Background
The Gram stain , developed by Christian Gram in 1884, is the premier differential stain of bacteriology. A differential stain is one that uses 2 or more dyes to distinguish between groups of microbes. The Gram stain is differential based upon the chemical composition of Gram positive and Gram negative cell walls.
Gram positive cell walls are composed of many layers of the heteropolymer, peptidoglycan strengthened by technoic acids. In contrast, the Gram negative cell wall has only a thin layer of peptidogylcan coated internally and extermally by dou;lbe membranes that contain lipopolysaccharide (LPS). This difference is not only critical to the mechanism of the Gram stain, but in other areas of bacterial physiology.
The mechanism of the Gram stain
is described as "differential solubilities". The primary dye (crystal
violet) and the mordant (Grams' iodine) react similarly in both cell types.
The decolorizer, ethyl alcohol, is the most crtitical step. Ethyl
alcohol is a nonpolar solvent, and thus penetrates the cell walls of Gram
negative cells more readily and removes the crystal violet-iodine complex.
However, caution must be used since applying the decolorizer too long will
remove dye complexes from the Gram positive cells as well.
Procedure
1. Apply the primary dye, crystal violet to heat-fixed smears for one
minute.
Wash with water.
2. Apply the mordant, Gram's iodine, for 1 minute. Wash with water.
3. Decolorize with 95% ethyl alcoholand wash immediately with water.
4. Counterstain with safranin for 1 minute, wash with water, blot dry,
and observe.
Results
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