Use of flow cytometry for enumeration of Mycoplasma mycoides subsp. mycoides large-colony type in broth medium

Abstract

Aims: The potential of using flow cytometry (FC) in combination with a fluorescent dye (SYBR green-I) for rapidly estimating Mycoplasma mycoides subSPS. mycoides large-colony type (MmmLC) in broth culture was examined.Methods and Results: The FC analysis was performed by staining the MmmLC cells with a fluorescent dye, SYBR green-I (SYBR), and the results were compared with plate count method (colony forming units, - CFUs). There was a good correlation (linear regression, r(2) = 0.93) between mycoplasma counts determined by FC (cells ml(-1)) and by traditional plate count method (CFU ml(-1)). The lowest bacterial concentration detected by FC and traditional plate count was of the order of 10(4) cells ml(-1) and 10(3) CFU ml(-1), respectively. FC method allowed results in 20-30 min, whereas at least 24 h were necessary to obtain results with the traditional plate count method (CFU).Conclusion: Growth rates of MmmLC in broth medium determined by FC were highly reproducible and correlated well with mycoplasma counts assessed by the plate count method.Significance and Impact of the Study: These findings suggest that FC could be a good alternative to replace other time-consuming techniques that are currently used to enumerate mycoplasma in broth medium, such as plate count method (CFU).