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Decolorizers are often the mixed solvent of ethanol and acetone. This step is known as “solvent treatment.” The slide is rinsed with water in 5 seconds. To prevent excess decolorization in the gram-positive cells, stop adding decolorizer as soon as the solvent is not colored as it flows over the slide. The decolorizing agent, (ethanol or an ethanol and acetone solution), interacts with the lipids of the membranes of both gram-positive and gram-negative Bacteria. The outer membrane of the gram-negative cell is lost from the cell, leaving the peptidoglycan layer exposed. The decolorizer, ethyl alcohol, is the most critical 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.
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