Enumeration of mycoplasmas after acridine orange staining

Abstract

Samples of liquid mycoplasma cultures were mixed with equal parts of a 0.01% solution of acridine orange and placed on agar plates. The number of fluorescing organisms per field was counted in an epifluorescence microscope at an X1,000 magnification. When the number of fluorescing organisms per field was related to the number of colony-forming units per milliliter during the growth cycle, highly significant correlation was found in cultures with ≥106 colony-forming units per ml during the exponential growth phase. The counts were weakly correlated during the stationary phase and not correlated during the death phase. This technique provides a mean to enumerate mycoplasmas in liquid cultures.