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Indian Ink Staining
This is a negative staining procedure for the demonstration of bacteria which are capsulated. The capsule protects the bacteria from the stain thus remaining in contrast with the Indian ink-stained background when viewed under a microscope’s objective power 10 and power 40.
The reagent used in this staining procedure is Indian ink which stains the background making the capsulated bacteria appear as a halo on the slide under a microscope. In capsule staining, Negrossin can give better results since it spreads easily and gives a more even background. This test is done to identify cryptococci such as Neisseria gonorrhea.
The other capsule staining procedure is B. Anthony’s Stain Method which uses crystal violet as the primary stain and 20% copper sulfate as both the decolorizing agent and counterstain. The copper sulfate washes the primary stain off the capsule but not from the cell, and therefore the result is a faint blue halo and purple cells.
Mycobacteria, also known as acid-fast bacteria, are covered with mycolic acid which is waxy and therefore resistant to normal bacteria staining (gram stain). It is therefore subjected to heat during staining with Carbol fuchsin of which the mycolic acid forms a complex that cannot be decolorized by a weak mineral acid or acid alcohol. the background is then stained blue by methylene blue or green by malachite green.
The complex formed between the primary stain and mycolic acid upon heating is red in color and thus the bacteria will appear red on a blue or green background when viewed under a microscope. decolourization is done by 20% sulfuric acid or 3% acid alcohol.
This is a technique that was developed by a danish bacteriologist, Hans Christian Gram, to differentiate between bacteria based on the thickness of their cell walls which influence the bacterial staining behavior. The gram-positive bacteria have cell walls with a thick peptidoglycan layer while that of the gram-negative bacteria is thinner, and therefore when the former is subjected to the primary stain, crystal violet, it gets trapped within the thick layer and thus cannot be washed off by the decolorizer making them purple when viewed under the microscope as they do not pick the counter stain.
The latter, however, does not hold the primary stain, it is decolorized and retained by the counterstain, therefore it appears pink or red when viewed under a microscope.
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