His To Pathology Department

HISTOPATHOLOGY DEPARTMENT
I - INTRODUCTION:
Cells are the building blocks of all living things. Groups of

these cells unite to perform a specific function, these are called
tissues. Microscopic study of individual cells in smears is called
cytology and study of tissue is called histology. Histology is a
branch of anatomy that deals with minute structure, composition,
and function of tissues. Histopathology means study of diseased
tissues. The pathologist responsible for the diagnosis of diseased
tissues is dependent on the technical skill of a histotechnologist /
cytotechnologist, who prepares microscopic slides of the
specimen. These specimen slides should be well sectioned and
properly stained to provide detailed information of the tissues
under examination. Histopathological studies have proved to be
one of the most effective in diagnosing tissue abnormalities,
benign and malignant conditions.
Histopathological specimens are mostly collected by a

surgeon in an operation room. The specimens in the form of
pieces of tissues are submitted to the histopathology section of
pathology department. Each specimen is immediately placed in a
proper fixative and then it is entered in a log book (logging),
labeled and given identification number. Histopathology request
(Form No. 37) is completed in all respect by the consultant /
physician and the nurse concerned. A morphological description
of the tissue is noted by the pathologist and the gross examination
of the tissue, a portion of tissue is trimmed into suitable sized
blocks and handed over to the histotechnologist for further
processing.
TARIQ AZIZ / LAB APP OF HISTOPATHOLOGY DEPARTMENT

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II – RESPONSIBILITIES OF A TECHNICIAN:
a.) Specimen preservation
b.) Specimen logging
c.) Preparation of specimen to facilitate their gross and
microscopic examinations to be performed by
histopathologist
d.) Filing and preservation of records for future reference.
III – BASIC STEPS FOR TISSUE PROCESSING:

a.) Fixation
b.) Embedding
c.) Microtomy
d.) Staining
e.) Mounting
IV – LABORATORY REQUIREMENTS:
a.) General glassware – pipettes, flasks, reagent bottles, etc.
b.) Specimen containers – various sizes.
c.) Couplin staining jars
d.) Microscope slides and coverslips
e.) Reagent bottles
f.) Fixatives
g.) Various organic solvents
h.) Decalcifying solutions
i.) Embedding materials
j.) Various staining solutions
k.) Various dilutions of ethyl alcohol
l.) Mounting media etc.
V – EQUIPMENT AND INSTRUMENTS:

a.) Microscopes h.) Slide washing tray
b.) Microtomes i.) Balance
c.) Manual/Automated tissue processor
d.) Paraffin oven j.) Automated staining
e.) Tissue floating bath machine
f.) Embedding oven
g.) Slide warmer

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HISTOPATHOLOGY TECHNIQUES AND PROCEDURES
FIXATION:
Good fixation is the most important factor in the production
of satisfactory slides. If the tissue is well fixed one rarely has
difficulty with the later stages; if it is badly fixed, good sections
are absolutely impossible. The three essentials are: fresh tissue,
proper penetration of fixative and the correct choice of fixative.
Surgical specimens should be placed in fixative immediately after
they are taken and should be sent to the laboratory as soon as
possible so that the pathologist can supervise their proper
fixation. In postmortem cases, proper refrigeration of the body
will do much to retard autolysis and permit reasonably good
sections if delay is unavoidable. Even very early autolysis renders
tissue abnormally fragile and such tissues should be handled as
gently as possible to avoid artifact.
Inadequate penetration of the fixative is one of the
commonest causes of bad results. The maximum thickness that
can be penetrated is about 10mm for loose tissues and 5mm for
compact or cellular tissues (e.g. lymph nodes or spleen). For
dealing with specimens bigger than this the following methods
are recommended:
• Solid organs: Cut slices as big as necessary but not thicker than5mm.
• Hollow organs: Either open out or fill with fixative or pack lightly with wool
soaked in fixative.
• Large Specimens that require dissection: Inject fixative along vessels (or
bronchi, in case of lungs).
CHOICE OF FIXATIVE
The purposes of fixation are:
1. To inhibit autolytic enzymes and to kill the organism of
decompositions.
2. To preserve tissue as nearly as possible in its original form.
3. To protect the tissue against subsequent damage during
embedding.
4. To render the various constituents receptive to the proposed
stains.
The choice of fixative will depend on the nature of the tissue and
the type of staining to be employed.

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The following substances are commonly used in fixation:
1. Ethyl Alcohol
Used as 90-100% alcohol. Precipitates albumin and globulin
but not nucleoprotein. Shrinks and hardens tissue and displaces
cytoplasmic contents. Destroys mitochondria. A reducing
agent, and therefore incompatible with chromic acid,
chromates or osmium tetroxide. Preserves glycogen. Useful for
histochemistry.
2. Formaldehyde
Sold as formalin (a 40% w/w solution of gas in water). Used as
10% or better 15% of formalin in normal saline, or calcium
chloride solution. Penetrates rapidly but fixes slowly. Does not
precipitate protein but combine with NH2 groups to form an
insoluble gel. Preserves practically all elements including fats.
Renders phospholipids insoluble in fat solvents. Does not
shrink but allows shrinkage during embedding.
3. Mercuric Chloride
Used as saturated (about 70%) or half saturated aqueous
solution. Oxidizing agent. Penetrates rapidly. Penetrates
protein by combining with it. Causes no shrinkage by itself and
subsequent embedding causes only minimal shrinkage. Fixes
chromatin well and enhances its subsequent staining. Preserve
nearly all elements. Forms precipitates in tissues. Corrodes
metal containers. Rarely used along but it is valuable
constituent of many other fixatives.
4. Acetic Acid
Used as 1-5% aqueous solution. Precipitates and fixes
nucleoprotein but not cytoplasm and is, therefore, valuable for
nuclear fixation. Penetrates extremely rapidly. Tends to swell
tissue and does not harden. Destroys mitochondria and
cytoplasmic granules. Rarely used alone.

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5. Picric acid
Used as saturated aqueous solution (about 1%). Penetrates
poorly and causes shrinkage but does not harden. Precipitates
protein by combining with them. Preserves glycogen and
nearly all other elements. Permits nearly all stains. Not used
alone.
6. Chromic acid
Used either as a pure chemical or as a mixture of dichromate
and acetic acid (e.g. Zenker). An oxidizing agent and therefore,
incompatible with formalin or alcohol. Fixes by both
denaturing and precipitating proteins. Causes moderate
shrinkage but does not harden badly. Penetrates slowly.
Preserves most elements. Tends to weaken nuclear staining by
dissolving nucleoprotein.
7. Potassium dichromate
Used as 2-3% aqueous solution. A weak oxidizing agent and
cannot therefore, be kept with formalin for long. Fixes by
making proteins insoluble in water but without actually
precipitating them. Tends to dissolve chromatin and therefore
weakens nuclear staining. A good cytoplasmic and bad nuclear
fixative. Fixes lipids and mitochondria well. Causes no
shrinkage but allows no shrinkage during embedding. Gives
chromaffin reaction.
8. Osmium tetroxide
Used as 1% or 2% aqueous solution. Expensive and unstable
with an irritating vapor. Powerful oxidizing agent and
therefore incompatible with formalin or alcohol. Penetrates
very badly. Fixes by setting proteins in an insoluble gel
without precipitating them. Preserves fats and gives black
precipitate of osmium dioxide with unsaturated fats. Also
preserves very fine cell detail e.g. Golgi bodies. Used mainly
for electron microscopy.
(Store in dark bottle with 1 drop of saturated aqeous HgCl2 to
each 10cc to prevent oxidation).

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TYPES OF FIXATIVE:
The number of features available is almost infinite. The following
list gives only those in regular use in this department but is
sufficient for all ordinary purposes .
Note: If dichromate is used it is better to wash the tissue in water

before embedding to prevent the mercury corroding instruments.
Mercury must be removed from the cut sections with iodine
before staining but there is no need to add iodine to the
dehydrating alcohol, it does not help.
1. Formol Saline
• Commercial formalin 15ml
• 0.85% aqueous solution of NaCl 85 ml
(Keep over marble chips or calcium hydroxide chalk to
absorb acid).
The acid only affects the staining and adds to quantity of

formalin pigment formed with fre Ebgin tissues.
Formalin is a very cheap and very popular fixative but it has

certain definite disadvantages.
Its advantages are as follows:

1. It is the only fixative foe specimens destined for tissue
mounting photography.
2. It is the only fixative which leaves tissues suitable for dissection.
3. By virtue of its cheapiness it is desirable for big specimens.
4. It is the best fixative for frozen sections.
5. It is the only fixative for many of the silver impregnation methods.
e.g., For neurologia or for spirochaetes, though silver reticulin and
Bodian can be done in other good fixatives, very good for myelin.
Its Disadvantages are as follows:

1. It acts slowly and requires several days to achieve complete fixation.
2. It allows considerable shrinkage during subsequent embedding.
3. It forms a black pigment with laked blood if the pH is less than 6.8
(e.g. in stale or autolysing tissue).
Note: The shiny pale blue glittering nuclei indicate poor fixation.

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2. Formol Calcium
• Commercial formalin 15ml
• 1% aqueous solution of calcium chloride 85ml
Used where the preservation of small quantities of fat is important. Not as
good as formol-saline. Used only for analytical histochemistry.
3. Formol-Mercury
(Formalin prevents protein flocculation caused by Hg).
• Mercuric chloride 70g (3.5%)
• Commercial formalin 350ml (17.5%)
• Water 2 liters
(We add Edicol pea green to aid recognition)
The color also helps keep track of tiny biopsies.
The original formula (Lendrum,1941) gave saturated mercuric chloride (7%
approximately) and 10% formalin. Our modification causes less hardening
and is cheaper.
This is probably the best routine fixative. It is rapidly acting and causes less
shrinkage and gives much more brilliant staining and sharper detail than
formol saline. It is equally useful for fresh biopsies or stable autopsies. It
permits all ordinary stains including the silver reticulin methods.
Time of fixation: 6-24 hours depending upon size.
Prolonged time does no harm.
4. Helly’s fluid (Modified by Maximow)

Also known in American literature as Formol-Zenker
• Potassium dichromate 2.5g
• Mercuric chloride 5.0g
• Sodium sulphate 1.0g
• Water 100 ml
(Add 10ml of commercial formalin before use)
This is an excellent fixative and unlike Zenker’s, it preserves cytoplasmic
granules well. We use it for all hemopoietic tissues or other tissue which
needs to be stained by Giemsa, Leishman, etc. Improved in routines.
Time of fixation: 3-18 hours, according to thickness. More than 24 hours is
harmful.
Zenker contains acetic acid. This produces chromic acid which is not a good
fixative and is therefore not recommended. The formalin in Helly’s allows
chromate to remain.
Potassium dichromate if applied for long periods interferes with nuclear
stain. For human tissues optimum time is 18-20 hours while for rat tissues 6
hours.

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5. Bouin’s fluid
• Saturated aqueous picric acid 75ml
• Commercial formalin 25ml(25%)
• Glacial Acetic acid 5ml
This is an excellent cytological fixative which preserves all detail well. It is useful
for small biopsies of cellular tissue but is not satisfactory for large specimens or
as a general fixative because it penetrates badly and ruins red blood cells. It is
a poor fixative for autopsy tissue. Bad for brain tissues. Blocks have to be thin.
Preliminary fixation formol.
Time of fixation: 6-24 hours. Unduly long fixation (several days) is harmful.
Transfer to alcohol not water).
Saline prevents the bad effects of Bouin and this gives
excellent results.
6. Heidenhain’s Susa
• Mercuric chloride 45g 4.5%
• Sodium chloride 5g 0.5%
• Trichloracetic acid 20ml 2%
• Glacial acetic acid 4ml 4%
• Commercial formalin 200ml 20%
• Water 800ml
This is a very good general fixative. It gives brilliant staining and very sharp
nuclear detail. It tends to dissolve cytoplasmic granules and makes the
cytoplasm rather transparent. It penetrates and fixes rapidly and neither shrinks
nor hardens. It permits nearly all stain, including silver reticulin methods. It
preserves red cells badly and is rather expensive. Useful when space is
essential. Nuclear stain is excellent and is good for mitotic fig. Studies and
hence ideal for tumors.
Time of fixation: 6-12 hours. Transfer to alcohol 90-96% which further saves time
7. Carooy
• Alcohol 60ml
• Chloroform 30ml
• Glacial Acetic acid 10ml
This fixative gives sharp clear staining but not fine detail. It preserves glycogen
and is better general fixative than alcohol but not as good as Bouin, It
hemolyzes red cells but leaves the empty envelops intact. It dehydrates as well
as fixing and is useful if speed is essential. Very poor fixative but contains no
water. Good for Mucin MPS.
Time of fixation: 12 hours depending on size. Longer does no harm. Transfer to
alcohol only.
8. Muller’s fluid
• Potassium dichromate 2.5g
• Sodium sulphate 1.0g
• Water 100ml
Not now used as a fixative. See under Marchi method.

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SPECIAL HISTOCHEMICAL FIXATIVES
Modified Gendre fluid (for glycogen)
• Saturated picric acid in 96% alcohol 85 parts
• 40% formaldehyde 100parts
• Glacial acetic acid 5parts
Time of fixation: (Small blocks only)
18 hours at 73o ± 12 hours at –20oC.
The lower temperature is preferable if diffusion
artifacts is to be avoided.
Cold Formalin/ Acetone (for phosphatases and esterases)

1. Fix thin slices of tissue in 10% neutral formalin (buffered to ±
pH 7.0 with either sodium acetate or diphasic sodium
phosphate) for 12-16 hours at 0 to –4 oC
2. Wash for 6-24 hours in running cold water.
3. Dehydrate in 2 changes of absolute acetone at 0 to
–4oC in 1 hour in each.
4. Continue dehydration in absolute alcohol at room temperature
for 1 hour.
5. Clear in toluene.
6. Embed in paraffin wax, preferably at 52-54oC in vacuo. Time 1
hour or 10 minutes in vacuo.
The following fixatives are recommended for fixation of cryostat

sections before carrying out histological or histochemical
procedures.
Fixative Temperature Time Methods
o
Acetone 4C 1 hr Phosphatases
o
Formol Saline 4C 1 hr Esterases
o
Formol Saline 20 C 5-60 min Histological Methods
o
Formol Calcium 4C Block 18 hrs A good block fixative
for histochemistry
1-3 hours
(especially lipids) also
good for
phosphatases and
esterases.
Formol Alcohol 20oC 3-6 hours Glycogen and mucins
1 part in 9 parts (also can be used as
a rapid fixative for
histology).
Wolman 20oC 5-30 min Nucleic Acid
5% Acetic

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PRACTICAL GUIDE TO CHOICE OF FIXATIVES
It is difficult to give general rules because the choice depends on
the nature of elements to be demonstrated and the stains to be
employed. The following list of method in use in this Department
is meant to be a guide.
• Autopsy tissues (do not allow sandwiching of blocks)
Thin slices ( not more than 5mm thick) are dropped into an
excess of formal saline. Blocks are trimmed to shape and
refixed in formol mercury (F.M.) in the tissue processor.
• Small cellular biopsies (Tumour biopsies, cytology
endometrium).
F.M.
• Large Surgical Specimens
Place the whole in formol saline in theatre. Dissect and select
blocks. Refix in F.M. in tissue processor.
• Tissue for frozen sections
Formol Saline (F.M. is possible but difficult)
Freeze unfixed: block cut cryostat section. Take one direct
to stain, 2nd fix it in an alcohol other
mixture for 1 minute. To fix as the
permanent section. Unfix will decompose.
• Nervous tissue
Formol Saline
• Specimens for museum mounting
Kaiserling’s solution
• Haemopoietic tissue
Helly
• Histochemical tests
Look up appropriate fixative
Formol Saline followed by Bouin’s or by Helly’s is best on
formol mercury.
Gelatin blocks can be used to serve as a base for very tiny

tissues on frozen section. Gelatin molten is poured in dish
allowed to set and fixed in formalin. Cut into blocks and stored
in the fridge.

