Mycotrim® RS - Irvine Scientific

6. Optional Use of Dienes Stain
Dienes stain should only be used when the flask will no longer
be incubated. The Dienes stain kills M. pneumoniae. Once
stained, a flask may be stored for several months in the
refrigerator.
To apply Dienes stain:
a. Open the flask and remove the broth with a pipet. Dispose
of the broth in a suitable infectious waste container.
b. Taking care not to wash the growth off of the surface of the
agar, gently apply two or three drops of stain to the agar
surface. Recap the flask.
c. Let the flask stand agar side down for 30 minutes to allow
the stain to diffuse into the agar.
d. Examine the flask microscopically for colonies. M. pneumoniae
will retain the stain while bacteria will decolorize
and not appear stained.
INTERPRETATION OF RESULTS
A positive result is indicated when the color changes (in the absence
of gross contamination) from red to yellow-orange and colonies are
observed on the agar.
Unlike bacteria and fungi, M. pneumoniae does not cause turbidity
in the broth, even at maximum titers. Gross turbidity or visible
colonies on the agar indicate bacterial or fungal overgrowth,
making the color change unreliable and possibly hiding M.
pneumoniae colonies.
A negative result is reported if there is no color change within 10
days after reinoculation (13 days total incubation).
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