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I had today a class in microbiology and my teacher said that the Decolorizer component is the critical one and cannot be more than 30 seconds. The decolorizer has alcohol in high concentration, my lecturer says 95%. I know that alcohol dissolves the lipid membrane, more exactly here for RBC which should be a similar situation here, not completely sure however.

What component is the critical one that you cannot keep alcohol on your bacteria samples more than 30 seconds?

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My guess is that this is because of its toxicity to the bacteria. –  Bitwise Oct 16 '12 at 15:04
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@Bitwise: if you're doing a Gram staining you most probably don't care about killing the bacteria. –  nico Oct 16 '12 at 17:39
    
@nico I was referring to the cells losing their structural integrity (the cells obviously need to be visible). –  Bitwise Oct 16 '12 at 18:20
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The crystal violet stain will be removed if the decolorizer is left on too long. I'm assuming that the crystal violet complexes that are retained in the gram positive bacteria's peptidoglycan layers end up being washed away (perhaps from overdehydrtation).

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