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PARAFFIN EMBEDDING
Blocks of tissues are trimmed to suitable size after fixation. This
should be 3-5mm thick and preferably small enough to fit under a
standard (7/8” x 7/8”) cover-slip. There is no advantage and much
disadvantage un using unduly small blocks since they give an
inadequate sample of tissue. If the tissue has been fixed in
bichromate and has not already been washed, the blocks should
be washed overnight in running water.
• Dehydration: The usual method of dehydration is the use of ascending
concentration of ethyl, isopropyl, or butyl alcohol; we use the first. It is
important to avoid sudden changes from water to high concentrations
alcohol because this causes the tissue to shrink and harden and
consequently makes it difficult to cut. Dehydration should be in
graduated steps through multiple baths.This can be done easily and
cheaply by using old used alcohol for the earlier baths.
• Clearing: The more useful agents are xylene, benzene, kerosene, and
chloroform. Xylene and Benzene are cheap and rapid but tend to harden
the tissue badly (benzene is the better of the two). Chloroform is slow
and more expensive but does not harden tissue nearly so badly. It can
be used several times. Kerosene is cheap and does no harden but will
not mix with alcohol unless it is over 99%. We use a mixture of 3 parts
chloroform to 7 parts kerosene.
• Wax impregnation: Paraffin wax with a melting point of about 56oC is
generally used and kept in an oven at 58-59 oC. It is important to see that
the oven temperature does not rise above this because temperature of
much over 60 oC will harden tissues badly. It is an advantage to keep the
tissues in the paraffin oven for the minimum time consistent with
complete impregnation. This is best achieved by passing the tissue
through a series of baths of wax; the clearing agent diffuses out into the
wax quite rapidly but takes so many hours to evaporate. A vacuum
embedding oven, if available, is very rapid.
• Casting: When the tissues are impregnated they are cast in fresh wax.
They can be cast in boxes of folded paper metal containers or boxes
made with a metal or glass plate and brass angle pieces. The tissue is
carefully oriented sot that the surface to be cut is downwards and the
wax is then rapidly cooled to prevent it s crystalling. If, inspite of rapid
cooling the wax crystallizes, it can be mixed with a small quantity of
beeswax (about 2g in a pint of molten wax); this will usually prevent
crystallization.
• Labeling: The most serious error which can occur in biopsy diagnosis is
the muddling of specimens. It is, therefore, essential to use a labeling
technique which is as nearly as possible foolproof. The best method is to
use slips of cardboard or cartridge paper (1 x 4cm) with the number
written in black lead and carry these through the whole process with the
specimen.

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Embedding Technique
The embedding technique used in this department is as follows:
The fixed specimens are examined, their morbid anatomy is
described and blocks are selected about tea time (this allows for
specimens arriving late in the day). The blocks and their
numbered labels are placed in the automatic tissue processor. This
is set to run from 5:30pm to 9:15am giving the following changes
of reagent:
• Formol Mercury for further fixation ………...2½ hrs
• Alcohol 4 times used…………………………¾ hrs
• Alcohol 3 times used………………………...1 hr
• Alcohol twice………………………………..1 ¼ hrs
• Alcohol once used…………………………...1 ¼ hrs
• Fresh alcohol (74 O.P.)……………………...1¾ hrs
• Kerosene-chloroform mixture(7:3 parts)……1 hr
• Kerosene-chloroform mixture……………….2½ hrs
• Pure chloroform……………………………..1 hr
• Paraffin wax (M.P. 54oC)……………………1 hr}*All 3
baths
• Paraffin wax (M.P. 56oC)……………………1 hr}*kept
at
• Paraffin wax (M.P. 56oC)……………………1 hr}*59-
60oC
This tissues are cased in the morning and cut in the afternoon.
Autopsy tissues are processed by hand if the machine is too full

of biopsy specimens. The technique is as follows:
The blocks together with their numbered labels are placed in old
(4 times used) alcohol (about 70% alcohol) overnight. Next
morning they are transferred to three times, used alcohol and
subsequently to twice used, once used, and finally fresh alcohol
(74 O.P.) These 4 baths take about 6 hrs and in the afternoon the
tissues are transferred to used kerosene chloroform (i.e. kerosene-
chloroform containing some alcohol). At the end of the day they
are transferred to fresh kerosene chloroform where they remain

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overnight. The following mornings they are put through 4 baths
of wax in three hours and then cast in fresh wax.
Common Technical errors:

• Incomplete fixation: This usually affects the center of the block. If
severe, the tissues crumble on cutting and will not adhere to slide. With
less severe degree the nuclei look homogenous and take only a pale
blue stain.
• Incomplete dehydration: If xylene or benzene are used for the
clearing, this error can be recognized because the center of the block
fails to become translucent. With chloroform, which does not render
tissue transparent, it cannot be recognized until the block is cut, when it
will be found that the middle of the block is soft and not impregnated
with wax.
• Incomplete impregnation: the middle of the block is soft and smells
of the clearing agent.
• Crystallization of the wax: The wax block should be homogenous and
translucent. It is streaky and opaque the wax may have crystallized due
to slow cooling or unsuitable wax.
• Unduly hard blocks: The block may be so hard that it cannot be cut
properly. The tissues commonly affected are dense collagen, bone,
skin, eye and colloid goiters. The commonest causes are: incomplete
decalcification, bad fixation, sudden jumps from water to high grade
alcohol, too long in xylene or benzene (not chloroform), too long in
paraffin oven, too high temperature in the paraffin oven.
It is sometimes found the very fatty tissues (e.g. lipoma) crumble and fail to
cut properly. This may be due to failure to remove all the fat. If this occurs,
give the tissue an extra bath of clearing agent.
The nomenclature of the different forms of alcohol is muddling.

The following data may be helpful.
¾ Proof spirit: Originally the mixture of
alcohol and water when used to wet gun
powder; would just permit it to burn. Now
defined as that mixture which at a room
temperature of 60oF will weigh 12/13 of the
weight of an equal volume of water. In
practice this is 49% of alcohol by weight and
60% by volume.
¾ Under-proof and over-proof: A spirit that
is X under proof will become proof spirit

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when X parts of pure alcohol are added to
100 parts of it. A spirit that is X over proof
becomes proof spirit when X parts of water
are added to 100 parts of it.
¾ Methylated spirit: (64 O.P.) consists of
rectified spirit (approximately 95% ethyl
alcohol) to which 10% of wood spirit (about
95% methyl alcohol) has been added. To this
pyridine and a dye are added before sale to
the public.
¾ Absolute Methylated spirit: (74 O.P.)
consists of a 10:1 mixture of ethyl and
methyl alcohols with about 1% of water.
¾ Absolute Ethyl alcohol: is pure ethyl
alcohol and is nearly anhydrous (about 0.3 to
0.5% of water).
CALCIFIED TISSUES
Bone
For ordinary specimens of bone the following technique gives
reliable results in a reasonable length of time.
• Fix in formol saline or formol mercury.
• Trim the pieces of tissue to a reasonable size and a
thickness of about 3mm (a fret-saw is useful for this).
Refix if necessary.
Decalcifying solution: Formic acid 15 ml
Sodium citrate 5g
Water 100 ml
• Place tissue in an excess of this solution and keep
it agitated. (We use the tissue processor, this is
essential to shorten the time). Test for complete
decalcification either by testing for calcium in the
solution or by x-rays.
• When completely decalcified wash in running
water overnight.
• Embed in wax in the ordinary way.
• Stain as desired.

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Calcified tissues
Calcified tissues, e.g. heart valves, are treated as above. The time
needed in acid varies with the tissue..
Undecalcified bone
To distinguish between bone and osteoid the most reliable method
is to cut undecalcified sections and stain the calcium by Kossa’s
method. This is not unduly difficult and the distinction between
bone and osteoid.
• Fix in formol saline.
• Cut out a small piece of cancellous bone about 5 x 5 x 2 mm. (This
can be done with a sharp knife).
• Dehydrate in the ordinary way as far as absolute alcohol.
• Immerse in 3% nitrocellulose in methyl benzoate (8 hrs)
• Transfer to 6% nitrocellulose in methyl benzoate (8 hrs)
• Transfer to 12% nitrocellulose in methyl benzoate (8 hrs)
• Transfer to 20% nitrocellulose in methyl benzoate (overnight)
• Drain off the excess nitrocellulose and immerse the tissue with its
adherent nitrocellulose in Benzene (2 changes of ½ hour each).
This doubly embedded block can be cut on a sledge-type microtome using a
heavy razor without damaging the latter unduly but the sections break to pieces
in the process of cutting and the fragments can not be picked up. To prevent
this a strip of “Sellotape” is pressed on to the top of the block so that when the
next section is cut it remains stuck to the “Sellotape”. The strip of “Sellotape”
can be moved fprward with each cut until sufficient sections have been
obtained.
The individual sections are cut with scissors and attach to slides by pressing
them on to a warmed, albuminised slide (sticky side to glass), The tape can be
floated off by wetting it with wet blotting paper for 10 minutes. The adhesive can
then be dissolved off in warm benzene leaving the section of bone attached to
the slide.
• Stain the calcium by Von Kossa’s method and counterstain with Van
Gieson’s stain for 30 seconds only.
• Dehydrate, clear and mount.
Result : Bone - Dark brown to black

Osteoid - Red
ADHESION OF SECTIONS TO SLIDE

PARAFFIN SECTIONS
Sections that are well fixed adhere well. Badly fixed sections
tend to float off. Thick sections tend to float off more easily than
thin ones. All sections float off dirty or greasy slides. Prolonged

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staining methods or any method involving ammoniacal solutions
(e.g. silver) tend to loosen sections.
While well fixed sections will generally adhere to clean slides
without aid, it is a wise precaution to use some adhesive. The
following two methods are in use in this department and work
well.
1. Glycerine Albumin: This is a mixture of glycerine and egg white

and can be made or bought. A very tiny drop (pinhead or less) is
rubbed over the surface of the slide before floating the section on
to it from the hot water bowl. If you are too slow in picking up the ,
the solution gets washed away. This method depends on the
albumin being precipitated by alcohol when bringing the section
down to water.
2. Chromate-Gelatin: Dissolve a fragment of leaf gelatin about ½”

square and a crystal of potassium dichromate about as big as a
split pea in the hot water on to which the sections are floated. The
gelatin dissolved in the water is denatured by the chromate in the
presence of light and becomes insoluble in water after the section
has been dried.
FROZEN SECTIONS:
Gelatin 0.5g
Merthiolate 15mg (preservative)
Distilled water 100ml
• Warm to 56oC and dip clean slides in the solution to coat them
• Dry coated slides at room temperature under cover to avoid dust.
Store in boxes for use.
• Float frozen sections on to slide and flatten out.
• Blot carefully.
• Place slide in Couplin jar with a few ml of commercial formalin for 1-2
minutes for vapor to denature gelatin.
• Wash in tap water to remove formalin.
• Stain sections in the ordinary way.
MOUNTING MEDIA
The choice of a suitable mountant is important to avoid fading of
sections.
1. D.P.X.
Dibutyl phthalate 5ml
Xylene 35ml
Mix and add distrene 80
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• D.P.X. can be bought ready made.
• A good general mountant. Does not cause fading as
badly as balsam. Shrinks a good deal. Must be in a
thin layer or it distorts the microscopic image. Dries
very rapidly. Used as a routine in this department.
2. Canada Balsam:
A very old mountant. Easily becomes acid and causes
fading of sections.
3. Chrome Glycerine Jelly:

Glycerine 20ml
Powdered gelatin 3g
Chrome alum 0.2g
• Dissolve the chrome alum (chromium potassium

sulphate) in 30ml of water. Dissolve the gelatin (edible
gelatin from the grocer’s) in 50ml of water. Mix and
add the glycerine. Test pH with indicator and
neutralize with NaHCO3 if acid.
• This mountant does not cause fading like ordinary
glycerine jelly and also sets much harder, obviating
the need to ring sections.
4. Apathy’s aqueous medium

Gum acacia (gum Arabic) 50g
Sucrose (cane sugar) 50g
o o
• Dissolve at 55 C – 60 C with shaking. Top up to
100ml. If necessary, add 15mg of merthiolate. Place in
vacuum oven while warm to get rid of bubbles.
• Add: Potassium acetate 50g
or NaCl 10g
- to prevent violet dye leaching out of amyloid.
(This medium sets hard and does not need ringing).

