The simple staining procedure, makes it possible to see bacteria clearly, but it does not distinguish between organisms of similar morphology.
In 1884, a Danish pathologist, Christian Gram, discovered a method of staining bacteria with pararosaniline dyes. Using two dyes in sequence, each of a different color, he found that bacteria fall into two groups. The first group retains the color of the primary dye: crystal violet (these are called gram positive). The second group loses the first dye when washed in a decolorizing solution but then takes on the color of the second dye, a counterstain, such as safranin or carbol fuchsin (these are called gram negative). An iodine solution is used as a mordant (a chemical that fixes a dye in or on a substance by combining with the dye to form an insoluble compound) for the first stain.
The exact mechanism of action of this staining technique is not clearly understood. However, it is known that differences in the biochemical composition of bacterial cell walls parallel differences in their Gram-stain reactions. Gram-positive bacterial walls are rich in tightly linked peptidoglycans (protein-sugar complexes) that enable cells to resist decolorization. Gram-negative bacterial walls have a high concentration of lipids (fats) that dissolve in the decolorizer (alcohol, acetone, or a mixture of these) and are washed away with the crystal violet. The decolorizer thus prepares gram-negative organisms for the counterstain.
The Gram stain is one of the most useful tools in the microbiology laboratory and is used universally. In the diagnostic laboratory, it is used not only to study microorganisms in cultures, but it is also applied to smears made directly from clinical specimens. Direct, Gram-stained smears are read promptly to determine the relative numbers and morphology of bacteria in the specimen. This information is valuable to the physician in planning the patient’s treatment before culture results are available. It is also valuable to microbiologists, who can plan their culture procedures based on their knowledge of the bacterial forms they have seen in the specimen.
The numerous modifications of Gram’s original method are based on the concentration of the dyes, length of staining time for each dye, and composition of the decolorizer. Hucker’s modification, to be followed in this exercise, is commonly used today. The choice of decolorizing agent depends on the speed wanted to accomplish this step. The slowest agent, 95% ethyl alcohol, is used in this exercise to permit the student to gain experience with decolorization. Acetone is the fastest decolorizer, while an equal mixture of 95% ethyl alcohol and acetone acts with intermediate speed. The acetone-alcohol combination is probably the most popular in diagnostic laboratories.
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