A fluorescent staining procedure for determining the viability of mycobacterial cells

A fluorescent staining procedure has been developed which rapidly, accurately, and economically measures the viability of mycobacterial cells. M. smegmatis and M. phlei have served as prototype organisms to establish conditions which ensure optimal staining. The staining method incorporates the use of the fatty acid ester fluorescein diacetate (FDA) and ethidium bromide (EB). Non-polar, non-fluorescent FDA enters live cells where it is enzymatically hydrolyzed by acetylesterase to polar, fluorescent fluorescein which rapidly accumulates in the cytoplasm. These cells appear green when viewed under incident ultraviolet illumination. Ethidium bromide enters dead cells and intercalates between the bases of DNA molecules. These cells appear red-orange under UV illumination. Live cells are, therefore, identified on the basis of possessing acetylesterase and their ability to exclude EB; whereas dead cells are identified on the basis of lacking acetylesterase and their inability to exclude EB. The feasibility of applying the staining procedure of M. leprae has been investigated and the results are encouraging. Our findings reveal that armadillo-derived M. leprae possess acetylesterase and, therefore, stain green. M. leprae cell suspensions exposed to adverse physico-chemical conditions give rise to high proportions of red-stained cells as would be expected if the cells are being killed. An alternative means of determining the viability of M. leprae appears to be feasible.