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STAINS FOR NUCLEI
Three groups of stains are available: haematoxylin, celestine blue
and a group of red dyes.
1. HAEMATOXYLIN
This is an extract of a tree (log wood) and is bought as a
brown powder. Used alone it is quite useless but it has the
property of combining with various heavy metals to form
dyes known technically as “lakes”. The metals most
commonly used are iron, aluminum, tungsten, and lithium
but others have been used. As a general rule the iron lakes
give intense grey-black or blue-black nuclear staining which
is resistant to subsequent decolorization; these dyes are used
when the counterstain chosen is liable to decolorize more
labile haematoxylin or where very sharp definition of the
nuclear chromatin is required, as in the study of
chromosomes. Examples are Weigart’s and Haidenhan’s
hematoxylin.
The aluminum “lakes” (known as haemalum) give a lighter,
more transparent blue color but are labile and change color
to red in the presence of acids. They are very simple to use
and are, in fact, the common routine stains for nuclei.
Examples are Erlich’s, Harris’ and Delafield’s haemalum.
Tungsten is used as phosphotungstic acid and the lake

produced is used to stain tissue components as well as
nuclei.
Lithium-hematoxylin lake can be used to stain myelin

sheaths.
2. CELESTINE BLUE:
Celestine blue ( a synthetic oxazin dye) combines with iron
to form a lake which stains nuclei blue. If, however, sections
are stained for a few minutes in this lake and then restained in
haemalum the resulting stain is as resistant to subsequent
decolorization as an iron-hematoxylin stain. This method,

18
being rapid and easy, has become a popular substitute for
Weigart’s iron-haematoxylin.
3. RED NUCLEAR STAINS:

Red nuclear stains are sometimes necessary as a contrast to
some other blue or black dye such as Perl’s stain for iron or
Masson’s method for melanin. The three common ones are
Alum-Carmine, Carbol-Safranin, and Neutral red.
FORMULA AND TECHNIQUES
HAEMATOXYLIN AND HAEMALUM

1. Heidenhain’s Iron Hematoxylin:
Sol. A 5% (approx) aqueous iron ammonium
sulphate (use lilac crystals.
Sol. B 0.5% (approx) aqueous hematoxylin (dilute
a stock 10% alcoholic solution with water)
• Bring paraffin sections down to water.

• Mordant in Sol. A for 1 hour at 37oC or up to 12
hours at room temperature.
• Rinse (do not wash for long).
• Stain in Sol. B until quite black (about the same
time as for mordant).
• Rinse
• Decolorize in saturated alcoholic picric acid until
staining is correct.

19
• Wash well in running water. (All picric acid must
be removed).
• Counterstain if desired, dehydrate, clear and mount.
2. Weigert’s Iron Hematoxylin:

Sol. A 1% alcoholic haematoxylin
Sol. B Liq. Ferri. Perchlor.(B.P.) 4ml
Conc. HCl 1ml
• Mix equal volumes of Sol. A and B in test tube and

pour the mixture on to the slide. (The mixture will
only keep for a few hours).
• Stain for 15-30 minutes
• Rinse
• Decolorize in acid alcohol
• Wash, counterstain, etc.
3. Erlich’s Haematoxylin:
2% alcoholic haematoxylin 100ml
Water 100ml
Glycerine 100ml
Glacial acetic acid 10ml
Potassium alum To excess
• Plug bottle with wool (not cork) and leave in the light
to ripen for a few weeks. The stain is usually fit for
use when it has darkened to a rich Burgundy color.
4. Mayer’s Acid Haemalum:

Haematoxylin 1g }
Sodium iodate 0.2g }Dissolve overnight
Potassium alum 50g }then add:
Distilled water 1000ml}
Chloral hydrate 50g
Citric acid 1g

20
• Boil 5 minutes. Ready for use when cool.
5. Harris’ Haematoxylin:
10% alcoholic haematoxylin 10ml
10% aqueous ammonium alum 200ml
• Mix and bring to boil. When boiling, add 0.5g yellow
mercuric oxide. When the solution turns purple,
plunge flask into water and cool rapidly. When cold,
filter and add 4ml glacial acetic acid. This stain is
artificially oxidized and can be used immediately.
• Good as nuclear stain in frozen section for fat as well

as in cytology smears.
6. Delafield’s Haematoxylin:
Saturated aqueous ammonium alum 40ml
Haematoxylin 4g
Absolute alcohol 25ml
• Mix and ripen for a few days and then add 100ml of
glycerine and add 100ml of methyl alcohol. Ripen for
a further six weeks.
Note:
Liq. Ferri. Perchlor is an aqueous
solution of ferric chloride.
Iron ammonium alum is iron ammonium
sulphate.
Potassium alum is potassium aluminum
sulphate.
Ammonium alum is ammonium
aluminum sulphate.

21
• It is very convenient to make up a
10% alcoholic solution of
haematoxylin as a stock solution from
which all the above can be made by
diluting with alcohol or water.
• Such a stock hematoxylin slowly
ripens on its own.
7.Cole’s haematoxylin:
Haematoxylin 3g in 25ml of 75 OP spirit. Add this to 500ml
of warmed D.W., dissolve and 100ml of 1% alcoholic
iodine. Add 1,400ml of saturated aq. am. alum (mordant).
• Bring to boil – must boil – cool and filter
• Use: No need for ripening.
COUNTERSTAINS
Most pathologist use Eosin as a general counterstain, but some
prefer more complex mixtures such as phloxine-tartazine. Van
Gieson or even Masson’s trichrome. In this department, Eosin is
used as a routine and other counterstains are only employed to
demonstrate particular tissue elements.
Eosin is used as a 1% aqueous solution of the water soluble
yellowish variety. If staining is found to be weak, the dye can be
improved by adding 2% calcium chloride or a trace of acetic acid.
Too much acid causes precipitation. The dye will usually grow
molds but these are harmless and can be filtered off.

22
To get good results, tisuues should be heavily overstained with
eosin and then differentiated in running water. This also improves
the keeping properties of the section. In a good preparation, red
cells, muscle and connective tissue should be in three distinct
shades.
ROUTINE HAEMALUM AND EOSIN STAINING:

Any fixative. Paraffin Sections.
1. Warm slide and treat with xylene to remove wax.
2. Treat with alcohol to remove xylene.
3. Rinse in water.
4. Treat with Lugol’s iodine to remove mercury if necessary 5
minutes.
5. Treat with 5% sodium thiosulphate till white.
6. Rinse in water.
7. Stain in any haemalum till overstained.
8. Rinse in water.
9. Differentiate in acid alcohol till nuclei only are stained.
10. Rinse.
11. Scott’s tap water substitute 5minutes.
12. Wash in water for a few minutes.
13. Stain in Eosin till section is bright red (5-15 minutes).
14. Wash in running water till eosin is differentiated.
15. Dehydrate in alcohol.
16. Clear in xylene.
17. Mount in D.P.X.
NOTES: If desired, sections may be stained for 2-5 minutes in
celestine blue and rinsed in water between stages 6and
7. If this is done, 5 minutes in haemalum usually
suffices.
• Lugol’s iodine: Potassium iodide 2, water 100.
• Acid alcohol: Conc. Hydrochloric acid 9ml.
Absolute methylated spirit 99ml
OTHER NUCLEAR STAINS:

1. Celestine Blue:

23
• Dissolve 2.5g of iron ammonium alum in 50ml of
distilled water.
• Add 0.25g of celectine blue B.
• Boil, cool and add 7ml of glycerol
2. Alum Carmine: (Carmalum)

Carminic acid (BDH) 1g
5% aqueous ammonium alum 200ml
Salicylic acid 0.2g
• It is impossible to overstain with this. If the result is
too weak, rinse and stain for 1 minute in neutral
red.
3. Carbol-Safranin:
• Melt 0.5g of phenol in a dry flask under the hot
tap.
• Mix in 0.1g of safranin to make dark red sludge.
• Grind together 0.25g of starch and 0.25g of
destrine.
• Add 50ml of water, with further grinding.
• Heat to 80oC. Cool, filter and dissolve the
carbolsafranin sludge in this filtrate.
• Stain for 10 minutes and differentiate in the
dehydrating alcohol.
4. Neutral red- Carbol fuchsin:

1% aqueous neutral red 15 parts
Carbol fuchsin (Ziehl Neelsen) 1 part
• Stain for ½ to 2 mins (alcohol tends to remove the
stain).
Notes:
Scott’s tap water substitute:
Potassium bicarbonate 2g
Magnesium sulphate 20g
Distilled water 1 liter

24
¾ This neutralizes the acid and makes the
section blue without loosening the section
from the slide. It is not necessary if tap water
is alkaline but does save time.
Alcohol: For all dehydrating of tissues and sections we use
absolute methylated spirit which is 74 overproof and
consists of methyl alcohol with 90% a small impurity
of methyl alcohol. It is about 99% alcohol and is as
good as and cheaper than absolute alcohol. For making
up solutions however, pure absolute ethyl alcohol
should be used.
Xylene: Some batches acid. If so, they will cause fading of slides.
This can be cured by standing the stock xylene over
marble chips.
FROZEN SECTIONS
Indications:
1. To stain fats.
2. Rapid diagnosis during an operation.
3. Some special stains (e.g. neurologia or histochemical tests).
4. To minimize artifacts.
5. To cut very thick sections.
Fixation:
• Formalin: All other fixatives make tissues too brittle.
For speed, small blocks may be fixed for 2 minutes in
formol-saline at 80oC, but sections obtained in this
way are of poor quality.
• Embedding in gelatin is useful for friable tissues.
Cutting:
• Set the microtome at 15μ and start with the knife
about 1mm (not less) from the surface of the chuck.

25
• Face the fixed block carefully, lay the flat surface
(moist but not wet) downwards on the chuck, and
press firmly so that it makes contact all over,
especially at the corners.
• Freeze, with short sharp burst of CO2 until a thin white
line of frozen tissue appears along the lower margin of
the block. The block is now fixed to the chuck and the
knife should be brought across to slice off the upper
part of the block before the plane of section freezes.
The flat surface for sectioning is now prepared.
Continue freezing until the new upper surface of the
block just balances, then let it thaw slightly.
• Start cutting. The ideal is to cut through tissue half
thawed, supported by frozen tissue a few μ below.
• Wipe each section off the blade with the little finger
of the left hand wet in 50% alcohol: the knife is
thereby kept wet with alcohol and sections run up on
its surface much more easily. Float each section off
the finger in a dish of water. The first few sections are
usually useless. Once one good section is obtained a
series out at intervals of about 5 seconds will usually
stay in the right zone of semi-thawing. Do not forget
an occasional blast of CO2 to keep the bottom of the
block frozen to the chuck (the next section after this is
usually a failure). Do not cut right down into the
chuck.
Liver and kidneys are the easiest tissues to cut. Necrotic

material and normal lung the hardest. With practice
favourable tissues may be cut at 10μ. The nearer the chuck
the knife is set the less time it takes to freeze the tissues and
the better one’s control of the temperature at the cutting
surface, but do not set at less than 1mm without a good
practice.

26
Fat Stains:
See the section on Sudan III
Gelatin embedding for friable tissues:

• Wash out formalin with running water for 6-8
hours.
• Place washed block in a covered pot containing the
following mixture for 4-6 hours at 37oC.
Gelatin 15g
Glycerine 15ml
Thymol A crystal
• Transfer block to a fresh lot of gelatin mixture in a
small mould (e.g. paper) and allow to cool.
• Harden the block of gelatin in 10%-20% formol saline
overnight. Trim off all excess gelatin and re-immerse
block in formol saline for 8 hours (or till convenient).
Gum for freezing:

Gum arabic (ground to powder) 50g
Cane sugar 20g
Thymol 0.05g
Mix and filter through muslin is necessary.
RAPID FROZEN SECTIONS DURING AN

OPERATION:
• Select and trim a suitable thin block to tissue about
3mm thick.
• Press on to a wetted microtome chuck.
• Freeze rapidly with CO2
• Place on the microtome of the cryostat and cut
sections.
• Pick up sections while still frozen on the side of the
knife with a coverslip. (They thaw and adhere).
• Place on a warm plate (e.g. microscope lamp) till just
drying.
27
• Stain the unfixed section for 1 minute in 1%
polychrome methylene blue.
• Rinse in water till the depth of staining is right.
• Rapidly dehydrate, clear and mount.
• Place on a slide face down avoiding bubbles and
examine. (This unfixed section cannot be kept).
Result:
Nuclei - Blue
Cytoplasm -Very pale blue
Fibrous tissue - Pink
Elastic tissue - Unstained but refractile and greyish
Mast cells - Purple
Further sections can be stained and kept permanently as follows:
• Dry section in air (NOT on warm plate) 15 seconds.
• Fix in the following solution for 1 minute.
Formalin (commercial) 10ml
Acetic acid 3ml
Alcohol (Abs. Meth. Spirit) 87ml
• Rinse in tap water.
• Celestine blue 1 minute.
• Rinse.
• Haemalum 1 minute.
• Rinse.
• Acid alcohol rapidly.
• Scott’s tap water substitute until blue.
• Rinse.
• Rosin 5 seconds.
• Rinse.
• Dehydrate, clear and mount in D.P.X.

28
SPECIAL STAINING METHODS
METHODS FOR CONNECTIVE TISSUE FIBERS:
The proper recognition of connective fibers and their relationship
to the other structures is often of vital importance in the diagnosis
of lesions. Numerous methods are available and an understanding
of their properties is necessary before the right one can be
chosen.
Collagen fibers:
• Van –Gieson’s stain: is traditional and easy to apply but it
will only stain relatively coarse fibers and it readily fades.
It is still useful for the study of coarse fibers such as
leiomyoma, breasts and arteries (after elastic stains).
• Mallory’s Trichrome: (Picro-Mallory). This stains finer
fibers than does Van Gieson and will pick out in blue the
basement membranes and interstitial connective tissue in
liver, kidney, heart, etc. Its greatest value in this respect is
the staining of interstitial tissue of the glomerular tuft. In
addition to its use for collagen it stains firbrin red. It can
also be used to differentiate the cells of the pituitary.
• Mason’s Trichrome: This has similar properties to
Mallory’s stain but stains connective tissue green. It is less
useful for kidney but it is easier to use than Mallory’s. It
will stain the striations of muscle.
• Silver Impregnation: This is undoubtedly the best method
for demonstrating the finest fibrils and will distinguish
them from coarser collagen by color (black and brown
respectively). It is especially valuable for demonstrating
the structure of tumors and lymph nodes and will also
show the finest fibrils in early organization. This method is
now reliable, rapid and applicable to nearly all fixatives
and is rapidly replacing the earlier dyes. It cannot be used
for glomerular basement membranes.
• Periodic acid Schiff: Carbohydrate in fine connective
tissue is colored red. Used for glomerular basement
membranes.
29
• Period acid methenamine silver (Jones): This method
demonstrates the connective tissue carbohydrate in black.
The color is intense so sections must be very thin. Only
used for glomerular basement membranes. (See under PAS
methods).
Elastic fibers:
Four methods are available:
• Verhoeff: This method is rapid easy and reliable. It
fails to demonstrate the finest fibers. It can be easily
combined with Van Gieson’s to give a very useful
stain for vessels.
• Weigert: The stain is difficult to prepare and ready
made commercial samples are unreliable. When good,
the stain picks out the finest fibrils and is superior to
the rest.
• Orecin: The natural dye is poor. The synthetic dye
(G.T. Gurr) is a good stain. It is better than Verhoeff
but not quite good as Weigert.
• Aldehyde fuchsin: A recent, reliable and easy method.
Comparable to Weigert in sensitivity.
VAN GIESON’S STAIN:

