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How do I perform a Gram stain?

One of the most powerful and widely used diagnostic tools of the microbiologist, this technique requires a few simple steps:

1. Thin smears of bacterial cultures should be air-dried and flame-fixed on clean glass slides. 18- to 24-hour cultures will give the best results.

2. Apply Gram crystal violet stain from eye-dropper or dropper bottle. Slide should be flooded with stain and let stand for one minute. Gently rinse slide with water from a dropper or by dipping in a beaker or cup of clean, distilled water.

3. Flood smears with Gram iodine (serves as a mordant, or dye retainer) and let stand for one minute. Rinse with clean water as above.

4. Decolorize smears with 95 percent ethyl alcohol by applying one drop at a time-just until no more color runs off-for approximately 30 seconds. It is very important not to overdecolorize in this step. Rinse with water as above.

5. Apply Gram safranin counterstain and let stand for 30-45 seconds. This step serves to stain those bacteria (Gram-negative) not holding the crystal violet after step 4. Rinse with water as above. Air or blot dry the slides and examine under oil-immersion.

Note: Do not allow any of the stains to evaporate to dryness on slides prior to rinsing. Spring-type wooden clothespins make excellent slide holders during flaming, staining and rinsing

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