![]() Do not smear.īacteria have a cell wall made up of peptidoglycan. Blot gently and allow the slide to dry.However, the purple gram-positive color is not altered by the presence of the counter-stain, it’s effect is only seen in the previously colorless gram-negative cells which now appear pink/red. The counterstain stains both gram-negative and gram-positive cells. 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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