Use any fixative and paraffin sections.
1. Bring sections down to water and remove
mercury if necessary.
2. Stain nuclei with an iron hematoxylin (e.g.
Weigert) 15 minutes or with celestine blue
followed by haemalum 10 minutes. Rinse and
differentiate 10 min. Tendency should be to
under-differentiate.
3. Wash in water.
4. Rinse in alcohol.
5. Stain for 1-3 minutes in Van Gieson’s
mixture.
6. Rinse quickly in distilled water or pour the
stain off. Blot off.
7. Dehydrate rapidly in absolute alcohol.
30
8. Clear in xylol.
9. Mount in DPX.
Formula : 1% aqueous acid fuchsin 10ml

Saturated aqueous solution
of picric acid 90ml
• Dilute this with an equal
volume of water and boil
for 3 min. to ripen.
Results:
Nuclei - Black to dark brown
Collagen - Red
Other tissues - Yellow
Note:
• The fuchsin is removed by water and the
picric acid by alcohol. It is often better
to omit step 5 and dehydrate as quickly
as possible. Alkaline water removes
fuchsin very quickly.
• Picric acid decolorizes alum
haematoxylin, hence a more resistant
one must be used.
• In place of acid fuchsin in Van Gieson,
one may use poncean or sirdus red.
These are good for amyloid.
PICRO-MALLORY STAIN
Any fixative. Fix paraffin sections on albuminized slides.
1. Stain nuclei with iron haematoxylin or celestine blue
and haemalum.
2. Rinse in tap water.
3. Differentiate in picro-orange (1) until only nuclei are
stained (3-5 minutes).
4. Wash in water till only R.B.C. are yellow.
5. Stain in acid fuchsin mixture (2) till tissue is red (5-10
min.)
31
6. Rinse in 2% acetic and differentiate in red
differentiator (3) till only required elements are red
(up to 5 min.).
7. Wash in tap water (10 seconds).
8. Stain in aniline blue (4) till connective tissue is well
stained (10 minutes).
9. Rinse in 2% acetic.
10. Differentiate in blue differentiator (5) till only
connective tissue is stained blue (not less than 1-2
minutes).
11. Dehydrate rapidly, clear and mount in D.P.X.
SOLUTIONS:
1. Picro-orange:
80% alcohol saturated with picric acid 100ml
Orange G 0.25g
2. Acid- Fuchsin Mixture:
Acid fuchsin 0.5g
Ponceu Red 0.5g
1% acetic 100ml
3. Red differentiator:
Stock differentiator* 40ml
95% alcohol 40ml
Water 20ml
4. Aniline Blue solution:
• Aniline blue dissolve in 100 ml
boiling distilled water.
• Add 2.5 ml glacial acetic acid.
Cool and filter.
5. Blue differentiator:
Stock differentiator* 20ml
Water 80ml
*Stock differentiator: Absolute alcohol 100ml

32
MASSON’S TRICHROME STAIN:
Any fixative. Paraffin sections.
1. Bring sections down to water.

2. Stain nuclei by either iron hematoxylin or celestine
blue and haemalum, rinse and decolorize.
3. Wash in tap water.
4. Stain for 1-5 minutes in Masson’s Ponceu-fuchsin.
5. Rinse in acid water*(tap water washes the dye out).
6. Treat with 4% aqueous phosphomolybdic acid till
collagen is not darker than pale pink.
7. Rinse in acid water.
8. Stain 2-5 minutes in Masson’s light green **(till
collagen is green).
9. Rinse in acid water.
10. Dehydrate, clear and mount.
Results:
Nuclei - blue-black
Cytoplasm - light red
Muscle - dark red
Red cells - bright red
Hyaline and fibrin - bright red
Collagen and mucin - green
*Ponceu-fuchsin:
Ponceu 2 R 0.7g
Acid fuchsin 0.35g
Glacial acetic acid 1.0ml
**Light green:
Light green 2g
Glacial acetic acid 2 ml
HEIDENHAINS MODIFICATION OF
MALLORY’S ANILINE BLUE METHOD

33
(The following technique is taken directly from Mallory’s book)
Staining solutions:
• Azocarmine
Azocarmine B 0.25-1cm
Acetic acid, glacial 1cc
If azocarmine g is used instead of B add 0.1g to the 100ml
water, bring it to a boil, cool to room temperature and filter
through coarse filter paper in the paraffin oven at 51oC. After
cooling add the 1cc of glacial acetic acid.
• Aniline blue:
Aniline blue 0.5g
Orange G 2g
Acetic acid, glacial 8ml
Boil and filter after cooling. For staining, dilute the stock
solution 1:3 with distilled water.
Method of staining:
1. Stain in the azocarmine solution in a glass-covered
dish in paraffin oven at 51oC to 55oC for 45-60
minutes, then cool at room temperature 5-10 minutes.
2. Wash in distilled water.
3. Differentiate in an alcoholic solution of aniline made
up as follows:
Aniline 1cc
Alcohol, 90% 1000cc.
4. Rinse in acetic acid alcohol made up as follows for 30
seconds to 1 minute:
Acetic acid, glacial 1cc
Alcohol, 95 percent 100cc.
5. Mordant in 5 percent aqueous solution of
phosphotungstic acid 1-3 hours.
6. Wash quickly in distilled water.
7. Stain in the aniline blue solution 1-3 hours.
8. Wash very quickly in water.
9. Differentiatee in 95% alcohol followed by absolute.
10. Clear in xylol and mount in balsam.

34
Results:
Collagen - deep blue
Chromatin - red
Muscle tissue - reddish to orange
Erythrocytes - red
Neuroglia - reddish
Mucin - blue
Pituitary alpha granules - red
Pituitary beta granules - blue
GORDON AND SWEET’S

SILVER IMPREGNATION OF RETICULUM
This method can be used after all ordinary fixatives
including Helly’s.
1. Cut paraffin sections and attach firmly to slides.
2. Take sections down to water (remove mercury if necessary).
3. Oxidize for 1-7 minutes in acid permanganete solution.
0.5% aqueous potassium permanganete 50ml
3.0% sulphuric acid 2.5ml
4. Wash in water.
5. Bleach until white in 1% oxalic acid or 10% hydrobromic
acid (about 1 minute).
6. Wash in two changes of glass distilled water.
7. Mordant for 2-15 minutes in 2% aqueous iron alum.
8. Wash in 2 to 3 changes of distilled water.
9. Impregnate for 5-7 second in Wilder’s silver bath.
10% silver nitrate 5cc
- Add ammonia drop by drop until precipitate is not quite
dissolved.
- Add 3% sodium hydroxide 5cc

35
- Titrate with ammonia until not quite clear.
- Make up to 50cc with double distilled water.
- Filter into a bottle.
10. Wash briefly in distilled water.
11. Reduce in 10% aqueous formalin* in tap water (about ½
min.)
12. Wash in water. (If the sections are over-impregnated,
repeat the process from step 7).
13. Tone in 0.2% yellow gold chloride 1-3 minute (optional)
15. Fix in 5^% sodium thiosulphate 5 minutes.
16. Wash well in tap water.
Notes:
• All silver solutions should be made up in chemically
clean glassware. The Wilder’s silver bath will keep for
3-6 months.
• If desired a light counterstain can be used between
steps 16-17. Carmalum or neutral red-fuchsin are
recommended.
• Steps 3-5 constitute “Mallory’s bleach”. It can be
repeated; this is said to increase the sensitivity of the
method.
*This formalin may be neutral or slightly alkaline but not acid.
WEIGERT’S ELASTIC STAIN
The whole success of this method depends on preparing a good
batch of stain. Endless formula have been published but much
depends on the making of the batch and factors necessary for a
good batch are not all known. A good batch keeps for about a
year. We have found the following method works well:
Crystal-violet 2.5g
Basic fuchsin 2.5g
Dextrin 1.0g
Resorcin 10.0g
30% sol. Ferric chloride 62ml
(B.I. anhydrous)
36
• Heat the distilled water to nearly boiling in a large
evaporating basin. Mix the dyes and dextrin and
dissolve in the hot water. Add the resorcin and bring to
the boil. When boiling add slowly the freshly prepared
ferric chloride solution, stirring continuously with a
glass rod. It is important to keep the mixture boiling,
though not too vigorously. Continue boiling and stirring
for a further 2 minutes or so to coarsen the precipitate.
Cool and filter through a Buchner funnel and filter flask
attached to the section pump. Wash the deposit with
distilled water until the drips are colorless and the bulk
of the filtrate a clear azure blue. Usually 8-10 liters is
sufficient. The filter paper is now removed and dried
overnight in the incubator, when the deposit is scraped
off and dissolved in 550ml of absolute ethyl alcohol, to
which has been added 1 ml of conc. hydrochloric acid,
by simmering on an electric hot plate or water bath for
30 minutes or so. Cool and filter, then add 19ml conc.
hydrochloric acid and allow to stand 24-28 hour before
use, when the color should be a dark greenish-blue.
Staining Solution:
Stock solution 35ml
70% alcohol 30ml
(These amounts may have to be varied slightly
with each fresh batch of stain).
Technique of Use:
1. Xylol
2. 2 changes of absolute alcohol.
3. Blot and dip in 0.5% colloidin (as this is a long
staining method)
4. Harden colloidin in 70% alcohol for 5 minutes.
6. Remove mercury (as described under routine
haemalum and eosin staining.

37
7. Treat with permanganete and oxalic acid as in silver
reticulin method).
8. Wash in alcohol.
10. Stain in Weigert’s stain for 8-24 hours (time depends
on the strength of the batch.
11. Wash in alcohol (74 Op or ordinary acid alcohol
until only elastic tissue is stained.)
12. Remove colloidin with ether-alcohol mixture or
acetone.
13. Wash in water.
14. Follow with full procedure for Van Gieson’s stain.
Result:
Elastic fibers - dark blue-black
VERHOEFF’S ELASTIC STAIN:

2. Stain in Verhoeff’s mixture* till black (about15 mins.)
3. Rinse in water.
4. Differentiate in 2% ferric chloride until only elastic
fibers and nuclei are stained.
5. Wash in water.
6. Rinse in distilled water.
7. Counterstain with Van Gieson ½ to 1 minute.
8. Rinse in distilled water, dehydrate, clear and mount.
*Verhoeff’s stain:
5% unripened alcoholic haematoxylin 10cc
10% aqueous ferric chloride 4cc
Lugol’s iodine 4cc

38
(Neither the haematoxylin nor the ferric chloride should be
old; mix in the order stated).
Result:
Elastic tissue - black
Nuclei - grey-black
Collagen - red
Other structures - yellow
- This is an excellent, easy and reliable combination of stains.
Its only fault is the failure of very fine fibrils to stain but for
most practical purposes it is perfectly adequate.
- Prolonged staining in Van Gieson removes the elastic stain.
SYNTHETIC ORCEIN STAIN FOR ELASTIC TISSUE:
Synthetic orcein 1g
Dissolve in 80% alcohol 100ml
Add conc. HCl 1ml
(The stain keeps well.)
1. Bring sections down to water and remove mercury if
necessary.
3. Stain in orcein for ½ to 1 hour (longer does no harm).
4. Rinse out excess stain with acid alcohol.
5. Wash well in water (this improves the contrast).
6. Counterstain with safranin, neutral red, or Van Gieson
(½ minute only) as desired.
Result:
Coarse elastic fibers - reddish-brown
Fine elastic fibers - dark-brown
Other structures - counterstain only
ALDEHYDE FUCHSIN STAIN FOR ELASTIC TISSUE:

Preparation of the stain:
• Add 1ml of conc. HCl and 1 ml of paraldehyde to
100ml of a 0.5% basic fuchsin in 60-70% alcohol.
Keep at room temperature until the mixture darkens
to a deep violet (about 24hours). The stain gradually
alters its properties with age, staining more rapidly
and strongly when fresh.

39
METHOD:
1. Bring sections to water.
2. Oxidize sections in Lugol’s iodine for 10 minutes to 1
hour.
3. Remove iodine with 5% thiosulphate for 1 minute.
4. Immerse in aldehyde-fuchsin for 5 minutes to 2 hours,
depending on the tissue component to be stained.
5. Rinse in 60-70% alcohol.
6. Counterstain if desired (orange G, light green, fast
green).
7. Dehydrate in alcohol.
8. Clear in xylene.
9. Mount in D.P.X.
Times of staining:
Elastic tissue 5 minutes
Pancreatic β-cells 15-30 mins.
Pituitary β- granules 30 mins to 2 hours
Mast cells granules 5-10 mins.
Color: Deep purple
STAINING METHODS FOR DEMONSTRATING

PARTICULAR CELLS:
There are certain cells in structure that are difficult to recognize

or find and need special stains for their demonstration. The
following methods are the ones that we have found most useful:
Striped muscle: Lendrum’s “Phostox” or P.T.A.H.

Smooth muscle: P.T.A.H. Lissamine fast red.
Mallory’s or Masson’s trichrome

40
Bone marrow cells: Giemsa
Eosinophils: Phloxine Tartrazine
Plasma cells: Pappenheim
Mast cells: Thionine, toluidine, or
Polychrome methylene blue.
Aldehyde fuchsin.
Pituitary cells: P.A.S. or Heidenhain’s Mallory
Method
Enterochromaffin cells: Diazo method
Bile canaliculi: P.T.A.H.
Islets of Langerhans: Aldehyde fuchsin (β cells)
MALLORY’S PHOSPHOTUNGSTIC ACID

HAEMATOXYLIN (P.T.A.H.)
1. Bring sections down to water. If necessary remove mercury
with iodine and remove iodine with alcohol (hypo. ruins the
stain).
2. Postchromate* for ½ hour. Wash in water.
3. Differentiate the chromate for 1 minute in acid
permanganete.
4. Wash in water.
5. Treat with 1% oxalic acid till white.
6. Rinse in water and transfer to Mallory’s stain* for 12-24
hours.
7. Shake off excess stain (do not wash, water removes the red
component).
8. Dehydrate in alcohol (this differentiates the blue
component).
9. Clear and mount.
*SOL. A. 10% HCl in methylated spirit.
SOL. B. 3% aqueous potassium dichromate.
Mix 1 part of SOL. A. and 3 parts of SOL.B. and pour on slide.
*Mallory’s stain:
Haematoxylin (or haematin) 0.1g
Phosphotungstic acid 2.0g

41
- Dissolve separately and mix. Leave to ripen for several
months. Artificial ripening has not proved satisfactory in
our lands. When ripe, the stain keeps for several years.
Result:
Fibrin and neuroglial fibrils and red cells- dark blue
Nuclei - light blue
Collagen - Rose red
Note:
The balance of red and blue colour depends on the
postchromating and the removal of the chromate by
permanganete; the more chromate left in, the darker the blue.
The times given above are average ones.
LENDRUM’S “PHOSTOX”
1. Bring down to water.
2. Mordant in Lugol’s iodine for 1 hour.
3. Pour off and without washing treat with permanganete and
oxalic acid, as above.
5. Stain in Mallory’s stain till muscle is stained blue (a few
hours).
6. Dehydrate, clear, and mount as above.
PHLOXINE TARTRAZINE
Fix in formalin or F.M., avoid chromate or Bouin.
1. Bring down to water and remove mercury if necessary.
2. Stain nuclei with haemalum.
3. Differentiate in acid alcohol.
5. Stain for ½ hour in phloxine.

