Gram Stain Procedure

The performance of the Gram stain procedure is a standard laboratory technique to adequately visualize microscopically the organism under study. Primarily you need to have the necessary equipments and materials on hand, these are: microscope glass slides, glass cover slips, copper wire loop, Bunsen burner, solutions (crystal violet stain, Gram's iodine stain, 0.25% safranin stain, and 95% alcohol), and a dropper for each solution. Using the Bunsen burner, heat the copper wire loop until it luminesce, allow it to cool for 2-3 minutes then get a sample of the specimen, and smear it on the microscope glass slide adequately covering the central most area. Heat fix the smear so the water will evaporate, then allow it to cool. Stain the microscope slide as follows: (1) Using a dropper, flood the microscope slide with crystal violet for one minute then pour off excess dye and wash it gently in with tap water and drain, then dry the slide with a drying paper. (2) Using a dropper, flood the slide with the Gram's iodine for one minute, then pour off the excess dye again wash with tap water and drain but this time do not dry with paper. (3) Using a dropper, wash with 95% alcohol for 30 seconds, to decolorize the stains, then wash with tap water and drain. (4) Finally, using a dropper, counterstain the smear with the 0.25% safranin for 30 seconds, and again wash with tap water and drain. Dry blot with paper. You are now ready to visualize the slide under the microscope. You can appreciate the bacterial cells of its morphology, grouping, and relative sizes.