Histochemical stains-II

 MR G.P. TIWARI
 Tata Memorial
Hospital

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 What is special stain?

 Special stains are “special” because they

are not routine. They are applied to tissue
section in addition to Haematoxylin and
Eosin(H&E) stain to answer several
questions which is not possible with H&E
alone
 Significant
Special stains remain an important tool for
many pathologists and technologists providing a
powerful complement to immunohistochemistry,
flow cytometry, in situ hybridization, and other
diagnostic technologies that define a patient’s
medical profile.

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Importance of histochemical stains

 In identification of organisms (GMS ,AFB).

 In evaluation of kidney disease (PAS, MTS,
Congo red).
 In examination of liver biopsies.
 Useful in muscle biopsies.
 Identifying pigments.
 Used in the diagnosis of various tumors.

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 Stains which are discuss

 AFB
 PAS
 GMS
 MUCIN
 RETIC

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Outline of the lecture
 Introduction
 Stain principle
 Results/Technical aspects &
application

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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
CONTROL: Any tissue containing acid-fast
organisms.
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 PRINCIPAL

Acid fast bacilli ZNCarbol fuschin SOL

DECOLORISED
1%acid alcohol

COUNTERSTAIN
Acid fast bacilli METHYLENE BLUE

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 Results

Acid fast bacilli-bright red

Background-light blue
Improper decolorization

It will lead to false positive results.

To overcome this problem proper decorization must be
carried out
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 Application
It is used to demonstrate acid fast
bacilli which belong to genus
mycobacterium tuberculosis and
mycobacterium leprae
Gomori’s Methenamine
Silver (GMS)
It was first described by
Gomori in1937 later on it was
modified by Grocotts in 1955

CONTROL: Any tissue containing fungus.

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 GMS - Principle

Fungal cell wall (mucopolysaccharide)

Oxidation
Chromic acid
Aldehyde groups
Reduction
Silver stain

Black precipitate along fungal cell wall

 Results-

 Fungus-black
 Background-light green 4/29/2019 14
 Troubleshooting

Light staining if Dark staining if over-

incomplete reduction reduction

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Application Of G.M.S.

It is used widely as a screen

for fungal organisms.
 PERIODIC ACID SCHIFF( PAS )

The PAS stain was discovered by Joseph .F.

McManus, in 1944. Periodic acid-Schiff (PAS) is
a staining method used to detect
carbohydrates.

Control: Glycogen containing tissue like liver

Intestine
 PRINCIPLE OF PAS STAIN

Carbohydrates ( vicinal glycol group)

Periodic acid - oxidation

Dialdehydes
SCHIFFS REAGENT

MAGEMTA COLOUR COMPOUND

Esophageal candidiasis, PAS stain.
 Utility of pas
 PAS staining can be used to assist in the
diagnosis of several medical conditions:
 Glycogen storage disease ,Ewing's sarcomas
 Adenocarcinomas which often secrete
neutral mucins
 Paget disease of the breast
 Alveolar soft part sarcoma
 Stain macrophages in Whipple's disease
 Candida in lung
 Nodular Kidney disease
 PAS
How to test the reactivity of Schiff’s
reagent?

 Add few drops of reagent in water /

formalin.

 Magenta colour indicates reactivity.

 No colour indicates that reagent is to
be discarded.
 MUCIN STAIN

It was first applied by Mayer in 1896 later it

was modified by Southgate IN 1927.
It is used to detects mucins.
It is useful in certain intestinal carcinomas. It
is also detects fungi like Cryptococcus
neoformans. And useful in tumor site
determination.
 Mucin stains
PRINCIPLE: aluminum is believed to form a
chelation complex with the
carmine, changing the molecule to a positive
charge allowing it to bind
with the acid substrates of low density such as
mucins

 CONTROL: Small intestine

 RESULTS:

Mucin deep rose

Nuclei black
Other tissue elements yellow
 Reticulin stain

 In pathology the reticulin stain, is a popular

staining method in histology. It is used to
visualize reticular fiber and used extensively
in liver, Spleen, kidney histopathology.It was
discovered by Gordon & Sweet's in
 KMNO4

PRINCIPLE
Exposed reactive Aldehyde group of
reticulin fibre where silver deposit is
initiated
Na thiosulphate-removes
Reduces by Diamine silver unreacted silver
sol at ph 9

Silver is ppt in
metallic silver Renders the preparation
permanent & produce a
neutral black color of high
Reduced by formalin intensity

Black metallic silver Toning with gold chloride

 Technical Points

1. Make the silver solution fresh each time.

Ammoniacal silver solutions can be explosive when
allowed to dry. Immediately after use neutralize the
silver solution with saturated sodium chloride and
discard.
2. Clean glassware (used for preparing silver
solution) with glassware cleaning solution. Wash
thoroughly before use with tap water and use distilled
water for the final rinse.
3. It is important not to over dissolve the precipitate
at any stage as this will result in a decrease in
sensitivity.
basic important steps in retic
Ammonical silver solution ( Tollens' reagent )

10 % AgNO3 + 3%KOH sol

Precipitate appears.

Dissolve this drop wise with Conc. NH3

Add equal amount of D/W

Always use fresh solution

 procedure
KMnO4 & oxalic acid

Iron alum-washing (thorough)

Tollen’s reagent (AgNO3 + KOH)

10 % Formalin

Gold chloride

Na-thiosulphate
 Results-

Reticulin fibers ............................Black

Nuclei .......................................Red
 Conclusion
Even though special stains
have become less important
after introduction of
immunohistochemistry,
some of the special stains
remain indispensable
THANK YOU