Gram Stain Reaction: Click Here to View Images
The gram stain is one of the most useful methods in identifying bacteria. In
this procedure, the bacteria is stained and examined under the
microscope. Organisms are judged to be gram positive if they retain the
crystal violet after decolorization and will appear purple. Gram negative
organisms are decolorized and appear pink to red because they take up the
safranine counter stain.
Tips for Success:
Only perform gram stains on fresh cultures (24hr incubation). Using older
cultures often yields unexpected results. Be sure to stop the application of
decolorizer immediately after it runs clear to avoid over-decolorizing. Always
run positive and negative controls from known cultures.
Procedure:
The gram stain is one of the most useful methods in identifying bacteria. In
this procedure, the bacteria is stained and examined under the
microscope. Organisms are judged to be gram positive if they retain the
crystal violet after decolorization and will appear purple. Gram negative
organisms are decolorized and appear pink to red because they take up the
safranine counter stain.
Tips for Success:
Only perform gram stains on fresh cultures (24hr incubation). Using older
cultures often yields unexpected results. Be sure to stop the application of
decolorizer immediately after it runs clear to avoid over-decolorizing. Always
run positive and negative controls from known cultures.
Procedure:
- Place a loopful of sterile distilled water onto a microscope slide.
- Touch an isolated colony with inoculating loop and swirl it in the
drop of water on the slide. - Let the smear air dry at room temperature
- Heat fix the smear by waving the slide over a flame, being careful not
to overheat. - Flood the slide with crystal violet, and let stand for one minute.
- Wash the slide briefly with cold water.
- Flood the slide with gram's iodine; let stand for one minute; wash off
with water. - Decolorize until the solvent flows colorlessly from the slide.
- Flood the slide with safranine; let stand for 30 seconds; wash off
with water. - Blot the slide dry with bilbous paper.
- Examine the slide under the microscope for gram reaction (100x oil-
immersion objective) - Gram positive organisms will appear purple, while gram negative
organisms will appear pink.
| Gram Stain Procedure For The Identification of Bacteria |
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