PRINCIPLE OF GRAM STAINING.-As touched on previously, the crystal violet stain, the primary stain, stains everything in the smear blue. The Gram's iodine acts as a mordant, a substance that causes the crystal violet to penetrate and adhere to the gram-positive organisms. The acetone-alcohol mixture acts as the decolorizer that washes the stain away from everything in the smear except the gram-positive organisms. The safranin is the counter-stain that stains everything in the smear that has been decolorized: pus cells, mucus, and gram-negative organisms. The gram-negative organisms will stain a much deeper pink than the pus cells and mucus will stain even lighter pink than the pus cells.

MATERIALS REQUIRED FOR GRAM yes"> STAINING.-To Gram stain a smear, the following materials are required:

Gram stain kit, which consists of:
-Crystal violet stain
-Iodine or stabilized iodine (mordant)
-Acetone-alcohol decolorizer
-Safranin stain
Staining rack
Blotting paper or paper towel
PROCEDURE FOR GRAM STAINING SMEARS.-After smears have been dried, heat-fixed, and cooled, proceed as follows:

1. Place the slide on a staining rack. Then flood slide with primary stain (crystal violet). Let stand 1 minute.

2. Remove the primary stain by gently washing with cold tap water.

3. Flood the slide with mordant (iodine or stabilized iodine) and retain on slide for 1 minute.

4. Remove mordant by gently washing with tap water.

5. Tilt slide at a 45-degree angle and decolorize with the acetone-alcohol solution until the solvent that runs from the slide is colorless (30 to 60 seconds).

6. Wash the slide gently in cold tap water.
7. Flood the slide with counter-stain (safranin) and let stand for 30 to 60 seconds.

8. Wash slide with cold tap water.
9. Blot with blotting paper or paper towel or allow to air dry.

10. Examine the smear under an oil immersion objective.

Reading and Reporting Smears
Place a drop of oil on the slide and, using the oil immersion objective of the microscope, read the smear. All body discharges contain extraneous materials, such as pus cells and mucus. Of interest, however, are the types of bacteria that may be present. The stained smear reveals only two features: the morphology and the staining characteristics of the bacteria present. Positive identification requires cultures and further studies.

Hospital Corpsmen should report only what
they see. For example, "Smear shows numerous gram-negative bacilli." If two or more types of bacteria are seen in a smear, the rule is to report them in order of predominance. For example:

"1. Numerous gram-positive cocci in clusters
2. Few gram-negative bacilli"
Gram-positive organisms are easy to see because they stain a deep blue or blue-black. Gram-negative organisms stain a deep pink, but since the background material is also pink, minute and detailed inspection is necessary before reporting the results.

In the presence of gonorrhea, the smear will reveal large numbers of pus cells with varying numbers of intracellular and extracellular gram-negative, bean-shaped cocci in pairs. Such a finding could be considered diagnostic. It is important to point out that only a few of the thousands of pus cells on the slide may contain bacteria, and sometimes it requires considerable search to find one.