The Gram stain is based on the differential ability of cells to retain the dye crystal violet after treatment with a decolourizing solvent. Cells are initially stained with crystal violet and then de-stained with alcohol or acetone. Cells with a thick, Gram-positive cell wall type retain the crystal violet, whereas cells with a Gram-negative cell wall type, which have a much thinner peptidoglycan layer and an outer membrane, are easily decolorized. Colourless Gram-negative cells are then counterstained with a pink dye to make them visible. There are many modifications of this method.
(For reference, see Cowan & Steel, p. 24)
1. Prepare a heat-fixed smear: For liquid cultures, place a drop of culture on a clean slide. For cultures on solid media, place a drop of water on a clean slide, add a loopful of bacterial cells from a colony and stir to produce a smear. (Note: your smear should not be too dense). In both cases, allow the smear to dry and then pass it through a bunsen flame three times to heat-fix the cells.
2. Stain: To your smear, add a drop of crystal violet solution. Allow this to sit for 1 minute and then carefully rinse off the stain with water. Be careful not to wash your smear off the slide.
3. Fix: Add a drop of Gram’s iodine solution. Allow this to sit for 1 minute and then carefully rinse.
4. De-stain: Hold your slide tilted over a piece of paper towel. Add a drop of decolourising solution (ethanol, acetone, or a mixture of the two) and let it run down over the smear. You may see some purple colour coming off the smear. Add more decolourizing solution, drop by drop, until no more purple colour runs off. This should not take more than a few drops.
5. Counterstain: Add a drop of counterstain (basic fuchsin). Allow this to sit for 1 minute and carefully rinse it off with water.
6. Dry: Blot the slide dry in the areas away from the smear. You may need to leave it to air-dry for a few minutes to get the smear itself dry. Do not put immersion oil on a slide while it is still wet!
7. Examine: Examine the slide under bright-field optics. You can add a drop of oil to the slide and use the oil-immersion lens. Gram positive cells retain the crystal violet and appear purple; Gram negative cells lose the crystal violet and are stained pink by the counterstain. Results are sometimes ambiguous (Gram-variable); for example, old cultures of the Gram-positive genus Bacillus may contain cells which stain Gram-negative.