Differentiation Help

Judy and Bob Dilworth dilworth at megsinet.net
Mon Mar 1 22:28:25 EST 1999

Take care also when you decolorize.  Streptococci can decolorize fairly
quickly.  The suggestion for staining from a liquid medium is a good one that we
have used in clinical microbiology before, as the gram reaction determines the
antibiotic the patient gets with a positive blood culture (septic situation).
When we perform gram stains in the clinical lab, we fix the stain first with two
minutes of the decolorizer, then tap it off and put the crystal violet right
over the top of the decolorizer.  We add a 5% solution of baking soda in water -
one drop per slide with a dropper bottle on top of the crystal violet and stain
1-2 minutes.  Then rinse, and add your iodine 1-2 minutes (various texts list
different times).  The baking soda seems to help the gram positives stay gram
positive and the gram negatives show up gram negative.  Decolorize ONLY until
you just see the crystal violet loosen up on the slide, then rinse immediately.
Thicker smears take a little longer.  If you're staining colonies as opposed to
clinical material, decolorization takes place in seconds.  Don't tarry long with
this step.  Then finish with two minutes of safranin. The decolorizer fixative
step works well with clinical material also.  This won't be something you run
into in college classes (hopefully) but with female genital specimens,
trichomonads will show up quite nicely on acetone/alcohol fixed stains.

Have fun with your unknowns.

Judy Dilworth, M.T. (ASCP)
Microbiology 22 years

Mike wrote:

> I'm a university student in a microbio intro course and we're working with
> unknowns.  Many in the class, including myself, are having a difficult time
> differentiating between rods and cocci on student-prepared Gram stain slides
> at 1000X oil-immersion magnification.  Often they look like rods but also
> dividing cocci.  The little "bugs" are just so small.
> Any tips for the beginner?
> Thanks in advance for your help!
> Mike

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