42
6. Rinse in water.
7. Treat with tartrazine** in cellosolve till the desired
components are red and the residue yellow.
8. Dehydrate in alcohol, clear and mount.
- Water removes the tartrazine at once but it can be restained in a

few seconds so a rinse at stage 7 may be used to ascertain if
staining is correct. A few seconds more in tartrazine restores
the yellow background.
- Red phloxine is replaced by yellow tartrazine in the tissue

components in the following order and the process can be
stopped at any desired point.
• Collagen
• Cell cytoplasm
• Muscle
• Erythrocytes
• Fibrin
• Mast cells and eosinophils
• Nucleoli
• Inclusion bodies.
- This is generally useful and easy to counterstain.
*Phloxine solution:
Phloxine 0.5g
Calcium chloride 0.5g
**Tartrazine solution:
Saturated solution of tartrazine N.S., in cellosolve (ethylene
glycol monoethyl ether).
LENDRUM’S LISSAMINE FAST RED
FOR UNSTRIPED MUSCLE:
1. Bring section down to water and remove mercury if
necessary.
2. Stain 5 minutes in celestine blue mixture.
3. Rinse in water.

43
4. Stain in haemalum for 5-10 minutes, rinse in tap water and
differentiate if necessary.
5. Blue in Scott’s tap water substitute for 3 minutes then wash.
6. Lissamine fast red for 5 minutes (1% in 1% acetic acid).
7. Immerse in 1% ohosphomolybdic acid, 5 minutes in 56oC
oven or in a bowl of hot tap water (the solution takes some
time to warm up).
8. Rinse.
9. Tartrazine 5 minutes (1.5% in 1.5% acetic acid).
10. Brief rinse in 65% alcohol (optional).
11. Complete dehydration with absolute alcohol using
dropping bottle.
12. Clear xylol, mount in D.P.X.
Result:
Nuclei - blue
Muscle, red cells and some
cell granules - red
Background - yellow
Note:
The Lissamine fast red solution goes off in a matter of weeks.
Prolonged staining does not seem to help. The times given are
arbitrary and it may be desirable to use the phosphomolybdic
acid cold if the red comes out too quickly. A little further red
comes out in the tartrazine so do not push differentiation too
far and if necessary shorten the time in tartrazine.
This method utilizes the basic technique of Mallory’s

trichrome.
MAY GRUNWALD-GIEMSA STAIN ON

PARAFFIN SECTIONS:
Fix in Helly. Other fixative are not as good.
Embed in paraffin and cut sections as soon as possible.
1. Bring section down to water and remove mercury.

44
2. Wash well in tap water for 5 minutes, when wash in two
changes of buffered distilled water at pH 6.8.
3. Stain in a mixture of equal parts of May Grunwald solution
and buffered (pH 6.8) distilled water for 1 hour.
4. Without washing, transfer to 1 in 20 dilution of Giemsa in
buffered water for 2 hours.
5. Rinse in buffered distilled water.
6. Differntiate in glycerine-ether-alcohol mixture, controlling
under the microscope. This takes a few seconds only. Gurr’s
glycerine-ether mixture diluted 1 in 4 with pure absolute
ethyl alcohol is used and should be prepared fresh each
time.
7. Dehydrate rapidly in absolute alcohol.
8. Clear in xylene.
9. Mount in Gurr’s neutral mountant.
Result:
• With fixation in Helly, the stain should be like ordinary
Romanowsky’s stain on films. Material fixed in
formalin alone will not take up enough red stain. This
may be partly corrected by adding a trace of eosin (alc.
sol.) to the alcohol used for dehydrating.
Note:
Buffer tablets at various pH can be bought G.T. Gurr.
GIEMSA ALTERNATIVE (BARRET’S MODIFICATION)

1. Bring to water and stain nuclei lightly Mayer’s Haemalum
(3-5 minutes).
2. Differentiate and wash.

45
4. Stain for 15-20 minutes in Orange mixture 0.5ml
Buffer 0.5ml
Acetone 1.0ml
5. Rinse in water and then in distilled water.
6. Stain for 18-24 hours in Orange mixture 0.15ml
Blue mixture 0.15ml
Buffer 3.0 ml
Distilled water 57.0ml
7. Blot, dehydrate quickly in alcohol. Clear and mount in
D.P.X.
Orange mixture:
1% Erythrocin 1 part
1% Orange G 2 parts
Distilled water 2 parts
Blue mixture:
1% methylene blue 2 parts
1% Toluidine blue 1 part
Distilled water 17 parts
Buffers:
PH 5.8 pH 6.4
M/15 KH2O4 45ml 34ml
M15 Na2HPO4 5ml 16ml
Note:
The staining mixtures should be made up freshly from the stock
solutions of 1% dyes. The two buffers can be tried because so
tissues vary in their reactions. A satisfactory result can usually be
obtained with one.
METHYL GREEN-PYRONIN METHOD (PAPPENHEIM)

This method offers a distinction between deoxyribonucleic acid
(DNA) and ribonucleic acid (RNA) by making use of the high

46
affinity of the latter for pyronin and of the former for methyl
green.
It is important to avoid undue heating during processing. If a high
temperature wax is used for embedding, or if sections are fixed to
the slides on a warm tray at 85oC, alterations in the structure of
both nucleic acids occur. These are most important and obvious in
the case of DNA. RNA is readily lost by even early autolysis.
Fresh tissue and good fixation are essential.
Method:
1. Bring paraffin sections to water. Cryostat sections,
briefly post-fixed in 5% acetic-ethanol should be rinsed
in distilled water.
2. Stain for 6 minutes in methyl green-pyronin*.
3. Blot gently with filter paper.
4. Dehydrate rapidly in absolute acetone.
5. Rinse in equal parts of acetone and xylene.
6. Rinse in 10% acetone in xylene.
7. Clear in two changes of clean xylene.
8. Mount in D.P.X.
Result :
DNA - green to bluish green (or purple)
RNA - red
*Preparation of the stain:

• Make up a 2% aqueous solution of Pyronin Y
(not all samples will perform adequately).
• Make up a 2% aqueous solution of methyl green and
extract it with chloroform in a separating funnel until
no more violet enters the chloroform layer (this may
take very many washes).
• For use, mix 12.5ml Pyronin Y and 7.5ml methyl
green with 30ml distilled water. (Alternatively 30ml of
M/5 acetate buffer of pH 4.8 may be employed).
HISTOCHEMICAL METHODS

47
In histological diagnosis it is often necessary to find out the
nature of various substances in the tissues. Their presence may be
suggested by the appearance of the routine section or they may
be known to occur in the type of lesion diagnosed. In any case,
their presence should be tested and verified and not guessed.
In the following pages methods are described for recognizing the

substances most often encountered in diagnostic routine.
FATTY SUBSTANCES:
Ordinary paraffin sections are useless for the recognition of fats.
Sometimes, as in liver, they suggest that fat is present but in most
tissues they give no hint. It is therefore, essential to cut frozen
sections if there is any question of fat being present.
Many different fatty substances occur in disease and it is not

possible to distinguish them individually in tissue sections.
Nevertheless certain broad groupings are possible by utilizing
several different tests. The tests used and the types of fat
recognizable are as follows:
Types of fat recognizable:

1. Neutral fats: (glycerides of fatty acids).
Occur in adipose tissue, in fatty infiltration and in degenerative
lesions.
2. Free fatty acids:
Occur in old inflammatory and degenerative lesions and in fat
necrosis.
3. Cholesterol and its esters:
Occur in atheroma, old hemorrhages, old inflammatory lesions,
in sebaceous glands, adrenal, corpus luteum, testis.
4. “Myelin" fats
A group of complex fatty substances which includes the
phospholipids. Examples are normal myelin and Gaucher’s and
Niemann Pick disease.
Tests:

48
1. Stain frozen sections with Oil Red O or Sudan II
& IV.
2. Reaction to polarized light:
• Examine unstained frozen sections
mounted in water or jelly by a polarizing
microscope or by an ordinary microscope
with one “Polaroid” glass below the
condenser and the other above the
objective lens. Anosotropic substances
show bright when the field is blacked out
by rotating the glasses at right angles.
Isotropic substances do not.
Note: There must not be any prism between the polaroids, i.e.
no inclined unit or binocular unit.
3. Solubility:
Most of the group of “myelin” fats are rendered insoluble by
fixatives containing formalin. They then persist in paraffin
sections after chloroform, xylene, etc. and can be stained by
Sudan black (but not by Sudan II or IV or by Oil Red O).
Reactions of fats:
1. Neutral fats:
• Strongly positive staining with
Sudan or Oil Red O.
• Isotropic
• Totally soluble in fat solvents.
2. Fatty acid:
• Weak staining with Sudan II &IV or Oil
red O variable, weaker than neutral fats.
• Normally isotropic but may be anisotropic.
3. Cholesterols and its esters:
• Cholesterol gives a weak Sudan or Oil red
O stain but the esters stain more strongly.
• Anisotropic in cold but become isotropic
on heating and revert on cooling.
4. Myelin fats:
49
• Stain with Sudan III or IV or with Oil red
O.
• Anisotropic.
• After formol fixation, they are insoluble in
fat solvents and give Sudan black staining
in paraffin sections.
SUDAN STAIN FOR FATS
These dyes, Sudan II & IV (Scharlach R) and also Oil red O,
depend for their action upon their greater solubility in body fat
than in the solvent used. A saturated solution of one dye will still
dissolve the other and this is made use of in preparing the Sudan
staining solution. The method is applicable only to frozen
sections. It will work after most watery fixatives but formalin is
preferable because the sections are less brittle to handle.
1. Cut frozen sections and receive into distilled water or diluted

formalin (cut sections can be stored in the latter).
2. Rinse sections in 70% alcohol.
3. Stain for not more than 1 minute in Sudan
solution* taking care to avoid letting the section
fold over. (Folded sections stain unevenly-
prolonged staining will dissolve out fine droplets
of fat).
4. Rinse in 70% or 50% alcohol to remove excess
stain.
5. Rinse in water holding section under till alcohol
has diffused.
6. Counterstain for a few minutes in alum
haematoxylin diluted 1 in 2 or 1 in 4 with
distilled water (control depth of staining with
microscope).
7. Differentiate if necessary in 0.5% HCl in 50%
alcohol.
8. Wash well in distilled water to which a few
drops of strong ammonia have been added.
9. Mount in glycerine jelly (see mountants).
*Sudan stain:

50
Mix equal quantities of dry Sudan III and Sudan IV powder and
place them in a clean dry bottle. Fill the bottle with Herzheimer’s
mixture (equal parts of acetone and 70% alcohol) and shake well.
Leave solution for a few days to obtain a saturated solution. For
use, pipette off some of the clear supernatant fluid; this is cleaner
and more economical than filtering. When using keep vessels
covered to avoid evaporation and precipitation of stain.
Result:
Fat - Orange-red
Nuclei - Blue
OIL RED STAIN FOR FAT
Several dyes of the oil red series can be used as fat stains. The
best in our hands has been oil red. It is used in a partly aqueous
solvent which helps to prevent solution of small fat droplets
during staining.
Stock Oil red O:
• Make up a saturated (0.5%) solution of Oil red O
in isopropyl alcohol.
• Stock staining solution:
• For use, dilute 6ml with 4ml distilled water and
allow to stand for 24 hours. Decant the
supernatant. This may be kept in a tightly
stoppered bottle for up to 6 months, filtered as
necessary through a No.42 Whatman paper,
directly on to the sections.
Method:
1. Cut formalin-fixed sections or use unfixed cryostat
sections mounted on coverslips.
2. Rinse briefly in water.
3. Rinse in60% isopropyl alcohol.
4. Stain in Oil red solution for 10 minutes.
5. Differentiate briefly in 62% isopropyl alcohol*.
6. Wash in water.
7. Counterstain and mount as for Sudan stain avoiding
ammonia at stage 8.

51
*Keep tightly stoppered. Absorbed moisture may dilute to
below 60%. This will cause precipitation of Oil red.
The following alternative method is better for

small quantities of fat:
Triethyl phosphate 60ml} “T.E.P.”
• Saturate some of this “T.E.P.” with oil red O at
56oC overnight. Cool and filter.
Method:
1. Rinse frozen sections in T.E.P.
2. stain in Oil red O in T.E.P. 10 minutes.
3. Rinse out excess stain in T.E.P.
5. Stain nuclei in haemalum.
6. Rinse in water.
7. Differentiate in T.E.P. containing 1% HCl.
8. Wash, blue, and mount in glycerine jelly.
(Fine fat droplets will not dissolve out in T.E.P. this being an
aqueous solvent).
SUDAN BLACK B
Sudan black B is a fat soluble dye like Sudan III and Sudan IV
(Scharlach R) and will stain all the fats that can be stainedby
them. A number of other lipid substances are rendered insoluble
by formalin fixation and can be stained in ordinary paraffin
sections by Sudan black (but not by Sudan III or IV). These lipids
include red cell envelops, lipofuscin, kerasin (Gaucher’s disease)
and myelin sheaths. For the latter, Sudan black is a rapid
alternative to the slower Weigert-Pal.
Sudan black is soluble in xylene and sections must be mounted in
a watery medium (glycerine jelly or Aparhy’s medium).

