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Gram staining bacteria is a general technique applied to distinguish two big groups of bacteria according to their different cell wall constituents.
The process involves three steps:
1. First the cells are stained with crystal violet dye. After that, a Gram's iodine solution (iodine and potassium iodide) is added to create a complex between the crystal violet and iodine. This complex is a bigger molecule compared to original crystal violet stain and iodine and is insoluble in water.
2. A decolorizer for example ethyl alcohol or acetone is mixed to the sample, which dehydrates the peptidoglycan layer, shrinking and tightening it. The big crystal violet-iodine complex cannot penetrate in this tightened peptidoglycan layer, and is therefore entrapped the cell in Gram-positive bacteria. On the other hand, the outer membrane of Gram-negative bacteria is degraded and the thinner peptidoglycan layer of Gram-negative cells is incapable to keep the crystal violet-iodine complex, and the color is lost.
3. A counter stain, for example, the weak water soluble safranin, is mixed to the sample, staining it red. As the safranin is lighter compared to crystal violet, it does not disorder the purple coloration in Gram positive cells. Though, the decolorized Gram negative cells are stained red.Reference
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