S.Y B.PHARM SEMESTER III
PHARMACEUTICAL MICROBIOLOGY PRACTICAL
EXPERIMENT NO. 03
SIMPLE STAINING TECHNIQUE
REFERENCE: -
1. Experimental
Microbiology book (As Per PCI Syllabus) by Savita Mandan, Umesh Laddha, Sanjay
Surana, Published by Career Publication, First edition, Page No -. 30 - 32
2. Pharmaceutical
Microbiology (As Per PCI Regulation) by Prof Chandrakant Kokare, Published by
Nirali Prakashan, First Edition, Page No. – AI19 - AI20.
3. Practical pharmaceutical microbiology
book by Prof Rageeb, K.D Baviskar, N.G Patil, Published by S. Vikas And Company
(Medical Publisher), Edition 2018, Page No 36–42.
REQUIREMENTS:
CULTURES: Broth cultures of Escherichia coli or Bacillus
subtilis or Staphylococcus aureus.
STAIN: Methylene blue or crystal violet or carbol
fuchsin.
APPARATUS: Staining tray, glass slides, Bunsen burner,
inoculating loop, blotting paper, glass marker etc.
EQUIPMENT’S: Microscope.
PRINCIPLE:
When single staining reagent is
used to colour the bacteria is commonly called as monochrome staining
(mono-single, chrome-colour).
The surface of a bacterial cell
is acidic in nature due to having large amount of carboxyl groups located on
the cell surface. Therefore, when ionization of carboxyl groups takes place it
gives negative charge to the cell surface.
COOH -> COO + H+.
Therefore, basic dyes are commonly used in monochrome staining to stain bacterial cell. These dyes are available as a salt of acids. E.g. methylene blue chloride. When methylene blue rehydrates, it ionizes to form methylene blue and chloride ions. The positively charged ion has the colouring property.
Ionization
MB.CL MB + CL
Methylene blue chloride Methylene blue chloride
On addition of methylene blue for
staining, exchange of MB+ with Na+ on the bacterial cell surface takes place,
resulting into ionic bond formation between MB+ and cell surface. Thus, when
colouring agent forms ionic bond with cell or cell components, it results into
the staining of cell. The most commonly used basic stains are methylene blue (2
to 3 minutes), crystal violet (1 to 2 minutes) and carbol fuchsin (15 to 30
seconds).
SIMPLE STAINING PROCEDURE:
1. Prepare
bacterial smears of each microorganism and fix it by heat.
2. Place a
slide on the staining tray and flood the smear with one of the stains.
3. Allow the
staining solution to react with bacterial cell for 30 to 60 seconds.
4. Pour off
the staining solution and wash the slide in running tap water.
5. Dry the
slide between blotting papers and examine the stained smear under microscope using
oil-immersion objective.
OBSERVATIONS AND
RESULTS:
Bacterial species seen are blue,
violet or red depending upon the stain used against the colourless background.
Note the shape, size and arrangement of cells.
Staphylococcus aureus is ovoid or
spherical in shape, arranged in clusters (grape like). Escherichia coli and
Bacillus subtilis are small, rod shaped bacteria.
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