52
Method:
1. SOL. A. Sudan black B 1% in isopropyl
alocohol
SOL. B. Sodium borate 1% aqueous
- Mix equal parts of SOL. A. and SOL. B.,
stand for 10 minutes, filter.
3. Stain for 10 minutes.
4. Differentiate in 60% isopropyl alcohol in water.
6. Counterstain with a red nuclear dye (Carmalum is the best).
7. Wash in water.
8. Mount in glycerine jelly or Apathy’s medium.
Result:
Lipids stain black
*Sudan black can also be used as saturated solution in 70%
alcohol and differentiated in 70% alcohol but staining takes
much longer.
COPPER PATHALOCYANIN IN
METHOD FOR PHOSPHOLIPIDS:
Fix tissues in formalin or preferably, in formol-calcium. Embed in
paraffin the usual way.
Method:
1. Bring sections to absolute alcohol.
2. Stain in 0.1% Methanol Fast Blue 2G* in 90-100% alcohol
at 60o for 8-16 hours.
3. Rinse in 70% alcohol and bring to water.
4. Differentiate in 0.05% aqueous lithium carbonate for ½ to 2
hours.
5. Rinse in water.
6. Counterstain in 1% aqueous neutral red, up to 30 minutes
(the neutral red forms a deep blue complex with the
phophalocyanin-phospholipid compound. This is very
insoluble and, if preferred, staining with neutral red may be

53
done before stage 4 which subsequently needs only a very
short period.
7. Rinse in water.
8. Blot dry and transfer rapidly through 70 and 100% alcohol
to xylene.
9. Mount in D.P.X.
Result:
Phospholipids - purplish blue
(e.g. myelin sheaths and dark blue to red cell envelops)
Nuclei and nucleoli - red
Protein - pale sky blue or reddish
shades
N.B.:The presence of water in the alcoholic solution of
phthalocyanin increases the staining of non-lipid components
in the tissues, but also the speed of staining of lipids.
MUCOID SUBSTANCES
Mucin is not a chemical entity. The group of slimy fluids
ordinarily spoken of as “mucus” comprises a large number of
compounds which apart from their slimy physical character, all
contain carbohydrate of some form in chemical combination with
protein or aminoacid complexes and sometimes with sulphuric
acid. This group contains substances of great diversity and no
single stain will demonstrate all of them.
The following list gives the most useful methods and their
behavior with different mucins.
Mucin-carmine P.A.S. Alcian green Toluidine blue
Stomach ± or - + ± -
Duodenal mucosa + + + or ± -
Brunner’s glands - + - -
Colon + ± + -
Salivary gland + + ± -
Pancreatic duct ± + + -
Bile passages + + + or ± -
Bronchus + + + ±
Cervix + + + ±
Endometrium - + ± -
Bartholin’s gland + + + -
Prostate ± + + -
Seminal vesicles - + + -
Ovarian cyst + + + -

54
Cartilage + or ± + or ± + or ± +
Umbilical cord ± ± ± +
Aorta - - ± +
Myxoma ± + or ± + or ± +
Chordoma ± ± ± +
SOUTHGATE’S NUCLARMINE STAIN

Any fixative. Paraffin section.
1. Take down to distilled water.
2. Stain in Weigert’s iron haematoxylin 5 minutes of celestine
blue and haemalum.
3. Differentiate in 1% acid alcohol if necessary and blue again
in tap water.
4. Stain for 15-30 minutes in mucicarmine solution* diluted
1:5 with distilled water.
Result:
Mucin - reddish
Nuclei - blue
Mucicarmine solution:
Carmine 1g
Aluminum hydroxide 1g
• Add 100ml of 50% alcohol and then add 0.5g of
anhydrous aluminum chloride (beware of frothing
and use a 500ml flask).
• Boil for exactly 2 ½ minutes.
• Cool and filter.
Note:Cheap carmines seem to work better than purified ones. The
mixture frothes when being prepared.
PERIODIC ACID-SCHIFF TECHNIQUE (P.A.S.)

This method depends on the production of aldehyde from 1:2
glycol groups present in carbohydrates by oxidation with periodic
acid. The aldehydes are combined with Schiff’s reagent to form a
red compound dye in situ.

55
There are two different ways of using the technique, the first
(McManus variant) uses a 1% aqueous periodic acid, the second
(Hotchkiess variant) a 0.8% solution in buffered 70% alcohol,
following by a reducing rinse.
The McManus or watery P.A.S. gives the stronger reacting and

should be used to demonstrate fungi amoebae, basement
membranes etc. The Hotchkeiss or alcoholic P.A.S. gives a
weaker reaction and should be used to demonstrate mucin and
pituitary basophils. Both methods stain glycogen and if this is
likely to be present it should be removed by 20 minutes
incubation with saliva (or diastase) before staining.
Strong P.A.S. (McManus)

1. Bring section to water and remove mercury.
2. Treat with 1% aqueous periodic acid 5 minutes.
3. Wash briefly in water (diluted).
4. Schiff’s solution 20 minutes.
5. Wash in water.
6. Stain nuclei with haemalum.
7. Differentiate and blue in the usual way.
Result:
Basement membranes of kidney and other organs - deep red
Most fungi - deep red
Connective tissues - reddish pink
Hotchkiss Method:
1. Bring section down to water and remove mercury.
2. Rinse in 70% alcohol or meth. Spirit.
3. Immerse in Periodic acid solution at room temperature.
4. Rinse in 70% alcohol or meth. Spirit.
5. Immerse in reducing solution* for 1 minute.

56
6.Rinse in 70% alcohol or meth. Spirit.
7.Immerse in Schiff’s solution** for 4 minutes or more.
8.Wash in running water for 10 minutes.
9.Stain nuclei lightly with celestine blue and haemalu (about
2-3 minutes in each). Differentiate and blue as usual.
10. Counterstain in Orange G*** for 10 seconds (optional).
11. Wash in water till pale yellow (about 80 seconds).
12. Dehydrate, clear and mount in D.P.X.
Result:
Mucin - red
Nuclei - blue
Background - yellow
R.B.C.’s and aci - yellow
Period Acid Solution:

Periodic acid 0.4g
M/5 sodium acetate 5.0ml
(M/S=27.2g hydrated salt in 1000ml)
Abs. Ethyl alcohol 35.0 ml
o
(Keep between 17-22 C in dark. Keeps about 14 days).
*Reducing bath:
Potassium iodine 1.0g
Sodium thiosulphate pentahydrate 1.0g
Abs. Ethyl alcohol 30.0ml
2N Hydrochloric acid 2.5ml
(use 20% of conc. HCl)
¾ (Ignore any sulphur deposit. Keep between
17-22oC. Lasts up to 14 days.)
**Schiff’s reagent:

57
• Basic fuchsin 0.5ml
• Dissolve in boiling distilled water 100ml
o
• And cool to 55 C and add N/HCl 10ml
• Cool at temperature
• and add sodium meth. bisulphate 0.75g
o
• Leave 48 hrs. in dark at 4 C
(in tightly stoppered flask)
• Shake with granulated charcoal
for 1 minute to remove pink color. 2g
• Filter
• Store in tightly stoppered flask at 4oC
(Lasts about 6 weeks at 4oC)
*** Orange G
Orange G (G.I. 27) 2.0g
Phosphotungstic acid 5%
Aqueous 100ml
Stand for 24 hours and use supernatant.
PERIODIC ACID METHENAMINE SILVER METHOD
(Jones)
This method is based on the periodic acid-Schiff but uses
methenamine silver to detect the aldehyde. The staining is
extremely intense- too intense for ordinary 5-6μ sections. It s
practical use is the demonstration of glomerular basement
membranes but it is the only really satisfactory on sections 2 μ
thick.
Method:
Any fixative. Paraffin sections (2 μ).
1. Sections to water and remove mercury if necessary.
2. Rinse well in distilled water.
3. Oxidize in 1% aqueous periodic acid 15-20 minutes.
5. Wash well in tap water for 15 minutes.
7. Place in hexamine silver solution* for 1-2 hours.
Examine the section at ½ hourly intervals after the
first hour. When the glomerular basement membranes
is sharply defined proceed as follows:
58
9. Tone in 0.2% gold chloride for 5 mins.
11. Fix in 5% hypo for 2-3 mins.
13. Counterstain section by haematoxylin and eosin.
Result:
Nuclei - black
Basement membrane - black
Cytoplasm and muscle - pink-red
*Working Hexamine silver solution:
• Add 2ml of 5% borax to 25 ml distilled water.
Mix well.
• Then add 25ml of stock hexamine silver
solution.
Stock hexamine silver solution:
5% aqueous silver nitrate 5ml
3% aqueous hexamine 100ml
(Hexamethylene-tetramine)
ALCIAN GREEN (2 G%)
Method:
1. Bring paraffin sections to water.
2. Treat with 1% acetic acid for 2 minutes.
3. Stain in 1% alcian green in 1% acetic acid for 30
mins..
4. Wash in running water.
5. Counterstain nuclei as required, with carmalum or
haematoxylin.
6. Dehydrate in alcohols.
7. Clear in xylene.
8. Mount in D.P.X.
Result:
Acid mucopolysaccharide stain green.
*If any procedure involving treatment with acids is to follow this
stage, it is advisable to immerse the sections first in 0.3% sodium
carbonate for 30 minutes. This converts the bound alcian green
into an insoluble pigment.

59
METACHROMASIA
This is defined as the staining of a tissue component so that the
absorption spectrums of the resulting tissue-dye complex differed
sufficiently from that of the original dye, add from its ordinary
tissue complexes, to give a marked contrast in colour. For most
purposes it is limited to the effects seen with the thiazine dyes
(Toluidine blue, Azure A, Methyl blue, Thionin). With the first of
those a bright red change is called gamma metachromasia, and a
change to purple- beta chromasia. Gamma metachromasia
signifies the presence of free electronegative surface changes of a
certain minimum density. These are most commonly attributable
to the SO3H groups of sulphated mucopolysaccharides.
Method:
Fixation has a pronounced effect on the intensity of
metachromasia. Paraffin sections can be used, cryostat sections
postfixed if necessary, are preferable.
1. Bring section to distilled water.
2. Stain in 0.1% Toluidine blue in 30% ethanol for 5-20
mins.
3. Rinse in 95% alcohol.
4. Dehydrate in absolute alcohol.
5. Clear in xylene.
6. Mount in D.P.X.
Result:
• Sulphate containing mucopolysaccharides or lipids
will show gamma (red) metachromasia.
• Phosphate esters (nucleic acids) may show beta
(purple) metachromasia.
• An orange red filter may be used to change the red
blue sequence to green red. This is more easily
distinguished.
CARBOHYDRATE SUBSTANCES
GLYCOGEN (BEST’S METHOD)
Glycogen is water soluble but is largely retained in tissues by
being trapped in the proteins by ordinary fixation. To detect small

60
quantities, special fixatives should be used- Alcohol carnoy or
best ice cold Bouin. Glycogen is readily stained in P.A.S. but a
second slide must be stained after 20 minutes incubation with
saliva to show that the stainable substance is diastase labile.
Best’s glycogen carmine method is specific.
Method:
1. Fix in ice cold Bouin, dehydrate in alcohol.
2. Transfer to alcohol-ether (equal parts) 1-2 hours.
3. ½ or 1% celloidin in ether overnight (use “Nucol”
356A/9).
4. Shake off excess celloidin and harden block in
chloroform 1 minute.
5. Clear in benzene till translucent (½ to 2 hours).
6. Impregnate in paraffin wax – 3 changes in hours.
7. Cut sections, mount and bring down to water.
8. Stain in haemalin 5 minutes (do not prolong time).
9. Differentiate and rinse (do not wash).
10. Stain in Best’s stain* 15-30 minutes.
11. Without rinsing in water, differentiate** for 5-30
seconds and inspect under microscope.
12. Wash in 80% alcohol.
Result: Glycogen - red
Best’s Carmine Stock solution: (Lasts 3 months in ice chest)
Carmine 2g} Boil gently for 5 mins. Cool
Pot. Carbonate 1g} and filter. Add to filtrate
Pot. Chloride 5g} 20ml of ammonia.
Aq. dest 60ml}
*For staining:
Stock solution carmine 15.0ml}
Ammonia(.880) 12.5ml}Lasts 2-3 weeks
Methyl alcohol 12.5ml}
**Best’s differentiation:
Abs. alcohol 8ml
Methyl alcohol 4ml
Aq. dest 10ml

61
• If boiling period is insufficient the stain may be less
fast (more easily removed by the differentiator at stage
11).
PIGMENTS
The following colored materials may be encountered in sections:
• Haemosiderin
Golden to rusty brown granules. Gives positive iron reaction
with Berl’s stain.
• Haematodin (Bilirubin)
Reddish-brown crystalline deposits. Negative iron reaction.
See bilirubin below.
• Formalin pigment
Jet black granules present in stale tissues which in blood and
fixed in formalin. Can be removed by alcoholic picric acid
(Immerse slides for 2 hours).
• Melanin
Variable color from very pale brown to nearly black
negative iron reaction. Bleached out by acid permanganete.
Gives positive Masson’s silver bestand positive Schmori’s
stain.
• Carbon
Totally insoluble in anything. Jet black. Gives no reaction
with any test.
• Bilirubin
Greenish-brown granular masses. Negative iron reaction
Gmelin’s and Stein’s tests.
• Lipofuscins
Brown granules in parenchymous cells. Iron test may be
positive (due to associated iron). Positive satin for fat in
frozen sections. Stains by Schmorl’s method.
• Malaria pigment
Resembles formalin pigment and carbon. Soluble in conc.
sulphuric acid.
• Haemoglobin
Peroxydase stains with leuco patent blue.
62
PERL’S REACTION FOR IRON
Any fixative but chrome salts better avoided.
1. Bring paraffin sections down to distilled water.
2. Place in slightly warmed mixture of equal parts of
20% hydrochloric acid and 20% potassium
ferrocyanide 5minutes.
4. Counterstain in 1% aqueous neutral red, or other red
dye.
5. Wash rapidly in tap water.
Result:
Iron (ferric salts) - dark blue
Tissue - red
• To intensify the staining treat the slide after step 3

with hydrogen peroxide (10vols.) for 5 seconds, then
wash for 5-10 minutes in running water.
Note: To avoid false reactions use iron-free analytical reagents.

Make up the ferrocyanide reagent freshly each tome (the
strength need not be very accurate).
GMELIN REACTION FOR BILIRUBIN AND

HAEMATODIN
Method:
1. Bring section to water.
2. Apply a 50/50 mixture of conc. nitric and absolute
alcohol.
3. Apply coverslip and wipe off excess reagent.
4. Ring edge with hot paraffin wax (optional).
5. Examine under the microscope.

63
• A spectrum of colors from red to green spreads
out from masses of bilirubin or from haematodin
crystals.
STEIN’S METHOD FOR BILE PIGMENTS

Method:
2. Treat with Lugol’s iodine 2 parts, tinc. Iodine 1 part
mixed for 12-18 hours.
3. Wash in running water 5 minutes.
4. Treat with 5% aqueous sodium thiosulphate
30seconds.
5. Counterstain with Mayer’s carmalum 3-18 hours.
6. Wash in water, dehydrate, clear and mount.
Result: Bile pigment - dark greenish-black
SCHMORL’S METHOD FOR MELANIN,LIPOFUSCINS: etc.

Method:
2. Immerse in ferric-ferricyanide solution* for 5-10
minutes, wash in 1% acetic acid and examine under
microscope.
3. Repeat stage 2 if necessary, until melanin is dark blue
but background is clear.
4. Wash thoroughly in running water.
5. Counterstain nuclei with 1% aqueous neutral red..
6. Dehydrate rapidly, clear and mount in D.P.X.
Result: - Reducing substance - dark blue.
(This include melanin enterochromaffin granules,
lipofuscin, and SH groups).
- Nuclei - red
*Ferric-Ferricyanide solution
1% Ferric chloride 30ml}bothfreshly prepared
1% potassium ferricyanide 4ml }
Distilled water 6ml
Mix, fliter and use within 30 minutes.

