STAINING TECHNIQUES IN MICROBIOLOGY
STAINS (DYES)
·
Stains
are organic compounds carrying either positive charges or negative charges or
both.
·
Based
on the charge they have stains are categorized as follows
Basic
Dyes : stains with
positive charge (Methylene Blue, Crystal violet) – Background staining.
Acidic
Dyes : stains with
negative charge (India ink, Nigrosine)- Bacterial cell staining.
Neutral
Dyes : stains with both
charges (Eosinate of methylene blue, Giesma sain)- Stains nucleic acid and
cytoplasm.
·
The
word DYE is used to describe
the crude form of colouring agent used for general purpose and the word STAIN used to describe the pure
form of colouring agent commonly used for biological staining purpose.
·
In
a normal natural smear bacteria are not clearly visible under microscope
because they have nearly the same refractive index as water as the bacterial
cytoplasm is transparent. Therefore we are applying different types of stains
for make them clearly visible under microscope.
·
Every
stains used for staining have two common properties
I.
Stains
have a chromophore group which give the dye its colour.
II.
They
can bind with bacterial cell wall through ionic, covalent or hydrophobic bonds.
PURPOSE OF STAINING MICROBIAL CELL
·
To
understand the size and shape of a bacterial cell.
·
To
enhance the visualization of bacterial cells or specific structural component
of a cell.
·
To
highlight metabolic process of a cell.
·
Dead
and live cell differentiation from a given sample.
·
Detailed
study of internal or external structures of a cell.
·
To
differentiate different types of cells from a single source.
MAJOR STAINING TECHNIQUES
·
Staining
is simply explained as coloured chemical compounds used for making a contrast
between the specimen and the background.
·
Microbiologists
developed different types of staining techniques in microbiology for the clear
visualization of different microbes.
·
It
give specific colour to bacteria or its structures and make it clearly visible
under microscope – simply bacterial staining means colouring of bacteria to
make it clearly visible under microscope.
·
Basic
Requirements for Staining
i)
Clean
and grease free glass slide
ii)
Bacterial
culture to be stained.
iii)
Inoculation
loop for the transfer of culture from suspension to slide.
iv)
Bunsen
burner for sterilizing the inoculation loop and for heat fixation.
DIFFERENT TYPES OF STAINING AND THEIR PURPOSE
Sr.
No.
|
Types
Of Staining
|
Staining
Purpose
|
Chemicals
used/ Example
|
1
|
Simple
Staining
|
To
understand the size, shape and cell arrangements.
To
understand the total count of bacterial cell in a given specimen.
|
Methylene
blue
Crystal
violet
Carbol
fuchsin
|
2
|
Gram
Staining
|
To
differentiate between gram positive (purple colour) and gram negative (pink
colour) bacterial cells from a given sample.
|
Crystal
violet
Safranin
|
3
|
Acid
fast Staining
|
To
identify the bacteria which have huge amount of lipids in their cellwall.
|
Used
for the staining of Mycobacterium
tuberculosis and Mycobacterium
leprae
|
4
|
Special
staining
|
For
the identification of special structural features of bacterial cells.
|
Endospore
staining
Capsule
staining
Flagella
staining.
|
5
|
Iron-Heamatoxylin
Staining
|
Staining
of tissue components.
|
Staining
of myelin, elastic and collagenic fibres etc.
|
6
|
Wheatley
Trachoma Staining
|
Easiest
method of staining protozoa
|
Detection
and identification of intestinal protozoa.
|
7
|
Positive
staining of Viruses
|
To
observe and identify viruses under electron microscope.
|
Analysing
morphological characters of virus from an aquatic sample.
|
8
|
Negative
Staining Of Viruses
|
To
observe and identify viruses under electron microscope.
|
Used
to obtain high quality micrographs.
|
9
|
Gomori
Methenamine Silver (GMS) Staining
|
To
observe and identify fungi from tissue and incytology specimens.
|
Microscopic
identification of Aspergillus.
|
10
|
Periodic
acid-Schiff (PAS ) Staining
|
To
observe and identify fungi from tissue and incytology specimens.
|
Cryptococcus staining.
|
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