BSCI 424 — PATHOGENIC MICROBIOLOGY — Fall 2000

The Gram Stain

The Gram stain is the most important and universally used staining technique in the bacteriology laboratory. It is used to distinguish between gram-positive and gram-negative bacteria, which have distinct and consistent differences in their cell walls. Gram-positive cells may become gram negative through mechanical damage, conversion to protoplasts, or aging, in which autolytic enzymes attack the walls.

In the Gram stain, the cells are first heat fixed and then stained with a basic dye, crystal violet, which is taken up in similar amounts by all bacteria. The slides are then treated with an I₂-KI mixture (mordant) to fix the stain, washed briefly with 95% alcohol (destained), and finally counterstained with a paler dye of different color (safranin). Gram-positive organisms retain the initial violet stain, while gram-negative organisms are decolorized by the organic solvent and hence show the pink counterstain. The difference between gram-positive and gram-negative bacteria lies in the ability of the cell wall of the organism to retain the crystal violet.

Technique: Transfer a loopful of the liquid culture to the surface of a clean glass slide, and spread over a small area. Two to four cultures may be stained on the same slide, which can be divided into 2-4 sections with vertical red wax pencil lines. To stain material from a culture growing on solid media, place a loopful of tap water on a slide; using a sterile cool loop transfer a small sample of the colony to the drop, and emulsify. Allow the film to air dry. Fix the dried film by passing it briefly through the Bunsen flame two or three times without exposing the dried film directly to the flame. The slide should not be so hot as to be uncomfortable to the touch.

1. Flood the slide with crystal violet solution for up to one minute. Wash off briefly with tap water (not over 5 seconds). Drain.



2. Flood slide with Gram's Iodine solution, and allow to act (as a mordant) for about one minute. Wash off with tap water. Drain.



3. Remove excess water from slide and blot, so that alcohol used for decolorization is not diluted. Flood slide with 95% alcohol for 10 seconds and wash off with tap water. (Smears that are excessively thick may require longer decolorization. This is the most sensitive and variable step of the procedure, and requires experience to know just how much to decolorize). Drain the slide.



4. Flood slide with safranin solution and allow to counterstain for 30 seconds. Wash off with tap water. Drain and blot dry with bibulous paper. Do not rub.



5. All slides of bacteria must be examined under the oil immersion lens.

Note: To remove immersion oil from a slide without damaging the smear, lay a piece of lens tissue on the slide, add a drop or two of xylene and draw the lens tissue across the slide. Repeat if necessary.

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