64
MASSON’S SILVER METHOD FOR MELANIN
The argentaffin cells of the intestine also gives this reaction. Note
that this reaction differs fundamentally from silver impregnation
method in that no reducer is used. Reticulin stains do not show up
melanin because they are too rapid and because the oxidizers used
in most of them bleach the melanin.
MASSON’S METHOD:
Fix in any ordinary fixative, avoiding chromates.
1. Routine paraffin sections.
2. Bring to distilled water.
3. Leave overnight in Fontana’s ammoniacal silver* in
the dark in a covered jar.
5. Fix in 5% “hypo”,1-2 minutes.
6. Counterstain with carbol-safranin.
*Fontana’s silver solution:

• To 20cc of 10% AgNO3, add strong ammonia
drop by drop till only a few granules of the first
formed precipitate remain; if the mark is
overshop, add more AgNO3 to faint palescence.
• Add 20cc of distilled water. Allow to settle for a
day and decant into a dark bottle. Filter each jar
full before use and do not use for more than a few
sections. (Keeps in the bottle for a month or two).
• If it is intended to do any large number of
sections, commercial buffered hexamine silver
may be used. It gives less deposit and being nearly
neutral does not loosen sections from the slide but
we have found it less reliable.
1. Take 100ml of 3% hexamine.

2. Add 5ml of 5% silver nitrate. The
precipitate redissolves.
65
3. Add 5ml of borate buffer of approx. pH
(to 3% boric acid add a little
phenolphthalein, then NaOH till a pink
color is just precipitable).
4. Make up to 200 with distilled water.
LISON – DUNN TECHNIQUE FOR HEMOGLOBIN

1. Fix in neutral (preferably buffered) formalin for 24-48
hours but preferably not longer.
2. Cut paraffin sections and bring to water.
3. Stain in leuco patent blue* 5 minutes.
4. Rinse in water.
5. Counterstain with neutral red.**
6. Dehydrate, clear and ount.
Result:
Haemoglobin - dark blue
Oxidase granules - dark blue
Nuclei - red
*Leuco patent blue:

1% aqueous leuco patent blue 100ml
Add powdered zinc 10g
• Boil the mixture until it is pale straw color.
• Cool and filter. This is the stock solution.
For use, make up:

Stock solution 10ml
10 vol. Hydrogen peroxide 1ml
Filter before use.
**1% neutral red in 1% aqueous acetic acid.
DIAZO METHOD FOR

66
ENTEROCHROMAFFIN CELL GRANULES
Fix tissues in any formalin containing fixative. Oxidizing agents
such as dichromate and chromic acid, should be avoided if
possible.
Method:
2. Treat for 30 seconds with a dilute (1mg/ml) solution of
the stable diazotate of 5-nitro-anisidine (Fast red salt
B, I.C.I. 1bd) also known as Echtrotsalz B, in 0.1 H
voronal acetate buffer at pH 9.2.
3. Wash thoroughly in running water.
4. Satin nuclei with Mayer’s haemalum, 6-10 minutes.
5. Wash in running water for 30 minutes.
6. Dehydrate in alcohol, clear in xylene and mount in
D.P.X.
Result:
Argentaffin coll granules - fiery orange red
Nuclei - dark blue
Cytoplasmic structures - yellow
Note: Any one of a large variety of stable diazotates can be

used most satisfactory results are obtained with those
giving reddish azo dyes. The colors given with granules
in carcinoid tumors are more usually brownish-red.
MISCELLANEOUS SUBSTANCES
FEULGEN REACTION FOR D.N.A.

This reaction depends on the fact that aldehyde (potential
aldehyde) groups are produced by hydrolysis or deoxyribonucleic
acid in fixed tissues by N HCl at 60oC. After hydrolysis the
sections are washed and transferred to Schiff’s solution which
gives a red compound with aldehydes. The time of hydrolysis
varies with the fixative (5-15 minutes for most fixatives).

67
Method:
1. Bring sections to water (remove mercury if necessary).
2. Rinse briefly in cold N HCl.
3. Incubate in N HCl (pre-heated) at 60oC for the
optimum hydrolysis time (find by experiment if
necessary).
4. Rinse in col N HCl and then in distilled water.
5. Transfer to Schiff’s solution ½ - 1 hour.
6. Drain and rinse in three changes of freshly prepared
bisulphate solution (5ml 10% K2S2O5, 5 ml N HCl,
water to 100ml)
7. Rinse in water.
8. Counterstain (1% aqueous light green or fast green).
STAINS FOR FIBRIN

Fibrin stains red with picro-Mallory or Masson’s trichrome but
these are not specific. It stains dark blue with Mallory’s
phosphotungstic acid and this is the most reliable method. It can
be stained blue black by a modified Gram stain(below) but this
method depends on proper differentiation and is, therefore, not
completely reliable.
Method:
Any fixative, paraffin sections.
1. Bring to water.
2. Stain lightly with carmalum, eosin and carbol safranin.
3. Wash.
4. 1% aq. sol. Aniline methyl violet * 3-4 minutes.
6. Lugol’s iodine 3-4 minutes.
7. Wash, Blot well.
8. Decolorize and dehydrate in aniline xylol** until
cytoplasm is practically colorless and fibrin dark-blue.
9. Blot very thoroughly.
10. Wash well in xylol and mount.
Result: Fibrin - dark-blue
Nuclei - reddish-brown

68
(Gram Positive organisms are also stained by this method)
*Aniline methyl violet
Solution A: Methyl violet 2.5g } Mix and filter.
95% alcohol 12cc }
Solution B: Aniline water 100cc
Aniline water: Aniline 2cc }Shake well and filter.
Aq. distilled 98cc }
**Aniline xylol: Aniline 1 part
Xylol 2 parts
AMYLOID
Amyloid stains metachromatically with various violet dyes. It
stains red with congo red and the stained material is tropic.
It stains with Thioflavian T and the stained product fluoresces in
ultraviolet light.
It stains non-specifically with PAS (McManus)
METACHROMASIA WITH METHYL VIOLET

1. Paraffin sections take down to water.
2. Stain in 1% aqueous methyl violet (or crystal violet) 5
minutes, or better, stain for 1 hour or more in a very
dilute solution (a few drops of 1% solution in 50ml of
distilled water).
3. Differentiate in dilute acetic acid (4 drops of glacial in
100ml of water) or 0.5-1.0% oxalic acid until amyloid
is pink and other tissues violet.
4. Wash well in distilled water to remove acid.
5. Stand section for as long as feasible (more than 1
hour).
6. Without rinsing, in Apathy’s gum acacia mountant.
Result:
Amyloid - pink
Background - blue to violet

69
A more sensitive stain can be obtained with cryostat sections
of fresh tissue:
1. Fix cryostat section in 10% formol saline 10 mins.
2. Wash in water.
3. Stain in 1% methyl violet 5 minutes.
4. Wash in water.
5. Part differentiate in 1% acetic acid.
6. Wash in water.
7. Complete differentiation and counterstain in2%
aqueous methyl green (chloroform washed).
8. Wash well in water.
9. Mount in glycerine jelly or blot dry, rinse in xylol and
mount in D.P.X.
Result: Amyloid - red
Nuclei - green
Mast cells - blue
Background - clear
HIGHMAN’S MODIFICATION OF
BENNHOLD’S CONGO RED METHOD
1. Stain in Congo red (0.5% in 50% alcohol) for 1-5
minutes.
2. Differentiate in 0.2% potassium hydroxide in 80%
alcohol for 1-3 minutes.
3. Rinse in water.
4. Counterstain with alum haematoxylin.
5. Wash inwater.
Result: Amyloid - red
Nuclei - blue

70
THIOFLAVIN T STAIN
This is probably the most sensitive method.
1. Any fixative. Paraffin sections.
2. Bring to water.
3. Stain in haemalum if desired.
5. Stain in Thioflavin T* 3-5 minutes.
6. Wash off with 1% aqueous acetic acid.
7. Differentiate in 1% acetic acid 10-20 minutes.
8. Rinse in water.
9. Mount in Apathy’s medium.
10. Examine under ultraviolet light using exciter filter
BG 12 and barrier filter O.G.4 or O.G. 5 or
alternatively with exciter filter U.G.I. or U.G.2 with
a colorless U.V. barrier filter.
Result: Amyloid fluoresces yellow
*Thioflavin T 1% aqueous solution. Store in cold room (4oC)

and filter before use.
CALCIUM
Calcium deposits stain a dark blue black with haematoxylin but
the staining is due to trace of iron in the deposit and is not
specific. Von Kossa’s method stains calcium black but here the
reaction is due to the acid radical of the calcium deposits and is
also not strictly specific. It is, however, a very useful method.
Alizarin is a specific stain.
VON KOSSA’S STAIN
1. Any fixative that does not contain free acid.
2. Bring paraffin sections down to distilled water.
3. Place in 1.5% aqueous silver nitrate (freshly prepared
from a 10% stock solution) and leave in the light for 1
hour.

71
5. Remove excess silver the “hypo”.
6. Counterstain in 1% neutral red, or carbol safranin.
8. Dehydrate, clear, and mount.
ALIZARIN RED’S
1. Fixation as above. Paraffin sections.
2. Bring to water.
3. Stain in Alizarin red S solution* 1-5 mins.
4. Rinse rapidly in distilled water.
5. Blot section dry.
6. Counterstain in 0.5% toluidine blue 5-10 seconds.
7. Rinse rapidly in distilled water.
8. Blot.
9. Dehydrate in two changes of acetone.
10. Mount in D.P.X.
Result: Calcium - orange red
Nuclei - blue
*We use Revector Alizarin red S

(Sodium Alizarin sulphonate 5800S)
2% aqueous Alizarin red

• Add 10% ammonia (880 diluted 1 in 10) drop by drop
until the solution turns deep iodine color.
• Allow to stand for 5 minutes.
• Filter.

72
METHODS FOR ENZYMES
THE CALCIUM COBALT METHOD

FOR ALKALINE PHOSPHATASE (after Gomori)
PARAFFIN SECTIONS:
Thin blocks of tissue should be fixed, according to Gomori’s
method (1946) in two or three changes of cold absolute acetone at
4oC for 24 hours. There are many subsequent ways in which the
tissues may be embedded in paraffin wax, most of them being
minor variations designed to minimize destruction of the enzyme.
As a routine procedure the following method gives good results.
1. Transfer the blocks progressively at ½ hourly intervals

to absolute ethanol, absolute ethanol-ether with one or
two changes, and then to 1% celloidin.
2. Drain off excess celloidin and harden in chloroform.
3. Clear in benzene.
4. Embed in paraffin wax, avoiding prolonged exposure
to the high temperature of the wax bath.
5. Cut sections at 5μ and mount on albuminized slides.
6. Dry the slides for an hour at 37oC.
7. Store at 4oC until required for incubation.

73
METHOD PROPER AND CRYOSTAT SECTIONS:
(After fixation from step 3)
1. Remove wax from the slides by brief immersion in
right petroleum.
2. Pass tap water via absolute acetone.
3. Incubate for ½ hr. to 16 hrs. at 37oC in the following
medium:
10ml 2% sodium B-glucorophosphate
10ml 2% sodium diethyl barbiturate
20ml distilled water
2ml 2% calcium chloride
1ml 2% magnesium sulphate
4. Rinse in running water.
5. Treat with 2% cobalt nitrate or acetate 3-5 minutes.
7. Treat with a dilute solution of yellow ammonium
sulphide 1-2 minutes.
8. Wash in water, counterstain in 1% eosin, 5 minutes if
desired.
9. Dehydrate in alcohol, clear in xylene and mount in
canada balsam.
Result: Various structures are stained black or brownish-black
in tissue processing alkaline phosphatase activity.
FROZEN SECTIONS:
1. Cut sections 10-15μ thick and mount on clean glass
slides without any adhesive.
2. Dry in air at room temperature for 1-2 hours.
3. Incubate in the substrate solution for ½-4 hrs.
4. Wash in water treat with 2% cobalt solutions, wash
treat with dilute yellow ammonium sulphide.
5. Counterstain in 1% aqueous eosin 5 minutes
6. Wash in running water 5 mins.

74
7. Mount in glycocino.
THE LEAD-NITRATE METHOD FOR ACID PHOSPHATASE

Suitable material: Cryostat, post-fixed and paraffin sections.
1. Paraffin and post fixed sections to water.
2. Incubate at 37oC for ½-16 hours, 1-2 hours for cryostat
sections; longer for others) in 0.01M sodium b-
glycerophosphate in 0.05M acetate buffer (pH 5.0)
containing 0.004M lead nitrate.
3. Wash briefly in water.
4. Immerse in 1-5% yellow ammonium sulphide 1-2
minutes.
5. Wash in water.
6. Counterstain if required in eosin.
7. Wash well and mount in glycerine jelly.
Result: Acid phosphatase activity is shown by a black precipitate
of lead sulphide.
COUPLING AZO DYE METHOD

FOR ALKALINE PHOSPHATASE
Method:
1. Fix with thin slices of tissue in 10% neutral formalin at
4oC for 10-16 hours.
2. Cut frozen sections 10-15μ thick and mount on clean
slides without adhesive.
3. Allow to dry in air for 1-3 hrs. to ensure adherence.
4. Dissolve 10-20mg sodium a-naphthyl phosphate in
20ml 0.1M veronal acetate buffer (pH 9.2). Add 20mg
of the stable diazotate of 4-benzoyl amino-2: 5-
dimethoxyaniline and stir well. Filter on to the slides
sufficient to cover each section adequately and incubate
at room temperature. (17-22o) for 15-60 minutes.
(Alternatively use the same quantity of the stable
diazotate of 4-chloro-o-anisidine; or of 5-chloro-o-
toluidine, and proceed in the same manner.

