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Intro: 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 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 iodine mordant to fix the stain, washed briefly with 95% alcohol (destained), and finally counterstained with a pink dye (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.