In vitro methods for determination of viability of mycobacteria: comparison of ATP content, morphological index and FDA-EB fluorescent staining in Mycobacterium leprae.

Bacilli were purified from the 23 cases of multi bacillary type of leprosy. The ATP content of these bacilli was assayed by a firefly bioluminescent technique which is capable of detecting a very small number of cultivable mycobacteria as assessed by colony counts. The A TP content was compared with morphological index (MI) and FDA-EB staining of bacilli from the same specimens. It was observed that when MI was I % or more, the A TP content/solid bacillus was fairly constant in 1 5 cases studied . It ranged from 2·02 x 1 0 1 5 g to 5 ·60 X I S g/solid bacillus (mean 3 ·46 x 1 0 1 5 g) and was in the same range as A TP content of viable cultivable mycobacteria . In the same IS cases, when the green-staining bacilli was considered as 'supposedly viable bacil l i ' , ATP content/green-staining bacillus varied up to 9-fold (0·22 1 5 x g to 1 ·98 X 1 0 1 5 g/green-staining bacillus) and this did not correlate . The percentage of green-staining bacil l i (FDA-EB) and solid­ staining bacilli (MI) was different in all the cases. In 2 cases with 0% MI in which ATP levels were also zero, 7 · 5 % and 2 1 · 5 % green-staining bacilli were present which implies that the enzymes responsible for green-staining character may persist for some time after death . Three cases with 0% MI had also 0% green­ staining bacilli and zero A TP levels, whereas in another 3 cases with zero M I significantly high levels o f A T P were detected. It is inferred that solid-staining bacilli may be the viable bacilli but when MI is 0% (I % or less) sampling error or clumping may be responsible for missing out the solid bacilli in some cases. I t is concluded that the ATP content of M. /eprae appears to be an easy, rapid and sensitive tool for determining the viability for monitoring the therapy. On the other hand MI and FDA-EB staining appear to have their limitations.