Rinse the slide with a counterstain (safranin or carbol fuchsin) which stains all cells red.Rinse with water to stop decolorization.The decolorization of the cells is the most “operator-dependent” step of the process and the one that is most likely to be performed incorrectly. This step washes away unbound crystal violet, leaving Gram-positive organisms stained purple with Gram-negative organisms colorless. The important aspect is to ensure that all the color has come out that will do so easily. This can be done in a steady stream, or a series of washes. Decolorize the sample by applying 95% ethanol or a mixture of acetone and alcohol.This acts as a mordant and fixes the dye, making it more difficult to decolorize and reducing some of the variability of the test. Add iodine (Gram’s iodine) solution (1% iodine, 2% potassium iodide in water) for 1 minute.The heat-fixed cells should look purple at this stage. Allow to stain for approximately 1 minute. Crystal violet (a basic dye) is then added by covering the heat-fixed cells with a prepared solution.Do not let the glass become hot to the touch. Heat fix the slide by passing it (cell side up) through a flame to warm the glass. Fix the cells to the slide by heat or by exposure to methanol.Take a fresh culture-old cultures stain erratically.This could make gram-negative organisms appear to be gram-positive or gram-variable. Take a small inoculum-don’t make a thick smear that cannot be completely decolorized.The culture can come from a thick suspension of a liquid culture or a pure colony from a plate suspended in water on the microscope slide. First, a loopful of a pure culture is smeared on a slide and allowed to air dry.The importance of this determination to correct identification of bacteria cannot be overstated as all phenotypic methods begin with this assay. The method is named after its inventor, the Danish scientist Hans Christian Gram (1853-1938), who developed the technique in 1884 (Gram 1884). Gram staining is an empirical method of differentiating bacterial species into two large groups (Gram-positive and Gram-negative) based on the chemical and physical properties of their cell walls. This article first appeared in the PMF Newsletter of February, 2006 and is protected by copyright to PMF.
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