[Detection of viable or non-viable mycobacteria by hydrolysis of fluorogenic substrate]

Acid-fast staining is broadly used for the detection of mycobacteria in smears or pathological preparations, but it does not distinguish viable and non-viable bacteria. Enzymatic hydrolysis of fluorescein diacetate (FDA) was reported as a tool to detect viable mammalian cells or protozoa. We have applied this method to mycobacteria samples to detect viable or non-viable bacteria in the smear. Mycobacterium bovis BCG (Tokyo), 20 mg/ml suspension of standard vaccine, was used as the sample of viable bacteria. A part of the same suspension was heated at 100 degrees C for 30 min and used as the sample of non-viable bacteria. Three samples, viable, non-viable, and 1:1 mixture of the two, were stained with acid-fast staining and FDA-EB (ethidium bromide) mixture, respectively. For FDA/EB staining, stock solution of FDA (5 mg/ml acetone) was diluted 1:50 in PBS and 25 microliters of FDA and 25 microliters of EB (20 micrograms/ml PBS) were mixed with 50 microliters of bacterial suspension. Preparations were observed with either light or fluorescein microscope. Living bacteria were stained in yellow green in FDA/EB staining while non-viable bacteria were red. Mixed sample of live and dead bacilli showed differential staining with green or red in FDA/EB staining, but no difference was shown in acid-fast staining between viable and non-viable bacteria.