Gram Stain Procedure

1. Use a sterile slide or pass one side of a clean glass slide through a flame several times. Allow the slide to cool before smearing with a specimen.
2. For bacterial suspensions in broth: Apply 1 loop of broth onto the cleaned slide. Dilute heavy suspensions by applying less than one loop to a drop of sterile water or saline on the slide. Spread the fluid to form a film about one centimeter in diameter. Excessive spreading may result in disruption of cellular arrangement. A satisfactory smear will allow examination of the typical cellular arrangement and isolated cells.

For bacterial colonies: Use water or saline to emulsify a colony or portion of colony on the previously flamed side of the slide. Only a very small amount of material from an isolated colony is needed for a gram stain (a loopful of a colony is excessive).

For inoculum on swabs: Roll the swab over the cleaned surface of a glass slide. To avoid contamination of culture media, discard the swab used to make the slide and use a second swab containing the inoculum to inoculate media.

The most common flaw in smear preparation is application of too much material on the slide. Broth cultures with visible growth contain at least 10⁶ bacteria/ml. Similarly, depending on size, one bacterial colony can be equivalent to 10⁵ bacteria. Excessive material interferes with the passage of light through the specimen, prevents adequate decolorization, and interferes with the ability to view single cells and to determine the cellular arrangement present in the specimen or culture.

3. Air dry and heat fix the slide by passing it through a flame two or three times.

DO NOT OVERHEAT the slide as protein in the specimen can coagulate and cellular morphology may appear distorted from excess heat.

4. Using the slide holder provided (it looks like a big clothespin), clamp the slide and suspend it, with specimen side up, over the sink. Flood the slide with crystal violet and allow it to remain for 1 minute. Rinse the slide gently with cold tap water.
5. Apply Gram’s iodine and allow it to remain for 1 minute.
6. While holding the slide at a slight angle, allow drops of alcohol to run over the smear (3-4 seconds). Quickly rinse well with water.
7. Apply safranin and allow it to counterstain for 30 seconds. Rinse with water until all free stain is removed. Blot (DO NOT WIPE) the slide dry with bibulous paper.
8. Examine the smear under oil immersion (you must use oil). NEVER determine the morphology or staining reaction of bacteria with any objective other than oil immersion.