstain give excessive coloration of the
background? If so, make sure you use weak solutions of
such counter stains as Methylene Blue. Alternatively, use
shorter periods of counterstaining.
Check that stained bacteria are in the focal plane of the
section. If not, these acid-fast bacteria may be contami-nants
from the water supply.
V. GROCOTT’S METHENAMINE – SILVER NITRATE

FUNGUS STAIN
Purpose
The demonstration of fungal organism in tissue sections.
Principle
Polysaccharides in the fungal cell wall are oxidized to aldehydes
by chromic acid. Chromic acid is a strong oxidant, further oxidizing
many of the newly released aldehyde groups to breakdown products
that will not react; this helps suppress the weaker background
reactions of collagen fibres & basement membranes. Only substances
that possess large quantities of polysaccharides, such as fungal cell
walls, glycogen & mucins will remain reactive with the Methenamine
silver, reducing it to metallic silver. Methenamine gives the solution
the alkaline properties necessary for proper reaction & sodium borate
acts as a buffer. Gold chloride is a toning solution & the sodium
thiosulphate removes any unreduced silver.
Fixative
10% neutral buffered formalin is preferred.

Quality control
A section containing fungi must be used; if staining for
pneumocystis use a pneumocystis control as the timing in the silver is
different. Chemically clean glassware & non metallic forceps must
be used.
Reagents
5% Chromic Acid
Chromic Trioxide….. 50.0g
5% Silver Nitrate
Silver Nitrate….. 25g
Distilled Water….. 500ml
3% Methenamine Solution
Hexamethylenetetramine….. 27g
5% Borax Solution
Sodium Borate….. 5g
‘ Stock Methenamine-Silver Nitrate Solution
Methenamine, 3% Solution….. 900ml
Silver Nitrate, 5% Solution….. 45ml
A white precipitate will form but will immediately dissolve
when shaken. The clear solution will remain usable for months
if stored in a chemically clean amber bottle in the refrigerator.
Working Methenamine – Silver Nitrate Solution
Borax, 5% Solution….. 2ml
Mix & Add Methenamine-
Silver Nitrate Stock Solution….. 25ml

1% Sodium Bisulfite Solution
Sodium Bisulfite….. 10g
O.1% Gold Chloride Solution
Gold Chloride, 1% Solution….. 7ml
Sodium Thiosulfate….. 20g
Stock Light Green Solution
Light Green Sf….. 1g
Glacial Acetic Acid….. 0.2ml
Working Light Green Solution
Light Green Stock Solution….. 10ml
Procedure
1. Cut 4 -5 micrometers paraffin sections.
2. Deparaffinize sections & hydrate to distilled water
3. Oxidize sections in chromic acid solution for 1hr. After 40
minutes, begin preheating the silver. The chromic acid solution
may be reused until it turns dark.
4. Wash slides in running tap water for a few seconds.
5. Rinse in 1% sodium bisulfite for 1 minute to remove any
residual chromic acid
6. Wash in tap water for 5-10 minutes.
7. Wash with three to four changes of distilled water
8. Using non metallic forceps, place slides in preheated working
Methenamine silver solution in water bath at 56 - 58°C for

15 minutes or until sections turn yellowish brown (paper-bag
brown). Remove the control, rinse in distilled water, & check
microscopically for adequate silver impregnation. Fungi should
be dark brown at this stage. If impregnation is not sufficient,
return the slide to the Methenamine silver & check every 3-
5 minutes
9. Rinse slides in six changes of distilled water.
10. Tone in 0.1% gold chloride solution for 2-5 minutes. This
solution may be used until brown precipitate appears & the
solution is cloudy.
11. Rinse sections in distilled water
12. Remove unreduced silver by placing the slides in 2% sodium
thiosulfate solution for 2-5 minutes
13. Wash thoroughly in tap water
14. Counterstain with working light green solution for 1 ½
minutes
15. Dehydrate with two changes each of 95% & absolute alcohol
16. Clear with 2-3 changes of xylene & mount with a synthetic
resin
Results
Fungi….. sharply delineated in black
Mucin….. taupe to dark gray
Background….. green
Use positive controls known to contain fungi, to check that
staining solutions have not deteriorated. If necessary, prepare
fresh solutions.
Incubation times required vary with fixative and dura-tion of
fixation. Use microscopic control, aiming for dark-brown fungi
with a colorless background. (Guideline: Try incubating for
60 minutes in the first instance.)

