Special stain in histopathology

Special stain in HIstopathology
Presented by Dr. Mahesh
Guide: Dr. S. P. Hiryur
- Associate Professor

• Special stains are used to identify certain normal and
abnormal substance present in the cells and tissue,
which can not be identified on routine
Haematoxylene & Eosin staining or are better
appreciated on special stain.

Periodic Acid-Schiff (PAS) stain
• Principle:
Substance containing vicinal glycol groups
or their amino or alkylamino derivatives are
oxidized by periodic acid into dialdehydes which on
reaction with schiff’s reagent give insoluble purple-
magenta compound.
• Dyes : 1%periodic acid
Schiff’s reagent
• Control : Appendix
• Result :
PAS +ve substance : Purple –magenta color
Nuclei : Blue

PAS positive substances :
• Glycogen
• Neutral mucoprotein
• Glycoprotein
• Glycolipid
• Basement membrane
• All fungi
• Phosphorylated sugar
• Cerebrosides

• PAS Diastase reaction : Glycogen is diastase
sensitive, hence section containing glycogen
when pretreated with diastase, the enzyme
will digest the glycogen and will give
negative PAS reaction.
• The purpose of using PAS with diastase
staining procedure is to differentiate
glycogen from other PAS positive elements
such as mucin that may be present in the
tissue sample.

Pocedure :
•Dewax the section and bring to water level
•Dip the slide in copplin jar containing Per iodic
acid for 10 min
•Wash well in tap water for 15-20 min
• Dip the slide in coplin jar containing Schiff
reagent for 20 min
•Rinse in distilled water
•Counter stain with hematoxylene for 5-10
seconds
•Wash in tap water
• Clear in xylene and mount in DPX.

• Uses of PAS:
• PAS is used to demonstrate glycogen and neutral
mucoprotein.
• In diagnosis of poorly differentiated adenocarcinoma of
various tissue like stomach, pancreas, lung.
• In diagnosis of hepatocellular carcinoma.
• Seminoma, Dysgerminoma
• RCC- Renal cell carcinoma: Clear cell type
• To demostrate – intracytoplasmic crystal in alveolar soft
part sarcoma
• To demostrate basement membrane e.g. adenoid cystic
ca.
• To demostrate neutral mucin in gastrointestinal tract

• To demostrate fungi in tissue sample which are positive
due to high carbohydrate content in their cell wall
like. Candidiasis
Actinomycosis
Histoplasmosis
Blastomycosis
• It can be used to confirm the metastatic malignancy.
Finding mucin positive tumor cells in an area that does not
contain mucin producing cells would indicate the tumor
did not arise from that area.
• In diagnosis of ALL- block positivity and AML- diffuse
cytoplasmic positivity in M5, M3 and cytoplasmic
granular/block positivity in M6 and cytoplasmic granular
and blebs positivity of blast in M7.

Gastric signet ring cell carcinoma on PAS stain

• Presence of glycogen will be evidenced by loss
of staining after enzyme treatment when
compared to the untreated sections.
PAS StainPAS with Diastase

Mucin
• Mucins are high moleqular weight substance containing
acidic groups and divided into two major categories-
epithelial and stromal.
• Epithelial mucin (membrane bound or secreted) are
composed of central protein core and sialic acid
containing chains of carbohydrate(polysaccharides)-
that may be sulfated or nonsufated.
• Stromal mucin known as glycosaminoglycans contain
hyaluronic acid may be sulfated or non sulfated. They
are better known as myxoid substance.
• Their functions are lubrication, chemical barrier and cell
signaling.

The types of mucin are as follows:
Neutral - Found in glands of stomach and in prostate. They
stain with PAS but not with Alcian blue, colloidal iron and
mucicarmine.
Acid (simple, or non-sulfated) - Are the typical mucins of
epithelial cells containing sialic acid. They stain with PAS,
Alcian blue at pH 2.5, colloidal iron. They resist
hyaluronidase digestion. E.g. salivary glands.
Acid (simple, sulfated -mesenchymal) - These contain
hyaluronic acid and are found in tissue stroma. They do
not stain with PAS, but do stain with Alcian blue at pH 2.5,
colloidal iron, and metachromatic dyes. They digest with
hyaluronic acid. They can be found in sarcomas.

• Acid (complex, epithelial) - These are found in
adenocarcinomas of colon, breast,ovary, mucoepidermoid
carcinoma . PAS is usually positive. Alcian blue is positive at
pH 1, and colloidal iron, mucicarmine, and metachromatic
stains are also positive. They resist digestion with
hyaluronidase.
• Acid (complex, connective tissue) - Found in tissue stroma,
cartilage, and bone and include substances such as
chondroitin sulfate or keratan sulfate. They are PAS
negative but do stain selectively with Alcian blue at pH 0.5.

Stains for mucin
• Alcian blue
• PAS
• Mayer mucicarmine
• Hale colloidal iron stain

Alcian blue
• PRINCIPLE
• Alcian blue is a copper phthalcyanin dye and contains
positively charged groups capable of salt linkage with
certain polyanions. These polyanions consist of the
sulphate and carboxyl radicals of the acid mucins
and the phosphate radicals of the nucleic acids that
do not react. Consequently, only the acid mucins are
stained. By varying the pH of the solution of Alcian
blue more information can be gained concerning the
types of acid mucin present.

Alcian blue
• At pH 2.5 it stains – Acidic mucin that include acid-simple or
non-sulfated and acid –simple mesenchymal mucin.
• At pH 1 it stains acid-complex or sulfated mucin and at pH
0.5 it stains acid-complex connective tissue mucin.
• It does not stain neutral mucin.
• Dyes : 1%Alcian blue in 3%acetic acid
Results : Acid MPS – deep blue
Nuclei – faint blue
• PAS-Alcian blue is best ‘pan-mucin’ stain combination, since it
demostrates mucin both neutral, slightly acidic and highly
acidic.

REAGENTS:
 3% Glacial Acetic Acid
 Alcian Blue Solution:
3% glacial acetic acid-
100.0ml
Alcian blue -1.0 gm
Mix, adjust pH to 2.5, using
acetic acid. Filter, add a
crystal of thymol, label with
initial and date.
 Nuclear Fast Red
(Kernechtrot):
Aluminum sulfate 25.0 gm
Distilled water 500.0 ml
Nuclear fast red 0.5 gm
Dissolve the aluminum
sulfate in the water. Add
the nuclear fast red,
dissolve
with aid of heat. Filter,
add a crystal of thymol
SPECIMEN
Standard paraffin section fixed in neutral buffered formalin

PROCEDURE: ( Alcian Blue)
1. Hydrate slides to distilled water.
2. Stain with3% acetic acid, 3 minutes.
3. Stain with alcian blue solution, for 30 seconds.
4. Wash in running water for 2 minutes, rinse in
distilled.
5. Nuclear-fast red, 5 minutes, wash in tap water.
6. Dehydrate, clear, and mount.

USES
• It is useful to differentiate gastric metaplasia into
complete and incomplete. Complete metaplasia is
small intestinal type, shows well defined brush border
and well formed goblet cells. Whereas incomlete
metaplasia is colonic type and shows multiple
irregular mucin droplets of varying size in the
cytoplam and absence of brush border.
• It is useful in diagnosis of salivary gland pleomorphic
adenoma, mucoepidermoid carcinoma, in stroma of
chordoma at sacrococcygeal region.
• Excessive amounts of non-sulfated acidic
mucosubstances are seen in mesotheliomas, certain
amounts occur normally in blood vessel walls but
increase in early lesions of atherosclerosis.

The goblet cells of the gastrointestinal tract are filled with
abundant acid mucin and stain pale blue with this Alcian blue stain.

Colonic mucosa showing sialomucin that have acid and
neutral mucopolysaccharide stain purple on alcian blue-PAS.

Mixed salivary gland. Acid mucopolysaccharides in mucous
cells are stained blue (Alcian blue at pH 2.5).

Chordoma on alcian blue stain at ph 2.5

MUCICARMINE STAIN –SOUTHGATE’S – MUCIN
PRINCIPLE :-
Positively charged carmine-mucicarmine complex
bonds with the negatively charged acid mucin. The
carmine solution is highly complex. Strongly
sulphated mucins are variable in their reaction,
neutral mucin do not stain at all, while other acidic
mucin stain strongly.

REAGENTS:
• Southgate's Mucicarmine Solution:
Carmine, alum lake -1.0 gm
Aluminum hydroxide -1.0 gm
50% alcohol -100.0 ml
Aluminum chloride, anhydrous- 0.5 gm
• Mayer's Hematoxylin:

PROCEDURE: ( Mucicarmine stain)
1. Deparaffinize and hydrate to distilled water.
2. stain with mayer's hematoxylin for 2 minutes.
3. Wash in running tap water.
4. Differentiate in acid alcohol.
5. Rinse in tap water.
6. Blue in scott’s tap water substitute.
8. Stain with mucicarmine solution for 20
minutes.
10. Dehydrate, clear and mount.

Uses:
• Mucicarmine is useful to identify acidic mucin.
• It is used to identify adenocarcinoma particularly
of gastrointestinal tract.
• It also stains the capsule of fungus- cryptococcus
neoformans found in lung and nervous tissue of
immuno-compromised patient.

Colonic Mucosa showing sialomsucin --
- magenta
RESULTS:
Mucin - magenta
Nuclei - black
Other tissue elements- yellow

Cryptococcus neoformans in lung of AIDS patient
capsule stains red

HALE’S COLLOIDAL IRON STAIN
• Positive staining with hale’s colloidal iron
stain is considered diagnostic feature of
chromophobe renal cell carcionoma and has
been used as discriminatory feature to
differentiate it from other renal tumour.
Result
• Acid mucopolysaccharides: blue
• Nuclei: red

Reagents
• Acetic acid 12%
• Potassium ferrocyanide 2% aqueous
• Hydrochloric acid 2%
• Colloidal iron suspension
• Working colloidal solution
Colloidal iron suspension 1 vol
Acetic acid 1 vol
• Perls’ solution
2% potassium ferrocyanide 1 vol
2% hydrochloric acid 1 vol

Procedure: (Hale’s colloidal iron stain)
• Bring the section to water level.
• Place into working colloidal iron for 15-20 minutes.
• Wash with distilled water.
• Wash with running tap water for 5 minutes to remove all
traces of colloidal iron.
• Place into freshly made perls’ solution for 10 minutes.
• Counter stain with neutral red for 1 minute.
• Dehydrate, clear ad mount.

a.Classic chromophobe RCC
b. classic chromophobe RCC
on hale’s colloidal iron stain
c. Eosinophilic chromophobe
d. Eosinophilic chromophobe
on hale’s colloidal iron stain
e.oncocytoma
f. oncocytoma diffusely
negative cytoplasmic
reaction with hale‘s colloidal
iron stain

RETICULIN STAIN-GOMORI’S METHOD
• Reticulin stain demonstrate both reticular fibers
and basement membrane material.
• Reticular fibers consist of very thin fiber of type
III collagen which are widespread in connective
tissue throughout the body.
• Basement membrane are largely composed of
type IV collagen and laminin.

Principle:
Reticulin fibers have little natural affinity for
silver solutions. On treatment with potassium
permangenate it produce sensitised sites on fibers
where silver deposition can be initiated. The silver is
in the form readily able to precipitate as metallic
silver. The optimal ph for maximum uptake of silver
ions is 9.0. A reducing agent formalin causes
deposition of silver in the form of metal.

• Dyes used: Silver nitrate 10%
NaOH 10%
KMnO4 1% aqu.
oxalic acid 5% aqu
Iron alum 2.5%
Formalin 10%
• Control: Cirrhosis of liver
• Result: Reticular fiber – Black
Nuclei- Gray
Other elements- According to counter
stain used

Pocedure :
•Dewax the section and bring to water
•Treat with 1% potassium permanganate solution for 3 min
•Rinse in tap water
•Bleach in 5% oxalic acid solution for 2 minute
•Wash well in tap water
•Treat with 2.5% iron alum for 1 min
•Rinse in distilled water
•Impregnation with ammonical silver solution for 3 min
•Distilled water wash
•Reduce in 10% aqueous formalin with agitation for 3 minute
•Dehydrate ,clean and mount.

Uses of reticulin stain
1. Diagnosis of liver cirrhosis.
2. To distinguish epithelial neoplasms from non-
epithelial neoplasms.
Foci of carcinoma have reticulin around tumour
nest but not in between tumour cell, whereas in
most sarcomas and large cell lymphoma reticulin
separates single cells.
3. To differentiate between in-situ and invasive
carcinoma

Reticulin fiber- Black
Nuclei -Black

Trichrome stain
• This stain is mainly used to evaluate the type
and amount of extracellular material like-
collagen, fibrin, muscle and elastic fiber.
Various technique includes:
• Masson trichrome stain
• Van gieson stain
• Mallory, Phosphotungstic or phosphomolybdic
acid stain
• Verhoeff-Van Gieson(VVG) stain

The general rule in trichrome staining is that
the less porous tissues are colored by the
smallest dye molecule; whenever a dye of large
molecular size is able to penetrate, it will
always do so at the expense of the smaller
molecule.

Masson trichome stain
• This method is used for detection of collagen
fibers in the tissues such as skin, stomach,
intestine and lung.
• The collagen fibers will be stained blue and their
nuclei will be stained black.

Masson trichrome stain
Principle:
As per the name 3 dyes are used which
selectively stain muscle, collagen fibers, fibrin
and erythrocytes. As general rule in trichrome
stain less porous tissues are stained by small
dye molecule. Acid fuchsine stain all the
connective tissue, PMA( phosphomolybdic
acid) competes with fuchsine and gain access
to collagen displacing fuchsine. If reaction
stopped at appropriate time, collagen will be
free to be stained by Methyl Blue.

• Reaent used – Weigerts iron hematoxylin solution
- Phosphomolybdic- phosphotungstic
acid solution
- Biebrich scarlet acid fucshin solution
- Aniline blue solution
- 1% acetic acid solution
• Result - Nuclei : Black
Collagen : Blue
Cytoplasm, Muscle, RBC : Red
• Positive control : skin, lung, stomach, intestine

Pocedure: Masson trichrome stain
•Dewax the section and bring to water
•Stain with weigerts iron hematoxylene for 10 minute
•Rinse well in running tap water for 10 minute
•Wash in distilled water
•Stain in biebrich scarlet acid fuchsin for 10-15 minute
•Wash in distilled water.
•Differentiate in Phosphomolybdic-phosphotungstic acid
solution for 10-15 minute
•Transfer section directly(without rinse) to aniline blue
solution and stain for 5-10 minute

•Rinse briefly in distilled water and differentiate in1%
acetic acid solution for 2-5 minute
•Wash in distilled water
•Dehydrate through alcohol, clear in xylene and mount in
DPX.

Result
Collagen- Blue
Cytoplasm, RBCs- Red
• Membranoproliferative glomerulonephritis on masson
trichome stain

Uses of Masson trichrome stain
• It is used to differentiate between collagen and smooth
muscle in tumour.
• To identify increased collagen deposition in condition
like cirrhosis, keloid, benign prostatic hyperplasia,
membranoproliferative glomerulonephritis etc.

VAN GIESON STAIN
Principle
When using combined solution of picric acid and
acid fuchsin, the small molecules of picric acid penetrate all
the tissue rapidly, but are only firmly retained in the close
textured red blood cells and muscle. The larger molecules
of ponceau S displaces picric acid molecule from collagen
fibres, which has larger pores and allow larger molecules to
enter.
It is used for detection of collagen.
Result -- Nuclei : Blue / Black
Collagen : Red
Cytoplasm, muscle, fibrin, RBCs : Yellow

• Results:
• Collagen – deep red
• Muscle, – yellow
cornified epithelium
• Nuclei – blue to black

Phosphotungstic acid- Hematoxylene
test (PTAH) test:
• Principle
There is much more phosphotungstic acid in the
solution then hematein. The phosphotungstic acid binds all
the available hematein to form blue lake pigment. This lake
stains muscle cross striations, fibrin, nuclei, and other
tissue elements blue. The rest of phosphotungstic acid
stains the red-brown components, such as collagen.
USES
• Traditionally used for demonstration of muscle striations,
glial cells and fibrin.
• This technique has been largely replaced by
immunohistochemistry techniques

RESULTS
• Muscle, cross striations : Blue- black
• Fibrin and neuroglia – Deep blue
• Connective tissue: Pale orange-pink to brownish red
• Bone and cartilage- yellowish to brownish red

Skeletal muscle striation on mallory’s PTAH stain

Verhoeff-Van Gieson(VVG) stain :
• This method is used for identifying elastic fiber in tissue
such as skin, aorta etc.
• Result – Elastic fiber: Blue-black to black
- Nuclei: Blue to black
- Collagen: Red
- other tissue elements: Yellow

Vessel wall on Verhoeff van gieson stain

This section shows elastic cartilage and elastic fibers (arrow),
which are dark-stained linear structures embedded
in the cartilage matrix.

Fibrin
• Fibrin is formed by polymerization of smaller
soluble fibrinogen.
• Found in tissue damage and acute inflammation,
fibrinoid necrosis in vessel wall.
• Stained by : Mallory PTAH
MSB Stain

MSB (Maritus, Scarlet, Blue)Stain for
fibrin:
• This is trichrome method for selective demostration of
fibrin.
• Dyes Used :
Maritus Yellow
Crystal Scarlet
Methyl Blue, PTA.
• Results :
Fibrin : Red
RBC’S : Yellow
Muscle : Red
Collagen : Blue

Fibrin stains red, collagen stains blue, muscle stains
red, nuclei stains blue/black, red cells stain yellow

Fibrin stains red, collagen stains blue, muscle stains red,
nuclei stains blue/black, red cells stain yellow

Stain for Amyloid
Amyloids are insoluble fibrous protein. They arise
from inappropriately folded protein and polypeptides
present naturally in the body. These misfolded structure
alter their configuration and interact with each other or
other cell components forming insoluble fibrils. Abnormal
accumulation of amyloid fibrils leads to amyloidosis and
play role in various pathological disease.
Stains used to demostrate amyloid:
• Congo red
• Crystal/methyl violet

Congo red stain
Principle :
Amyloids are homogeneous and eosinophillic,
proteinaceous deposits, that are extracellular and
may become sufficiently large enough to cause
damage to surrounding tissues. When stained with
the congo red stain the amyloid will show
birefringence an apple green color, under the
polarizing microscope.

Reagent:
• Solution a - 0.5% Congo red in 50% alcohol
• Solution b - 0.2% potassium hydroxide in 80%
alcohol
• RESULTS:
Amyloid, elastic fibers, --- red to pink
eosinophilic granules,
Nuclei – blue

PROCEDURE: (Congo red stain)
1. Deparaffinize and hydrate to water.
2. Stain in Congo red solution for5 minutes.
3. Differentiate with alcoholic potassium hydroxide
solution for 3-10 seconds.
4. Wash in water, stain nuclei in alum hematoxyllin,
differentiate and blue.
7. Dehydrate, clear in xylene & mount.

It is used to demostrate amyloid deposits in
• renal amyloidosis- in patients on dialysis for
long time
• Medulary carcinoma of thyroid,
• Vessel wall in case of Alzheimer’s disease
• Cardiac arrhythmias, etc.

Positive red staining is present around the large central artery and
a smaller vessel to its upper right. The right panel shows the green
birefringence that is diagnostic of amyloid when the Congo red
stain is viewed with polarized light.
All amyloids have a fibrillar ultrastructure that gives this reaction.

Crystal/Methyl violet for amyloid
• Crystal violet or methyl violet stain are used for
metachromatic amyloid staining.
• They stain amyloid as purple-red in blue background.
Reagents used
• Crystal/methyl violet
• 95% alcohol
• 1% aqueous ammonium oxalate
• 0.2% acetic acid

Preparation of solutions
• Dissolve 2 gm crystal/methyl violet in 20ml 95%
alcohol and 80ml of 1% aqueous ammonium oxalate
dissolve using minimum of heat. Cool and filter

Procedure
• Stain in crystal/methyl violet solution for 5 minute.
• Wash and differentiate in 0.2% acetic acid controlling
differentiation microscopically arresting differentiation
in water and repeating untill good contrast is obtained
between amyloid and background.
• Wash and mount in DPX.
RESULT
Amyloid - Purple
Backgrond - Blue

Amylod – purple
Background - blue

Stains for Lipid
- Oil Red O
- Sudan Black B

Oil red o stain
PRINCIPLE :
Staining with oil-soluble dyes is based on
the greater solubility of the dye in the lipid
substances than in the usual hydroalcoholic
dye solvents.
Result
Lipid – Red
Nuclei- Blue

• REAGENTS :
• 60% isopropanol
• Alum hematoxylin:
• Glycerin Jelly mounting medium
• Oil Red O working solution:
o To make oil red O stock solution
• Oil red O - 0.5gm
• Isopropanol 100 ml
Dissolve the dye in isopropanol, using the very
gentle heat of water bath. This is the stock solution.
o To make working oil red O solution
• Dilute 30ml of stock stain with 20ml of distilled water
and allow it to stand for 10 minutes, filter into coplin jar
and cover immedeiately.
• Working solution should be made fresh each time.

PROCEDURE : (Oil red O stain)
• Fix slides in 10% formalin if fresh.
• Wash well in tap water for 1-10 minutes to drain off
excess water.
• Rinse with 60% isopropanol.
• Stain with freshly prepared oil red O working solution
for 15 minutes.
• Rinse with 60% isopropanol.
• Lightly stain nuclei with alum hematoxylin 5 dips.
• Rinse with distilled water.
• Mount with aqueous mounting media, Glycerin Jelly.

Fat emboli seen as red dot within capillaries of lung on Oil red
O stain

Uses
• Oil red O stain is used for staining neutral
triglycerides, lipids and lipoprotein.
• Tumors arising from fat cells (liposarcomas) can be
differentiated from other types of tumors.
• Fat occurring in an abnormal place, such as fatty
emboli that may develop after either a bone fracture
or an injury that crushes a fatty body area.
• Lipid storage disorder like nieman-pick disease
• To demonstrate fat or lipids in conidition like fatty
liver.
• In hematological condition like burkitt’s lymphoma
and sea-blue histiocytic syndrome

FAT - SUDAN BLACK B - PROPYLENE GLYCOL
PRINCIPLE :
Sudan Black B is a dye that is insoluble in water but
dissolves in fat. Therefore this dye will accumulate in fat
globules within cells.
• It is slightly basic dye and will combine with acidic
groups in compound lipids, thus staining phospholipids
also.
Result
Fat- Blue/ Black
Nuclei- Red

REAGENTS :
• 85% Propylene Glycol:
Propylene glycol -85.0 ml
Distilled water -15.0 ml
• Hematoxylin:
• Sudan Black B/Propylene:
Sudan Black B -0.7 gm
Propylene glycol -100.0 ml

PROCEDURE : ( Sudan black stain)
1. Fix slides in 10% formalin if fresh.
2. Wash well in tap water, rinse in distilled water, drain
off excess water.
3. Propylene glycol, two changes, 5 minutes each.
4. Stain with Sudan Black, 7 minutes, agitate.
5. Dip in 85% Propylene glycol, 3 minutes.
6 Rinse in distilled water.
7. Stain with Nuclear Fast Red, 3 minutes.
8. Wash in water.
9. Wash in tap water, rinse in distilled.
10. Mount with aqueous mounting media, Glycerin Jelly.

Uses
• Sudan black is used for staining neutral
triglycerides, lipids, lipoprotein and phospholipid.
• In hematological disorder it can be used to
differentiate blast as it stains the myeloblast, but
does not stain lymphoblast.

Myeloblast on Sudan black stain

MASSON FONTANA METHOD- FOR MELANIN
Melanin is a nonlipid, non hematogenous
pigment. It is a brown-black pigment present
normally in the hair, skin, retina, iris and certain
parts of the CNS.
PRINCIPLE:
 Melanin is insoluble in organic solvents but soluble in 1M
sodium hydrooxide.
 It is slowly bleached by strong oxidising agents.
 The solutions of ammoniacal silver nitrate are reduced by
melanin to black metallic silver this is the basis of Masson
Fontana method for demostrating melanin.

USES:
• To identify melanin and argentaffin
granules.
• In diagnosis of malignant melanoma
• Argentaffin granules are found in
carcinoid tumors.

Melanin pigment of skin showing
RESULTS:
Melanin, argentaffin
granules - Black
Nuclei -red

Melanin pigment in cells of malignant
melanoma, Fontana-Masson stain.

VON KOSSA METHOD FOR CALCIUM
PRINCIPLE:
Tissue sections are treated with silver nitrate
solution, silver is deposited by replacing the the
calcium and then it is reduced by the strong light
and visualized as metallic silver.
USE:
Abnormal deposits of calcium may be found in any
area of the body. With the H&E stain, calcium
appear deep blue-purple. On von kossa method it
appear black.

Coronary artery showing
calcified atheromatous plaque
RESULTS :
Calcium salts -black
Nuclei -red
Cytoplasm -pink

PERL’S- PRUSSIAN BLUE REACTION FOR
IRON
PRINCIPLE :
Dilute mineral acid hydrolysis releases ferric
iron from protein bound tissue deposits, which in
the presence of ferrocyanide ions, is precipitated as
highly coloured and highly water soluble complex,
potassium ferric ferrocyanide- prussian blue.
• Ferrous ion do not produce a coloured reaction.
• Tissue deposits containing ferric iron are invariably
hemosiderin

USE:
• To demonstrate ferric iron in tissue sections.
• Small amounts of iron are found normally in
spleen and bone marrow.
• Excessive amount are present in
hemochromatosis- with deposits found in the
liver and pancreas and hemosiderosis- with
deposits in the liver, spleen, and lymph nodes.
• To access the bone marrow iron content

REAGENTS:
• 2% Potassium Ferrocyanide:
Potassium ferrocyanide - 2.0 gm
Distilled water - 100.0 ml
• Neutral-fast Red:
Neutral red - 1gm
Distilled water - 100ml
Glacial acetic acid - 1ml
• 2% Hydrochloric Acid:
Conc. Hydrochloric acid, - 2.0 ml
Distilled water - 100.0 ml
CONTROL: Postmortem lung – high number of heart
failure cells that contain hemosiderin

PROCEDURE: ( Perl’s prussian blue)
1. Deparaffinize and hydrate to distilled water.
2. Stain with mixture of equal volume of aqueous 2%
potassium ferrocyanide and 2% HCl solution for 30
minutes.
3. Thorough wash with distilled water.
4. Counter stain with 0.5% Neutral red lightly.
5. Wash in tap water.
6. Dehydrate, clear, and mount.

Hemosiderin in liver
RESULTS:
Iron (hemosiderin) -blue
Nuclei -red
Background - pink

Staining for Microorganism
- Ziehl- Neelsen (ZN) stain
- Wade fite faraco stain
- Gomori Methenamine (hexamine) silver stain
- Warthin-Starry stain
- Giemsa stain
- Gram staining

Ziehl-Neelsen (ZN) Stain for Mycobacterium
Bacillus
Mycobacteria are difficult to demonstrate by the
Gram technique because they possess a capsule
containing a long chain fatty acids that make
them hydrophobic.
Phenolic acid or heat may be used to reduce the
surface tension and increase the porosity.

• PRINCIPLE
Mycobacterias (tubercle bacilli) have a lipid-rich cell
wall which is capable of taking up strong phenol dye
solutions (eg. carbol fuchsin solution) such that the
dye is retained upon subsequent differentiation in
acid or alcohol. They are said to be acid and alcohol
fast (AAFB = acid and alcohol fast bacilli).
Results
• Mycobacteria - Red
• Background - pale Blue

Method
• Flood sections with carbol-fuchsin and heat to steaming
by passing the flame of spirit swab underneath the slides
on metal rack till, vapour just being formed.
• Wash the slides in distilled water. Shake off excess liquid.
• Decolourise the slides with 20% H2SO4
• Wash well in tap water, till no more red colour runs off
the surface when the slides is dipped in water
• Wash thoroughly with water.

• Counter stain in methylene blue solution, 30
seconds.
• Blot and differentiate by alternate dehydration and
rehydration until the background is a delicate pale
blue.
• Examine microscopically screening at high power
and confirming all suspicious organism with an oil
immersion lens.

Acid fast bacteria seen on intestinal biopsy

Wade fite faraco stain
• This stain is used for staining leprocy bacilli in
tissue sample.
• Leprocy bacilli are much less acid and alcohol fast
than tuberculous bacilli. Therefore alcohol is
removed from hydrating and dehydrating steps
and 10% sulphuric acid is used as decolouriser in
place of acid-alcohol solution.
• The section are also deparaffinised using ground
nut oil and xylene mixture to protect more
delicate waxy coat of lepra bacilli.

PROCEDURE
• Warm section and deparaffinised using mixture of two part
xylene and one part of ground nut oil for 10 minutes.
• Blot dry and wash in water untill section is uniformly
wetted.
• Stain with carbol fuschin for 20-30 minutes.
• Wash in tap water and blot dry.
• Decolorize in 10% sulphuric acid till no more red colour
appear while dipping in water.
• Wash in tap water and counter stain with 0.2% methylene
blue for 5-10 seconds.
• Blot dry and do not mount.
• Smears should be seen in oil immersion lens.

Leprrocy and other mycobacteria- red
Background- blue

Warthin-Starry method for spirochetes
(Warthin & Starry 1920)
PRINCIPLE
Organisms are demonstrated by silver
impregnation technique.
Solutions
Acetate buffer. pH 3.6
Sodium acetate 4.1 g
Acetic acid 6.25 ml
Distilled water 500 ml
1 % silver nitrate in pH 3.6 acetate buffer.

• It can also be used to demostrate
- Helicobacter pylori in gastric mucosa
- Leptospira organism in renal biosy
- Cat scratch disease associated bacilli on
lymph node biopsy

H. Pylori on gastric mucosa on Warthin starry stain

Leptospira organism Renal biopsy

Result:
Organisms - black
Background - golden
yellow
Cat scratch disease bacilli in lymph node biopsy

Gomori methenamine silver stain for
fungi
• GMS staining is a silver staining technique for
demonstrating fungi in tissue sections.
• It is primarily based on staining the polysaccharides
in fungal cell walls, and can be used to demostrate
the basement membrane.
 PRINCIPLE
This method depends upon the reduction of the
silver by the aldehyde groups produced after
oxidation of fungal wall components with chromic
acid.

Pneumocystis carinii
Result:
Fungi, pneumocystis carinii,
melanin - black
Mucin and glycogen-
- grey-black
RBCs - yellow
Background - pale green

GMS stain for Cryptococcus neoformans

Basement membrane on GMS stain

Giemsa stain
• PRINCIPLE
This method is a modified version of the original
Giemsa technique used for haematological smears
and gives good results for sections. Giemsa is a
Romanowsky stain which contains azure B and eosin
Y and is capable of making subtle distinction in
shades of staining. The acidic groupings of the
nucleic acids and proteins of the cell nuclei
determine their uptake of the basic dye azure B and
the presence of basic groupings result in an affinity
for acidic dyes and their staining by eosin.

• It can be used for histopathological diagnosis of malaria
and some other spirochete protozoan, and blood
parasites.
Results
• Micro-organism, fungi - purplish-blue
parasites
• Nuclei- blue to violet
• Erythrocytes- salmon pink
• cytoplasm - light blue
• Collagen, muscle and bone - pale pink

Aspergillus fumigatus on giemsa stain

Gram method for Bacteria
• Gram staining differentiate bacteria into 2 classes
depending on their cell wall structure and composition
• Gram positive and Gram negative.
• PRINCIPLE
Crystal Violet stains the nucleic acids of the bacteria (and
background tissue) and after treatment with iodine, the
sections are differentiated in acetone and counterstained
with basic fuchsin. The tissue background and Gram-
negative bacteria lose their blue staining and are
subsequently stained with counter stain basic fuchsin.
Gram-positive bacteria resist the decolourisation and retain
the crystal violet/iodine blue staining.

Result:
Gram-positive organisms - blue
Gram – negative organisms - red.

Cutaneous anthrax – Gram positive Bacilli stained blue on skin
biopsy

References
• Rosai and ackerman’s surgical
pathology- 10th edition
• Textbook of microbiology-
Ananthanarayan and paniker’s
• Internet

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