CLINICAL LAB SCIENEC

CHAPTER 15: MICROBIOLOGY 397
MANUAL MICROBIOLOGY PROCEDURE #3
Performing a Gram Stain
Principles
The most frequently performed stain in the microbiology department is the Gram
stain. Staining of bacteria is necessary as it is virtually impossible to see and describe
the morphology of the organisms unless they are stained. Bacteria are small and
have little natural color for most species, so the Gram stain provides for differentiation
as to the shape of the organisms as well as providing further aid in identification
by labeling the organisms as either Gram-positive or Gram-negative. Differences
in the cell walls of bacteria are based on their chemical constituents, which in turn
accounts for the staining difference. Gram-negative cell walls contain more lipids
(fats), polysaccharides, and lipoprotein complexes (including amino acids) than do
those of the walls of a Gram-positive organism. The cell walls of both Gram-positive
and Gram-negative organisms will absorb crystal violet, but Gram negative organisms
will lose the crystal violet in the decolorizing step, and will then absorb the safranin,
which imparts a pink to orange color to them. The Gram positive organisms will not
lose their crystal violet during decolorizing and will appear purple in color.
The four-part Gram-staining procedure is outlined here and provided in detail
below. The heat-fixed smear is placed on a staining rack and is flooded with crystal
violet (the primary stain). After 30 seconds to a minute, the slide is gently washed
under a stream of water and returned to the staining rack. Gram’s iodine is then
poured over the smear. After 30 seconds to a minute, the slide is again gently washed
under a stream of water. The slide is then held at a 30- to 40-degree angle and
quickly rinsed with an acetone-alcohol mixture. This will decolorize the organism if
it is a Gram-negative species of bacteria. The decolorizing process only takes a few
seconds. All color should run from a slide containing predominantly Gram-negative
organisms. Finally, a counterstain (safranin) is poured over the slide. Gram-negative
organisms absorb and retain the counterstain.
Equipment and Supplies
1. Gloves, disposable paper towels, and disinfectant or other cleaning solution
2. Bibulous paper
3. Bacterial smears (slides) created in Procedure #2 (both Gram positive and Gram
negative species) (slides should be cool)
4. Clean tap or deionized water
5. Staining rack on sink and drying rack
6. Gram stain kit containing crystal violet, Gram’s iodine, acetone-alcohol decolorizer,
safranine
7. Electronic timing device or clock/watch with second hand
8. Disposal containers appropriate for biohazardous waste and sharps container for
disposing of slides
Procedure
1. Wash hands and don gloves.
2. Assemble necessary equipment and supplies.
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