This method is not specific for fungi, and other polysaccharide
containing materials (e.g. chitin, glycogen, mucins and starch)
will stain. Materials able to reduce sil-ver cations without
prior oxidation, such as melanin, will also stain. Deposits of
insoluble calcium salts may also blacken, especially if light is
not excluded.
If connective tissue elements stain, reduce the incubation
time.
If background remains, even after reducing incubation
times, check that the impregnation was carried out in the
dark.
If random deposits of stain occur (e.g. on the glassware)
ensure you use chemically clean glassware and avoid contact
with metal items (e.g. use plastic forceps for handling
impregnated slides), as such contamination can reduce silver
cations directly or by acting as catalysts.
Moreover, check that the Coplin jar is sealed during the
impregnation and that the temperature of the oven or water
bath does not go above 60°C, as the silver ammine complex
is unstable at higher temperatures.
If the staining of fungi is so intense that details of the hyphal
septae are obscured, reduce the incubation time. Identification
of fungi requires this detail, and is best seen in
underimpregnated sections.
Floating problem
If the sections lift off the slides, check that the tempera-ture
is not above 60°C, and that the lid of the Coplin jar is well
fitting. Some workers celloidinize their sections, but if you do
this it may be necessary to remove strongly stained celloidin
with acetone during dehydration.

VI. FREQUENTLY ASKED EXAM QUESTIONS
1. What is the principle of AFB on Mycobacterium leprae?
Acid fastness of the leprosy organism is enhanced when the
waxy capsule is protected by the mixture of peanut oil and xylene
and by avoidance of dehydrating solutions.
2. What is the principle of brown and brenn technique of
gram stain?
The gram positive and negative bacterial, cell wall is composed
of peptidoglycan, (the gram-positive has a thicker wall) and both
will take up the crystal violet. The gram-negative has a layer of
lipopolysaccharide external to the peptidoglycan wall, which is
disrupted in the acetone rinse, allowing the crystal violet to be
differentiated out. This allows the gram-negative bacteria to take
up the basic fuchsin stain.
3. What organisms is alcohol fast?
Mycobacterium tuberculosis. But mycobacterium leprae are not
alcoholic fast.
4. What is the staining for mycobacterium paratuberculosis?
They lose their acid fastness and when they are oxidised renders
chromophobic bacilli
5. What are the other acid fast species?
Nocardia species, but to demonstrate them modified fite stain is
useful;
a) Stain in carbol-fuchsin for 10 minutes
b) Decolorize in 1% aqueous sulphuric acid for 5 to 10
minutes, agitating the slides frequently to remove the
back ground color.
c) Wash well in tap water.
d) Follow the remainder of the Fite procedure, beginning
with step 8.
6. What are the other methods to demonstrate AFB?
AFB reacts with Auramine and Rhodamine, exhibits golden
yellow fluorescent.
7. What is the principle of Grocott’s Methenamine Silver
stain?
Polysaccharides in the fungal cell wall are oxidized to aldehydes
by chromic acid. Chromic acid is a strong oxidant, further oxidizing
many of the newly released aldehyde groups to breakdown
products that will not react; this helps suppress the weaker
background reactions of collagen fibres & basement membranes.
Only substances that possess large quantities of polysaccharides,
such as fungal cell walls, glycogen & mucins will remain reactive
with the Methenamine silver, reducing it to metallic silver. Toning
is done by adding gold chloride.
8. What is the difference between the Grocott’s & Gomori’s
Methenamine Silver stains?
Grocott’s used only one hour for incubation at 45 - 50°C where
as Gomori used 1 to 3 hours at 37 - 45°C or ½ - 1 hour at 58 -
60°C. Rest all the procedures are same in both methods. Both
have equal results.
9. What are the other species demonstrated by this stain?
Actinomyces, Nocardia asteroides, and certain encapsulated
bacteria
10. Which method is useful for demonstration of P.carinii?
Microwave Methenamine Silver Method, done on frozen section
and it is very rapid procedure.
11. What is the merit of GMS over PAS?
Morphology of the fungus is best demonstrated and the
background is clear (green) so it has good contrast than PAS.
(High chance to miss the fungus)

