DIRECT
METHODS OF
BACTERIAL DETECTION
DR . SHARMIN
OBJECTIVE
• Staining methods
• Principle
• procedure
• uses
STAINING TECHNIQUES
• Structural details of bacteria cannot be
seen under a light microscope due to lack
of contrast.
• Hence, staining methods are necessary to
produce color contrast and thereby
increase the visibility.
BEFORE STAINING SMEAR
FIXATION IS DONE
• Fixation is done by 2 methods
- Heat fixation method- air dried and heat fixed
-Methanol fixation
STAINING METHOD
• Simple staining-methylene blue , basic fuchsine
• Negative staining- Indian ink , nigrosine
• Impregnation method
• Differential methods: Gram stain
• Acid fast stain
• Albert stain
SIMPLE STAINING
• basic dyes such as methylene blue or basic fuchsine are used as simple stain.
• Bacterial smear is stained with one of the simple stains for one minute and
then the slide is rinsed with water and allowed to air dry and focused under
oil immersion objective.
• RESULT: they provide the color contrast, but imparts the same color to all
the bacteria in a smear.
GRAM STAIN
FIRST DEVISED BY HANS
CHRISTIAN GRAM
PRINCIPLE
• GRAM POSITIVE cell wall contain thick peptidoglycan with
numerous teichoic acid cross linkages
• GRAM NEGATIVE CELL wall consist of thinner layer of
peptidoglycan and an outer layer lipid bilayer makes them
permeable to secondary dye after decolorization with
organic solvents like acetone.
CELL WALL
IT IS A DIFFERENTIAL STAIN
-based on chemical differentiation of organism as a result
of the structural chemical components of the organisms
cell wall.
GRAM stain divides bacteria into 2 groups-
-Gram positive i.e those that take up basic dye , crystal
violet
- Gram negative those that allow crystal violet to wash out
easily with decolorizer alcohol or acetone
PROCEDURE
1) Fixation - specimen is air dried and then heat fixed.
2) Primary staining - crystal violet, methyl violet, gentian violet
(1minute) without drying and rinse with water
3) Mordent - grams iodine (forming dye iodine complex in the
cytoplasm). 1minute, rinse with tap water
4) Decolorisation - with organic solvent like ethanol , acetone, keep for
10 -20 sec. Rinse with tap Water
5) Counter stain – safranin , carbol fuchsin , neutral red. 30 sec without
drying. Rinse with water, Air dry. Examine under oil immersion lens.
INTERPRETATION:
• Gram positive bacteria resist decolorization
and retain the color of primary stain i.e. violet
E.g.. staphylococcus, streptococcus, corynebacterium diphtheria
• Gram negative bacteria are decolorized and
therefore take counterstain and appear pink .
Eg. Pseudomonas , Klebsiella , proteus
MODIFICATIONS OF GRAM STAINING
• Kopeloff and Beerman’s modification : primary stain and
counter stain used are methyl violet respectively
• Jensen’s modification: absolute alcohol as decoloriser and
neutral red as counterstain . Meningococci and gonococci.
• Brown’s and brenn modification: actinomyces
• Preston and morrell’s modification: iodine –acetone as
decolouriser
• Weigert’s modification : Aniline xylol as decolouriser, for
tissue section
USES
• To differentiate between gram positive and gram negative
• For identification
• To start treatment
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