Name: Mike
Status: Other
Grade: Other
Location: NJ
Country: United States
Date: January 2008
Question:
I performed a gram stain on an unknown culture and I followed
the steps according to the book. crystal violet for 1 minute and rinse
iodine for 1 minute and rinse decolorizer 15 seconds and rinse saffranin
1 min 30 sec and rinse........the only problem was that I had purple and
pink staining. the decolorizer was on for exactly 15 seconds. i repeated
the steps and got the same result. under the microscope I saw gram negative
bacilli but no gram positive . Do you know why I had both pink and purple
stains?
Replies:
If the amount of bacteria on the slide wasn't evenly spread on the surface
you probably had portions that were more heavily smeared with bacteria than
others. The decolorizing step is the most sensitive and tricky to get right.
The purple portions could be under-decolorized, in other words the alcohol
may not have penetrated all they way through the smear in the time you gave
it. It could also be over-decolorized if the smear was too thin. You will
have pink and purple areas on the same slide especially if the edges are
thinner than the middle. I usually try to observe the outer edges of a
slide where the smear is thinner. It's probably more important that you
saw bacilli vs. whether they are gram pos or gram neg. Very few bacteria
are actually gram pos. Can you separate them onto selective media such as
PEA (for gram pos) or EMB or MacConkey for gram negative?
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