GRAM STAINING OF BACTERIA

GRAM STAINING

The Gram stain is a differential  stain which allows most bacteria to be divided into two groups, Gram-positive bacteria and Gram-negative bacteria. The technique is based on the fact that the Gram positive cell wall has a stronger attraction for crystal violet when Gram's iodine is applied than does the Gram negative cell wall. Gram's iodine is known as a mordant. The procedure was discovered by Christian Gram in 1884.

The Gram stain is most commonly used staining procedure in microbiology. It is extremely useful in identifying bacteria. It is important that you understand the color changes that occur at each step in the Gram stain.


MATERIALS REQURED FOR GRAM STAINING:
3 microscope slides (precleaned) Gram stain reagents (crystal violet, Gram's iodine, 95% ethyl alcohol, and safranin)
24 hr. TSB cultures of Stapylococcus epidermidis, Pseudomonas aeruginosa
24 hr. TSA slant of Bacillus licheniformis
48 hr. TSA slant of Mycobacterium smegmatis


GRAM STAINING PROCEDURE:
1. Cover with CRYSTAL VIOLET for 20 seconds(PRIMARY STAIN)
2. Gently rinse off the stain with water and shake off the excess.
3. Cover with GRAM'S IODINE for one minute(MORDANT)
4. Pour off the Gram's iodine.
5. Run 95% ETHYL ALCOHOL down the slide until the solvent runs clear (about 10-20 seconds). THIS STEP IS CRITICAL! THICK SMEARS REQUIRE MORE TIME THAN THIN ONES. (DECOLORIZING AGENT)
6. Rinse with water to stop the action of the alcohol.
7. Cover with SAFRANIN for 20 seconds. (COUNTER STAIN)
8. Gently rinse off the stain with water. Blot with bibulous paper and clean off the bottom of the slide
with 95% alcohol.

HELPFUL SUGGESTIONS:
a) DO NOT make your smears too thick!
b) Be very careful when you decolorize.
c) Be sure your cultures are young, preferably less
than 24 hours old. Older cultures tend to lose the
ability to retain stains.

RESULTS:
Observe your smears in the microscope under oil immersion. Draw a few representative organisms from each smear in your lab report.