Gram Stain

Gregg Y. Ayakawa ayakawa at
Sun Jul 3 11:38:13 EST 1994

In article <2v2gck$5h8 at>, Norman Suguitan
<suguitan at> wrote: 

>When I flood a smear with crystal violet, is it better to just
>concentrate on the smear, and don't bother dropping crystal 
>violet on the whole slide?

I'm not sure it matters all that much, although concentrating on the smear
is less messy and wasteful.

>Secondly, how do I know when to stop decolorizing the stain.  Most
>of the time, I just count 30 seconds and stop decolorizing.  Sometimes,
>I could decolorize too much or not enough.  Do I decolorize the stain
>directly, or just drop the alcohol on the side of the slide?

The length of decolorization depends largely on the thickness of the smear
-- smears made from cells in broth generally require less decolorization
than those made from cells grown on solid media.  Therefore, using a fixed
time period for all smears is probably not the ideal procedure.  Here's
what I do: 

Hold the slide at an angle over a sink.  Slowly run the decolorizer from
one end of the slide, allowing the fluid to flow over the smear and run
off the other end into the sink.  Keep an eye on the drops as they run off
the edge of the slide.  If you have a thick smear, the drops will be
purple at first and will gradually lose their color.  Decolorization
should be stopped when the drops are colorless.  A thin smear may not show
any color at all -- in this case, a quick rinse with the decolorizer
should be sufficient.

Hope this is useful to you.


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