For Further Readings…
1. Luna L (1968) Manual of Histologic Staining Methods of the AFIP,

3rd Edition, McGraw-Hill, New York, United States of America.
2. Culling C F A (1974) Handbook of Histopathological and
Histochemical Techniques, 3rd Edition, Butterworths, London, Great
Britain.
3. Stanley S Raphael (1976) Lynch’s Medical Laboratory Technology,
3rd Edition, W B Saunders Company, Philadelphia, United States of
America.
4. Sheehan D, Hrapchak B. (1980) Theory and practice of
Histotechnology, 2nd Edition, Battelle Press, Ohio, United States
of America.
5. Carson F (1990) Histotechnology: A Self-Instructional Text, 1st
Edition, ASCP Press, Chicago, United States of America.
6. Crookham J, Dapson R (1991) Hazardous Chemicals in the
Histopathology Laboratory, 2nd Edition, Anatech, United States of
America.
7. Carman R H (1993) Handbook of Medical Laboratory Technology,
2nd Edition, Christian Medical Association of India, Bangalore,
India.
8. Lille R D (1993) Histopathologic Technique and Practical
Histochemistry, 2nd Edition, Mc Graw – Hill Book Co, Columbus,
OH, United States of America.
9. Horobin R W, Bancroft J D (1998) Troubleshooting Histology
Stains, 1st Edition, Churchill Livingstone, Edinburgh, United
Kingdom.
10. Shameem S (1999) Laboratory Techniques in Surgical Pathology,
1st Edition, Prism Books Private Limited, Bangalore, India.
11. Bancroft J D, Gamble M (2005) Theory And Practice Of Histological
Techniques, 5th Edition, Churchill Livingstone, Philadelphia, United
States of America.


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The Human Path Education

© The Human Path Education, India

First Edition @ 2010

Medical knowledge is constantly changing, as new information becomes available,

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The Lord who created us…

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For whom this book is written for?

S Suban Mohammed Gouse & S Sarojini 9

I. BASICS OF STAINING MECHANISM

S Suban Mohammed Gouse & S Sarojini 11

12 The notes on Histochemical Stains

S Suban Mohammed Gouse & S Sarojini 13

S Suban Mohammed Gouse & S Sarojini 15

16 The notes on Histochemical Stains

S Suban Mohammed Gouse & S Sarojini 17

II. STAINING METHODS

20 The notes on Histochemical Stains

22 The notes on Histochemical Stains

III. PROBLEMS AND SOLUTIONS

S Suban Mohammed Gouse & S Sarojini 23

24 The notes on Histochemical Stains

S Suban Mohammed Gouse & S Sarojini 25

IV. FREQUENTLYASKED EXAM QUESTIONS

1. What is the principle behind H & E staining?

26 The notes on Histochemical Stains

S Suban Mohammed Gouse & S Sarojini 27

28 The notes on Histochemical Stains

S Suban Mohammed Gouse & S Sarojini 29

30 The notes on Histochemical Stains

S Suban Mohammed Gouse & S Sarojini 31

32 The notes on Histochemical Stains

S Suban Mohammed Gouse & S Sarojini 33

34 The notes on Histochemical Stains

I. BASICS OF STAINING MECHANISM

S Suban Mohammed Gouse & S Sarojini 35

36 The notes on Histochemical Stains

II. PERIODIC ACID SCHIFF STAIN

38 The notes on Histochemical Stains

40 The notes on Histochemical Stains

III. PERIODIC ACID SCHIFF STAIN WITH DIASTASE

S Suban Mohammed Gouse & S Sarojini 41

42 The notes on Histochemical Stains

S Suban Mohammed Gouse & S Sarojini 43

IV. MAYER’S MUCICARMINE STAIN

44 The notes on Histochemical Stains

46 The notes on Histochemical Stains

V. ALCIAN BLUE STAIN