Technique DIENES STAIN for Mycoplasma
Method 1: Rinse a coverslip with alcohol to remove any grease.
2: Let the coverslip dry
3: Spread a drop of Dienes stain on the cover slip
4: Let the stain dry.
5: Press the stained face of the cover slip against the suspected Mycoplasma colonies
6: Examine microscopically using the low power and high-dry lenses
Results Mycoplasma colonies have a distinctive dense blue stained center and light blue peripheries
Positive control  
Negative control  
Reagents Dienes Stain solution
Methylene blue 2.50 gm
Azure II 1.25 gm
Maltose 10.00 gm
Sodium carbonate 0.25 gm
Distilled water 100 ml
Reference Dienes & Weinberger 1951 Bact Rev 15:245)