EVELYN HONE COLLEGE MANAGEMENT BOARD

ACADEMIC AND APPLIED SCIENCES

SCIENCE LABORATORY TECHNOLOGY
MICROBIOLOGY LABORATORY PRACTICAL
TITLE: GRAM STAINING OF BACTERIA.

THEORY

Staining procedures that make visible the differences between bacterial cells or parts of the
bacterial cell are termed differential staining techniques. They are slightly more elaborate than
the simple staining technique in that cells may be exposed to more than one dye solution or
staining reagent.

One of the most important and widely used differential staining techniques in microbiology is
Gram staining. This technique was just introduced by Christian Gram in 1884. In this process
the fixed bacteria smear is subjected to the following staining reagent in the order listed; crystal
violet, iodine solution, alcohol (decolorizing agent), and safranin or some other suitable
counter stain. Bacteria stained by the Gram method fall into two groups: Gram positive
bacteria, which retain the crystal violet and hence appear deep violet colour, and Gram- negative
bacteria, which lose the crystal violet, are counter stained by the safranin, and hence appear red
or pink in colour.

PROCEDURE

1. Using a sterile inoculating loop, prepare a bacteria smear on a clean slid.

2. Air dry and heat fix.
3. Cover the smear with crystal violet for 1 minute and wash it out gently with water.
4. Cover the smear with gram’s iodine for 30 seconds to one minute.
5. Wash off the iodine by tilting the slide and squirting water above the smear so that the
water runs over the smear;
6. Decolorize with 95% ethanol ( usually 10 -20 seconds); Immediately wash gently with
water;
7. Cover the smear with safranin for 30 seconds.
8. Wash the slide with water, drain, blot and air dry.
9. Mount in glycerine/ immersion oil and examine.