75
5. Wash in running water for 1-3 minutes.
6. Counterstain in Mayer’s haemalum, 4-6 minutes.
7. Wash in running water for 30-60 minutes.
8. Mount in glycerine jelly.
Result: The sites of alkaline phosphatase activity are colored
black with salt 2 or brick red with salts 7 and 9;Nuclei-
dark blue.
PARAFFIN SECTIONS:
Cold acetone-fixed, paraffin-embedded, or cold formalin-fixed
paraffin-embedded as given in method for esterase.
Method:
1. Bring sections to water via absolute acetone after
removing the paraffin wax with light petroleum.
2. Cover with freshly made and filtered substrate-
diazonium salt mixture as above.
3. Incubate for 30 minutes to 4 hours (Salt 2), or for up to
2 hours (salt 7), or up to 12 hours (salt 9).
4. Wash inwater, counterstain as above and blue in
running water.
• (The use of salt 9 is particularly
recommended).
Result:
¾ With salt 9, the sites of alkaline
phosphatase activity appear dark
reddish-brown localization is
excellent.
¾ Nuclei - blue

76
STANDARD COUPLING AZO DYE METHOD
FOR ACID PHOSPHATASE
Suitable material: cryostat sections postfixed, formol-fixed
frozen sections or freeze dried sections.
Method:
1. All sections to water.
2. Incubate at 37oC in the following solution.
• 10-20mg sodium a-naphthyl phosphate in 20ml 0.1
veronal acetate buffer (pH 5.0). Add 20mg fast
garnet GBC (salt 18). The solution is mixed and
filtered on the sections. Incubation times vary from
1-45 minutes.
3. Wash well in running water.
4. Counterstain if required in haematoxylin.
5. Wash in water.
NAPHTHOL AS – PHOSPHATE METHOD

FOR ALKALINE PHOSPHATASE
Material: Post fixed cryostat sections, pre-fixed frozen sections,
freeze dried sections, paraffin sections.
Stock solution:
Dissolve 25mg naphthol AS-MX (or B1) in 10ml N,N-
dimethyl formamide, adding 10ml water and sufficient molar
sodium carbonate (2-3 drops) to bring the pH to 8.0. After the
addition of 300ml water the volume is brought up to 500ml by

77
adding 0.2M tris buffer (pH 8.3). The slightly opalescent
solution is stable at room temperature for several months.
Method:
1. Sections to water.
2. Incubate in stock solution containing 1mg/ml fast red
T.R. (salt 9) for 10-30 minutes at 22oC.
3. Wash briefly.
4. Counterstain if required.
5. Wash well.
Result: Alkaline phosphatase activity - red
NARHTHOL AS – PHOSPHATE METHOD

FOR ACID PHOSPHATASE
Material: Post fixed cryostat sections, pre-fixed frozen sections,
freeze dried sections.
Solutions:
A. Pararosanilin HCl. Stock solution.
B. 4% sodium nitrite in distilled water.
C. Veronal-acetate stock solution
buffer pH 9.2
D. Dissolve 100mg Naphthol AS-TR
(or B.I.), phosphate in 10ml N,N-
dimethyl formamide.
Incubating medium:
Dilute 5ml of solution C with 12ml distilled water and
add 1ml of solution D. Mix 0.8ml each of solutions A
and B and add to the solutions. Adjust the pH of the
whole solution to pH 5.0 with N NaOH.
Method:
1. Sections to water.
2. Allow to dry.
3. Place in incubating medium for 30-90minutes at room
temperature.

78
4. Wash in water.
5. Counterstain in methyl green if required.
6. Wash in water.
7. Mount in glycerine jelly or D.C.M.
Result: Acid phosphatase activity - red
THE a-NAPHTHOL ACETATE METHOD FOR

ESTERASE
Method:
1. Use either cold-formalin fixed frozen sections, without
washing out the formalin, or paraffin sections fixed in
cold acetone or in cold-formalin, and dehydrated with
cold acetone.
2. Incubate for 1-15 minutes at room temperature in the
following medium: Dissolve 20mg a-naphthol acetate
in 0.25ml acetone and add 20ml 0.1M phosphate
buffer (pH 7.4)*.
3. Wash in running water 2 minutes.
4. Counterstain in Mayer’s haemalum 4-6 minutes.
5. Wash in running water for at least 30 minutes.
*Alternatively, add 0.2ml 1% a-naphthol acetate in acetone to 10

ml of the buffer.
Result:
Esterase - black
Nuclei - dark-blue

79
METHODS FOR MICRO-ORGANISMS IN SECTIONS
The routine method is Gram’s stain. This is admirable for Gram
positive organisms but not wholly satisfactory for Gram negative
ones.
GRAM’S STAIN:
1. Any fixative, paraffin sections.
2. Bring down to water.
3. Stain for 1 minute in Gram’s crystal violet*.
4. Rinse in water.
5. Mordant in Lugol’s iodine ½ minute.
6. Rinse.
7. Differentiate in acetone till no more clouds of stain
come out (about 3 seconds).
8. Rinse in water
9. Counterstain in neutral red-fuchsin.
10. Dehydrate quickly in alcohol (this takes some of the
counterstain out).
11. Clear and mount in D.P.X.
*Crystal violet:

80
• Dissolve 2mg of crystal violet in 20ml of 96%
alcohol. Add 80ml of 1% aqueous ammonium
oxalate.
¾ As an alternative method to this use the

modified Gram’s method given as fibrin
stain.
¾ If Gram negative organisms fail to stain

well, stain an additional section with simple
methylene blue. This shows all organisms
well but does not distinguish between them.
(Giemsa’s stain also stains organisms blue.
GROCOTT HEXAMINE SILVER METHOD FOR FUNGI

Stock hexamine silver solution:
5% aqueous silver nitrate 5ml
3% aqueous hexamine 100ml
(hemamethylenetetramine)
• Mix; a white precipitate forms but
dissolves on shaking. (Store at 4oC, keeps
for months).
Working silver solution:
5% aqueous Borax 2ml }Mix and add
Stock hexamine silver solution 25ml
Light green counterstain:
Light green 0.2g }
Distilled water 100ml}Mix and dilute
Glacial acetic acid 0.2ml }1 in 5 for use
Method:

81
1. Hydrate section.
2. Oxidize in 5% aqueous chromic acid 1
hour.
4. Treat with 1% aqueous sodium
metabisulfite 1 minute.
5. Wash in tap water 5-10 minutes.
6. Rinse in 3-4 changes of distilled water.
7. Place in working silver bath in paraffin
oven (56oC) until section is yellow to
golden brown (about 30-45 min).
8. Remove with wax coated forceps, rinse in
distilled water and examine. Fungi- brown
to black.
9. Rinse in 3 changes of distilled water.
10. Tone in0.1% gold chloride 2-5 minutes.
12. Fix in 2% sodium thiosulphate.
14. Counterstain in light green solution ½ to 1
minute.
15. Rinse, dehydrate, clear and mount.
Result: Fungi, mucin, melanin, glycogen - black
Background - green
ZIEHL-NEELSEN CARBOL FUCHSIN STAIN
(For tubercle bacilli in tissue sections).
This stain is merely a slight modification of that ordinarily
employed on bacteriological smears. Any fixative may be
used, but mercurial fixatives are best.
1. Bring paraffin sections down to water.

2. Flood slide with carbol fuchsin* and heat to
steaming (not boiling). Keep hot for 15
minutes.

82
4. Differentiate in acid alcohol until the section
is a faint pink when washed in water and the
red cells are still pink.
5. Wash in running water for 5 minutes.
6. Stain lightly in haemalum or in methylene
blue.
7. Dehydrate, clear, and mount.
*Carbol fuchsin:
Basic fuchsin 10g
Phenol crystals 50g
Alcohol 100ml
• It is important that the counterstain should not

be too dark, otherwise bacilli are difficult to
find.
WADE FITE METHOD FOR

MYCO. LEPRAE IN SECTIONS
1. Warm section to melt wax and blot.
2. Remove rest of wax in
Peanut oil 1 part
Xylene 2 parts
3. Blot dry.
4. When dry, place in water (avoid alcohol).

83
5. Remove mercury if necessary.
6. Stain 30 minutes in carbol-fuchsin at room
temperature.
8. Decolorize for 2 minutes only in 0.5% HCl in
70% alcohol.
9. Wash well.
10. Counterstain nuclei in Mayer’s haemalum, 5
minutes.
11. Blue in tap water.
12. Blot and dry (avoid alcohol).
13. Clear in xylene. Mount.
Result:
Myco. leprae - red
Nuclei - blue
SILVER STAIN FOR SPIROCHAETES

The method depends on the impregnation of the spirochaetes with
silver nitrate and its subsequent reduction to an insoluble black
oxide. There are several modifications of Levaditi’s original

84
technique. The present one has given the best results in our hands.
The tissue is stained on block.
1. Fix in formal saline (other fixatives will not work).

(Avoid any impurity of mercury).
2. Cut thin slices of tissues, 2-3mm thick.
3. Absolute alcohol, 24 hours.
4. Wash in aq. dist. Until block sinks (several changes).
5. Place in 100ml of silver bath* 24 hours at 37oC.
6. Add 0.25ml of glacial acetic acid to the 100ml of silver bath
and leave for four more days at 37oC.
7. Wash well (2-3 hours) in aq. dist. (several changes).
8. Place in reducing fluid** at room temperature for 2-3 days.
9. Wash 2-3 hours in aq. dist. (several changes).
10. Dehydrate in alcohols, clear, embed, cut sections and
mount on slides.
11. Remove wax with xylol and mount in balsam carefully; do
not press the coverslip as the section is brittle and will
disintegrate.
Result: Spirochaetes - black
Nuclei - Greyish
Background - yellow
Silver bath:
Silver nitrate 1.5g
Double distilled 50ml
Water
Abs. alcohol 50ml
**Reducing fluid:
Tanin 3g }
Gellic acid 5g }Solution should be at
Fused sodium acetate 10g }least 3 months old.
Aq. dist. 350ml}
FLUORESCENT MICROSCOPY
(For tubercle bacilli in tissue sections)
Use a monocular microscope with normal objectives and
eyepieces. The condenser should be of the simplest (Abbe) type

85
unless a quartz one is available. The high dry obhective (1/6th) is
usually sufficient – if an immersion lens is required, a
nonfluorescent medium such as glycerol can be used, ordinary
immersion oil causes no trouble, however, ultraviolet light,
filtered through wood glass and a layer of aqueous copper
sulphate* passes through the specimen and into the microscope.
In the ocular, a UV absorbing filter (Ilford minus blue, Wratten
2B) is placed to prevent ultraviolet light from reaching the eye.
Method:
1. Bring paraffin sections down to water.
2. Stain at 60oC for 10 minutes (on rack over water bath)
in Carbol –Auramine-Rhodamine.**
4. Decolorize in 0.5% HCl in 70% ethanol for 1-2
minutes.
6. Mount in 80% glycerol for viewing.
If permanent mounting is required, dry the slide at 56oC for 2
hours and mount in D.P.X. via dilute D.P.X. in xylene.
Result:
Acid fast bacilli - bright reddish-gold rods which fluoresce green
*Aqueous copper sulphate:

Hydrated CuSO4 8g
Conc. Ammonia (S.G. 0.88) 100ml
Aq. dest. 160ml
**Carbol-Auramine-Rhodamine
Auramine O 1.5g
Glycerol 75ml
RhodamineB 0.75g
Phenol liquefactum 10ml
Aq. dest. 50ml

86

His To Pathology Department

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HISTOPATHOLOGY DEPARTMENT

Cells are the building blocks of all living things. Groups of

Histopathological specimens are mostly collected by a

TARIQ AZIZ / LAB APP OF HISTOPATHOLOGY DEPARTMENT

III – BASIC STEPS FOR TISSUE PROCESSING:

V – EQUIPMENT AND INSTRUMENTS:

TARIQ AZIZ / LAB APP OF HISTOPATHOLOGY DEPARTMENT

TARIQ AZIZ / LAB APP OF HISTOPATHOLOGY DEPARTMENT

TARIQ AZIZ / LAB APP OF HISTOPATHOLOGY DEPARTMENT

TARIQ AZIZ / LAB APP OF HISTOPATHOLOGY DEPARTMENT

Note: If dichromate is used it is better to wash the tissue in water

The acid only affects the staining and adds to quantity of

Formalin is a very cheap and very popular fixative but it has

Its advantages are as follows:

Its Disadvantages are as follows:

TARIQ AZIZ / LAB APP OF HISTOPATHOLOGY DEPARTMENT

4. Helly’s fluid (Modified by Maximow)

TARIQ AZIZ / LAB APP OF HISTOPATHOLOGY DEPARTMENT

TARIQ AZIZ / LAB APP OF HISTOPATHOLOGY DEPARTMENT

Cold Formalin/ Acetone (for phosphatases and esterases)

The following fixatives are recommended for fixation of cryostat

TARIQ AZIZ / LAB APP OF HISTOPATHOLOGY DEPARTMENT

Gelatin blocks can be used to serve as a base for very tiny

TARIQ AZIZ / LAB APP OF HISTOPATHOLOGY DEPARTMENT

TARIQ AZIZ / LAB APP OF HISTOPATHOLOGY DEPARTMENT

Autopsy tissues are processed by hand if the machine is too full

TARIQ AZIZ / LAB APP OF HISTOPATHOLOGY DEPARTMENT

Common Technical errors:

The nomenclature of the different forms of alcohol is muddling.

TARIQ AZIZ / LAB APP OF HISTOPATHOLOGY DEPARTMENT

TARIQ AZIZ / LAB APP OF HISTOPATHOLOGY DEPARTMENT

Result : Bone - Dark brown to black

ADHESION OF SECTIONS TO SLIDE

TARIQ AZIZ / LAB APP OF HISTOPATHOLOGY DEPARTMENT

1. Glycerine Albumin: This is a mixture of glycerine and egg white

2. Chromate-Gelatin: Dissolve a fragment of leaf gelatin about ½”

3. Chrome Glycerine Jelly:

• Dissolve the chrome alum (chromium potassium

4. Apathy’s aqueous medium

TARIQ AZIZ / LAB APP OF HISTOPATHOLOGY DEPARTMENT

Tungsten is used as phosphotungstic acid and the lake

Lithium-hematoxylin lake can be used to stain myelin

TARIQ AZIZ / LAB APP OF HISTOPATHOLOGY DEPARTMENT

3. RED NUCLEAR STAINS:

FORMULA AND TECHNIQUES

HAEMATOXYLIN AND HAEMALUM

• Bring paraffin sections down to water.

TARIQ AZIZ / LAB APP OF HISTOPATHOLOGY DEPARTMENT

2. Weigert’s Iron Hematoxylin:

• Mix equal volumes of Sol. A and B in test tube and

4. Mayer’s Acid Haemalum:

TARIQ AZIZ / LAB APP OF HISTOPATHOLOGY DEPARTMENT

• Good as nuclear stain in frozen section for fat as well

TARIQ AZIZ / LAB APP OF HISTOPATHOLOGY DEPARTMENT

• Bring to boil – must boil – cool and filter

• Use: No need for ripening.

TARIQ AZIZ / LAB APP OF HISTOPATHOLOGY DEPARTMENT

ROUTINE HAEMALUM AND EOSIN STAINING:

OTHER NUCLEAR STAINS:

TARIQ AZIZ / LAB APP OF HISTOPATHOLOGY DEPARTMENT

2. Alum Carmine: (Carmalum)

4. Neutral red- Carbol fuchsin: