Special Stain PPT by G.P. Tiwari
Special Stain PPT by G.P. Tiwari
Special Stain PPT by G.P. Tiwari
MR G.P. TIWARI
Tata Memorial Hospital
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Outline of the lecture
Technical aspects of histochemical stains
and its application
Stain principle
Control source
Procedures and preparation
Its results
basic troubleshooting
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AFB
The Ziehl-Neelsen stain, also
known as the acid-fast stain, was
first described by two German
doctors; Franz Ziehl a bacteriologist
and Friedrich Neelsen a
pathologist. IN 1882
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Application
IT IS USED TO DEMONSTRATION
OF ACID FAST BACILLI BELONGS
TO GENUS MYCOBACTERIUM
TUBERCULOSIS AND M. LAPRAE.
CONTROL: AFB CONTAINING
TISSUE
Principle
Mycobacterium( mycolic acid and lipids)
acid solution
Preparation of reagents
1) Zeil Neelson carbol fuchsin solution
Basic fuchsin--- 1 gm
Absolute alcohol-10ml
5% phenol solution-100ml (5 grams phenol dissolved in
100ml of distilled water Mix well. Filter into brown
color bottle.
2)1% acid alcohol
3)1% Methylene blue
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PROCEDURE
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Results
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MODIFICATION
MYCOBACTERIUM LAPREY BACILL IS LESS
ACID FAST THEN AFB
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Remark
We must filter the ZNCF stain before staining
with watman filter paper
Proper decorization must be carried out
After this thorough washing is required to
remove acid.
We must filter methylene blue with watman
filter paper during counterstaine
Counterstaining should be light otherwise it
will mask the bacilli.
This procedure can be performed either using
heat or without using heat
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GMS
It was first described
by Gomori in1937
later on it was
modified by Grocotts
in 1955
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APPLICATION
This technique is
mainly used to
identify fungi in the
sections
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Principle
The mucopolysaccharide component of
fungal cell wall is oxidized to release
aldehyde groups. The aldehyde groups
react with silver nitrate and silver stain
precipitates along the fungal walls.
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SILVER INPREGNATION
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Preparation of reagents
Working solution
25ml stock solution + 25ml distilled water + 3ml 5%
borax solution.
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Gomori/ Grocotts Modification
Oxidize with chromic acid at room temperature for 1 hour or by
periodic acid for 10 min
Fungus-black
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Remarks
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application
1.Bone marrow – Iron deficiency
anemia, Myelodysplastic syndrome,
Refractory
anaemias.
2. Hemosiderosis-due to excess
therapeutic iron or blood transfusion,
demonstrated
in spleen, bone marrow, liver.
3. Hemachromatosis
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Principle
Ferric iron
of the hemoglobin
+
2% ferrocyanide in2% hcl
Ferric ferrocyanide
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Procedure
2%Potassium 30
2% conc. HCL
ferocyanide min
1% Eosin
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Prussian Blue
Demonstration of hemosiderin pigment.
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Alizarin Red S Method
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Alizarin Red S Method
It was first introduce by
mc.gee –Russell in1958
it is used to
demonstrated calcium
salts in the tissue
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Principle
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application
Alizarine red S
Alizerine red S
calcium complex (orange red is formed)
Birefringent in polarized
light
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Procedure
Alizarine red S
(5min)PH-4.2
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GIEMSA
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Principle
Giemsa-
1.Methylene blue-stains
acidic cell components
2.Eosin-stains basic cell
components
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Preparation of reagents
1 Gms Giemsa powder
+54 ml Glycerin
preheated 60 0 C.+ add
84ml Methanol. Mix well
and filter the stain
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Preparation of reagents cont
Buffer solution
Potassium dihydrogen phosphate—2.72 gms
Distilled water— 100 ml
Sodium hydroxide – 0.8 gm
Distilled water – 100ml
50 ml of Potassium dihydrogen phosphate is
mixed with 23.6 ml of NaOH. The pH of the
solution is adjusted to 6.8.
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Procedure
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Procedure for bone marrow
imprints &smears
Smears are fixed in methanol for 30 min.
Stain in working Giemsa solution 30
minutes.
Wash in water for 5 minutes.
Air dry smears.
Clear in Xylene , mount in DPX.
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Result – Bone Marrow Imprint
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RESULTS
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Gram Staining
The method is named after its
inventor, the Danish scientist
Hans Christian Gram who
developed the technique in
1884
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application
The purpose is to
demonstrate Gram negative
& Gram Positive organisms
in the tissue.
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Principle OF Grams stain
Gram +ve Insoluble lake
barrier
Gram +ve
Lipo poly
saccharide
pepidoglycan
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BROWN AND BRENN
Crystal violet (1 min)
Acetone (2dip)
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BROWN AND BRENN
ORAL FLORA
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Melanin
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application
For the detection of Melanin
pigments and Argentaffin
granules can be demonstrated by
this method. Melanin is an
intracellular pigment it is usually
located in skin retina substantia
nigra of brain and in hair.
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Principle
Melanin
reduction
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Procedure
Silver solution 600C for 1 hour.
2% Sodium thiosulphate
1% Eosin
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Prepration of silver nitrate
5ml 10% Silver nitrate
+
45 ml distilled water (1 hr at 600c )
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RESULTS
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Precautions
Check under microscope, if required
repeat
Bleaching step
Preferably use Poly-L- Lysine coated
slides.
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MELANIN BLEACH
PIGMENTED MELANOMA
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Chloroacetate esterase
Pararosaniline method
Introduction :
Chloroacetate esterase is the only enzyme
which can be demonstrated in paraffin
section.
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Principle
Leukocyte esterase
hydrolyses
Derivative of Naphthalene
+
Diazonium salt
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Preparation of reagents
1) substrate solution :
Naphthol As. D. Chloroacetate- 10 ml
N-N dimethyl formamide –1 ml:
Pararosanilin– 0.4gms, distilled water – 0.4ml, conc.
HCl – 1.6ml.
sodium nitrite – 0.4gms, distilled water – 10ml.
Concentrated HCl – 8.35 ml ,distill water 91.65 ml.
Sodium barbital powder – 1.03gm, distilled water –
100ml.
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Procedure
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Result
Reactive lymph node -neutrophils
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