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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
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