RESUMO
A buoyant density centrifugation procedure using Percoll was developed for the isolation and purification of Mycobacterium leprae from experimentally infected armadillo liver tissue. The method separates the bacteria from host adenosine triphosphate (ATP) and tissue debris and recovers 20-25% of the bacteria within 2-2 1/2 hours under controlled conditions. The mean ATP content (585 pg/10(6] of the purified bacteria was similar to cultivable bacteria. The organisms did not leak intracellular ATP when exposed to phosphate buffer. Temperature-dependent ATP synthesis was observed within minutes and could be inhibited by 2,4-dinitrophenol. Freeze-thawing M. leprae as purified suspensions in buffer damaged the organisms, resulting in decreased ATP levels and an accelerated loss of ATP upon incubation under defined conditions. In vitro treatment with the antileprosy drug clofazimine increased the rate of ATP decay directly proportional to drug concentration.