Mycoplasma colonies can be identified by observing typical fried egg colonies on culture medium. Visualization is facilitated by application of Diene’s stain directly to the agar surface. Diene’s stain is a nonspecific stain that imparts a contrasting appearance to Mycoplasma colonies on agar. It allows easy visualization of colony morphology and characteristics.

Composition of Diene’s stain
Methylene blue     2.5 gm
Azure II                 1.25 gm
Maltose                  10 gm
Na2CO3                 0.25 gm
Distilled water      100 ml
Diene’s stain working solution is prepared by diluting an aliquot of the stock solution 1:3 with distilled water.

Procedure for Diene’s stain:
• Flood the agar containing Mycoplasma with 1 ml of Diene’s stain working solution.
• Immediately rinse the agar surface with distilled water to remove the stain.
• Decolorise the medium by adding 1 ml of 95% ethanol for 1 minute and wash with distilled water.
• Rinse the medium with distilled water and allow it to dry.
• Observe the typical colonies under low power objective (50 to 100X).

Observation and Interpretation:
Mycoplasma with fired egg colony morphology will stain with a dark blue center and light blue periphery and will appear highly granular. The agar background will remain clear or slightly violet.
Mycoplasmas other than M. pneumoniae will remain stained; M. pneumoniae reduces the methylene blue after some time and will become colorless. If the isolate is from genital tract and produce typical colonies and staining, the isolate can be reparted as M. hominis.

Reference: Koneman’s Color Atlas and Textbook of Diagnostic Microbiology, Sixth Edition

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