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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:
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.
• Wash with distilled water.
• Place into freshly made perls’ solution for 10 minutes.
• Wash with distilled water.
• 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:
• Muscle, – yellow
cornified epithelium
• 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.
• 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
- Oil Red O
- Sudan Black B
Oil red o stain
PRINCIPLE :
Result
Lipid – Red
Nuclei- Blue
• REAGENTS :
• 60% isopropanol
• Alum hematoxylin:
• Glycerin Jelly mounting medium
• Oil Red O working solution:
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.
Result
Fat- Blue/ Black
Nuclei- Red
REAGENTS :
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.
RESULTS:
Melanin, argentaffin
granules - Black
Nuclei -red
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.
RESULTS :
Calcium salts -black
Nuclei -red
Cytoplasm -pink
• 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
Nuclei -red
Background - pink
Hemosiderin in liver
Staining for Microorganism
Results
• Mycobacteria - Red
• Background - pale Blue
Method
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
PRINCIPLE
This method depends upon the reduction of the
silver by the aldehyde groups produced after
oxidation of fungal wall components with chromic
acid.
Result:
RBCs - yellow
Pneumocystis carinii
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
Toxoplasma gondii
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.
Gram Positive
Gram Negative
Cutaneous anthrax – Gram positive Bacilli stained blue on skin
biopsy
References