a. Monochrome staining
Principle - In Monochrome staining, the bacterial smear is stained with a single reagent, which produces a distinctive contrast between the organisms and its background.
Mechanism - Basic stains with a positively charged chromogen are preferred because bacterial nucleic acid and certain cell wall components carry a negative charge hat strongly attracts and blinds to the cationic chromogen.
To purpose of simple staining is to elucidate the morphology and arrangement of bacterial cells.
Stains - The most commonly used basic stains are methylene blue, crystal violent, safranin and carbol fuchsin etc.
Procedure -
i) Preparation of a smear and heat fixing
* Using as sterilized inoculating loop, transfer loopful of liquid suspension containing bacteria to clean grease free microscope slide or transfer an isolated colony from a culture plate to a slide with a sterile water drop.
* Disperse the bacteria on the loop in the drop of water on the slide and make a thin and even smear on slide.
* Allow the smear to dry thoroughly .
* Hear-fix the smear cautiously by passing the underside of the slide through the burner flame two or three times. It fixes the cell in the slide. Do not overheat the slide as it will distort the bacterial cells.
ii) Staining
* Cover the smear with methylene blue and allow the dye to remain in the smear for approximately one minute (staining time is not critical here: somewhere between 30 seconds to 2 minutes should give you an acceptable stain, the longer you leave the dye in it, the darker will be the stain).
* Using distilled water wash bottle, gentle wash off the excess methylene blue from the slide by directing a gentle stream of water over the surface of the slide.
* Wash off any stain that got on the bottom of slide as well.
* Wipe the back of the slide and blot the stained surface with a clean filter paper.
* Place the stained smear on the microscope stage smear side up and focus the smear using the 10X objective.
* Choose an area of the smear in which the cells are well spread in a monolayer. Center the area to be studied, apply immersion oil directly to the smear and focus the smear under oil with the 100X objective.
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i. Preparation of a smear and heat fixing
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ii) staining
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Observation -
* The bacterial cells usually stain uniformly and the colour of the cell depends on the types of dye used.
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