Quantification of mycoplasmas in broth medium with sybr green-I and flow cytometry

Mycoplasmas are the smallest and simplest organisms known. They form a large group of bacteria that can infect humans, animals, and plants. Even though several techniques have been proposed to enumerate mycoplasmas in broth medium, the determination of mycoplasma growth still remains a difficult task. The potential of using flow cytometry (FC) for rapidly estimating several species of mycoplasmas, M. agalactiae (Ma), M. putrefaciens (Mp), M. capricolum subsp. capricolum (Mcc), M. bovis (Mb), M. capricolum subsp. capripneumoniae (Mccp) and M. hyopneumoniae (Mh) in broth medium was examined. The FC analysis was performed by staining the mycoplasma cells with a fluorescent dye, SYBR green-I (SYBR), and the results were compared with plate count (Colony Forming Units--CFU) or Colour Changing Units (CCU) methods, depending on the mycoplasma species. There was a good correlation between mycoplasma counts determined by FC (cells ml(-1)) and by traditional plate count (CFU) or CCU methods. A correlation of 0.841, 0.981, 0.960, 0.913, 0.954, and 0.844 was obtained for Ma, Mp, Mcc, Mb, Mccp and Mh, respectively. FC method allowed results in 20-30 min, while 24-72 h was necessary for plate count method and 15 days for CCU method. FC was found to be a very useful, practical and fast technique to count mycoplasmas. These findings suggest that FC can be a good alternative to replace other time-consuming techniques that are currently used to enumerate mycoplasmas in broth medium.