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Simple Staining

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29 views3 pages

Simple Staining

Uploaded by

madhumicrobio89
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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SIMPLE STAINING

 The simple stain can be used as a quick and easy way to determine cell shape, size and
arrangements of bacteria.
 Simple stain is a very simple staining procedure involving single solution of stain.
 Any basic dye such as methylene blue, saffranin, or crystal violet can be used to color
the bacterial cells.

Procedure:

Simple Staining Procedure

Preparation of a smear and heat fixing:

1. Using a sterilized inoculating loop, transfer loopful of liquid suspension containing


bacteria to a slide (clean grease free microscopic slide) or transfer an isolated colony from
a culture plate to a slide with a water drop.
2. Disperse the bacteria on the loop in the drop of water on the slide and spread the drop
over an area the size of a dime. It should be a thin, even smear.
3. Allow the smear to dry thoroughly.
4. Heat-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.
Staining:

1. 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).
2. Using distilled water wash bottle, gently wash off the excess methylene blue from the
slide by directing a gentle stream of water over the surface of the slide.
3. Wash off any stain that got on the bottom of the slide as well.
4. Saturate the smear again but this time with Iodine. Iodine will set the stain
5. Wash of any excess iodine with gently running tap water. Rinse thoroughly.
6. Wipe the back of the slide and blot the stained surface with bibulous paper or with a
paper towel.
7. Place the stained smear on the microscope stage smear side up and focus the smear
using the 10X objective.
8. 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.

Results

The bacterial cells usually stain uniformly and the color of the cell depends on the type of dye
used. If methyene blue is used, some granules in the interior of the cells of some bacteria may
appear more deeply stained than the rest of the cell, which is due to presence of different
chemical substances.

References:

1. Dr C.P. BAVEJA- Text book of Microbiology – Arya publications


2. www.microrao.com

Short answer type questions:


1. Simple staining

MCQ’S:

1. What are the reagents used in simple staining


a. Methylene blue
b. Crystal violet
c. Saffranin
d. All of the above

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