Ethyl Alcohol, 95% (Decolorizing agent)
serves a dual function as a protein-dehydrating agent and as a lipid solvent, its action is determined by two factors, the concentration of lipids and the thickness of the peptidoglycan layer in bacterial cell walls. in gram-negative cells it increases the porosity of the cell wall by dissolving the lipids in the outer layers. thus CV-I can be easily removed from the thinner and less highly cross-linked peptido layer, the washing-out effect facilitates the release of the unbound CV-I, leaving the cell colorless. the much thicker peptido layer in gram-positive cells is responsible for the more stringent retention of the CV-I complex, as the pores are smaller, due to the dehydrating affect of the alcohol. it is dificult to remove, cells remain purple.