- Before everything else, wear gloves and observe standard safety precautions and ensure the glass slide is absolutely clean. The bacteria sample should be healthy and active, preferably cultured in the laboratory. Applying the sample bacteria on the slide should be even and thin, as thick smears are hard to decolorize and read. A nickel-size sample should be sufficient to make handling easier and the decolorization thorough.
- Fixing is done by heating the slide sample either over a low flame or a slide warmer until the specimen is dry. Do not overheat it because this could kill or change the characteristics of the specimen. Another fixing method involves flooding the slide with methanol for a minute. This method is especially useful for a liquid specimen because it prevents the specimen from washing off, preserves blood cell properties and gives a clearer background.
- Staining involves the pouring of crystal violet over the specimen on the slide and letting it stand from 10 seconds to a minute, depending on the thickness of the sample. Once the sample is stained, rinse off the crystal violet with distilled water. Make sure to flood the entire slide with the stain mixture to ensure even coating; do not let it stand longer than the required time, and do not rinse excessively.
- Decolorization is the crucial step in the entire procedure; it determines if the specimen is gram-negative or gram-positive. Let the decolorizer flow over the smear as you hold the slide slightly tilted downward. Make sure that every portion of the sample is covered, but do not over-wash. This takes only a few seconds, and when left too long the decolorizer can remove the stain. Ethanol and acetone both function as decolorizing agents, but the former acts slower.
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