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Supravital staining

Sagar Sharma
B.SC.MLT
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Introduction
Staining
is an auxiliary technique used in
microscopy to enhance contrast in the
microscopic image.
Stains and dyes are frequently used in
biology and medicine to highlight
structures in biological tissues for viewing,
often with the aid of different
microscopes.
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Vital stain
A vital stain in a casual usage may mean a
stain that can be applied on living cells
without killing them
Vital stains have been useful for diagnostic
and surgical techniques in a variety of
medical specialties.
In supravital staining living cells that have
been removed from an organism, whereas
intravital staining is done by injecting or
otherwise introducing the stain into the body.
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Supravital staining
Supravital staining is a method of staining
used in microscopy to examine living cells that
have been removed from an organism.
supravital stain may have a greater toxicity, as
only a few cells need to survive it a short while.
As the cells are alive and unfixed, outside the
body, supravital stains are temporary in nature.
Either the smear is prepared from sediment or
directly sample is used .

Procedure
As per the sample taken .
Smear is prepared from sediment or whole
sample .
In some cases 1-2 drops of mixture of dyes
is taken , sediment is mixed and covered
with cover slip and observed under
microscope .
Reticulocytes count : 3-4 drops of
methyline blue solution+ venous or
capillary blood . Mix well and make a thin
smear , dry and observe under
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Types of supravital stain


New methyline blue
organic compound of thiazine class of
heterocycles.
It is used as a stain and as an
antimicrobial agent.
NMB is a staining agent used in diagnostic
cytopathology and histopathology.
Typically used for staining immature red
blood cells. It is a supravital stain
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Neutral red and James Green Stain


use for the differentiation of the
macrophages and leukocytes from
neoplastic and mesothelial cells .
Result : macrophages and leukocytes
are brilliant red
neoplastic and mesothelial cells are
not stained .
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Brilliant cresyl blue


Brilliant cresyl blue is an example of
supravital stain used for counting
reticulocyte.
Using Brilliant Cresyl Blue, the
reticulocytes will show a "reticular" (meshlike) network.
The mesh like network is of residual rRNA
that has not yet been extruded from the
cell.
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Crystal violet
is a triarylmethane dye.
The dye is used as a histological
stain and in Gram's method of
classifying bacteria.
In laboratory, solutions
containing crystal violet and
formalin are often used to
simultaneously fix and stain
cells grown in tissue culture.
It helps to preserve cells and
make them easily visible, since
most cells are colourless.
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Methyl violet
Methyl violet is a family of organic
compounds that are mainly used as dyes.
Depending on the amount of attached
methyl groups, the color of the dye can be
altered.
Medical use : as supravital stain .

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Nile blue
Nile blue (or Nile blue A) is a stain
used in biology and histology.
t may be used with live or fixed cells,
and imparts a blue colour to cell
nuclei.
It may also be used in conjunction
with fluorescence microscopy to stain
for the presence of
polyhydroxybutyrate granules in
prokaryotic or eukaryotic cells.
With the Nile blue derivative N-EthylNile Blue (EtNBA), normal and
premalignant tissues in animal
experiments can be distinguished

Nile blue in water.


Left to right: pH0,
pH4, pH7, pH10,
pH14.

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Toluidine blue
Toluidine blue is a basic thiazine
metachromatic dye with high affinity for
acidic tissue components.
It stains nucleic acids blue and
polysaccharides purple and also increases
the sharpness of histology slide images.
Used in renal pathology and
neuropathology.
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Major Uses of supravital staining in


laboratory
1. Reticulocytes count
.

The most common supravital stain is performed


on smear of human blood for reticulocytes by
using new methylene blue or brilliant cresyl blue
stains.
makes it possible to see the reticulofilamentous
pattern of ribosomes characteristically
precipitated in these live immature red blood
cells by the supravital stains.

2.Rate of red cell production from


bonemarrow
. By counting the number of reticulocytes

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3.Detection of
hemolytic anemia
When there is an increased
production of red blood
cells to overcome chronic or
severe loss of mature red
blood cells people often
have a markedly high
number and percentage of
reticulocytes.
A very high number of
reticulocytes in the blood
can be described as
reticulocytosis.

Supravital stain of a smear of


human blood from a patient
with hemolytic anemia. The
reticulocytes are the cells with
the dark blue dots and curved
linear structures (reticulum) in
the cytoplasm.
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4. Supravital staining of
spermatozoa
Supravital staining of human spermatozoa
is a useful technique to assess semen
quality.
relationship of eosin concentration to the
percentage of cells staining live.

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5.early diagnosis of oral mucosal


malignancies
Supravital staining of suspicious mucosal lesions in
oral cavity helps in the early diagnosis of malignant
lesions.
Toludine blue is the dye which is preferred for the
purpose of supravital staining.
This dye is acidophilic in nature and preferentially
stains acidic cellular components like DNA & RNA.
Malignant lesions of mucosa have large amounts of
acidic DNA & RNA due to the active cellular
proliferation which is occurring there.
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6.Flouresscence activated cell


sorting (FACS) analysis
Supravital staining can be combined
with cell surface antibody staining
(immunofluorescence) for
applications such as FACS analysis.

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References
John P.greer, John Foerster, George M Rodgers, Daniel
A. Arber Wintrobes Clinical Hematology.11th (ed.),
lippincot ,Williams and Wilkins , 2009
Wikipedia , staining: supravital staining 2013.
http://http://en.wikipedia.org/wiki/supravital_staining
Winifred Gray, Gabrijela Kocjan Diagnostic
Cytopathology.3rd (ed.), church hill Livingstone , 2010
drtbalu's otolaryngology, Role of Supravital staining
in early diagnosis of oral mucosal malignancies 2012

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