Viable M. leprae as a research reagent

Mycobacterium leprae remain a rare research resource. They cannot be cultivated on artificial media, and the only established means to quantify viability of M. leprae has been by its relative growth in the foot pads of conventional mice (MFP). The MFP method is technically difficult and requires several months to yield results. More effective methods are needed. We examined the association between M. leprae's ability to oxidize 14C-palmitate in axenic culture and the MFP growth results of a large number of suspensions. Oxidative activity was assessed by radiorespirometry (RR) using the Buddemeyer-type biphasic culture vessels containing 7H12 liquid medium and 14C-palmitate, or with commercially prepared BACTEC 12B vessels containing the same medium. The RR results were highly correlated (r = 0.71) with the growth level that each M. leprae suspension achieved by the MFP technique. In using this technique to examine the effects that many common laboratory practices have on M. leprae viability, we found that viability varies markedly between bacillary suspensions derived from different hosts and tissues. The highest viabilities were obtained with bacilli from moderately enlarged nude MFP (< 1 g). Viability tended to be lower among very large nude MFP or long-duration infections and from armadillo tissues. After their harvest from host tissues, leprosy bacilli lost viability quickly. Suspensions stored in 7H12 liquid medium retained < 1% of their viability within 3 weeks of harvest, and freezing bacillary preparations or incubating them at 37 degrees C resulted in nearly an immediate equivalent loss in metabolic activity and viability. M. leprae viability is maintained best when bacilli are stored for only short periods of time at 4 degrees C-33 degrees C. Palmitate oxidation is a rapid, reliable and objective means by which to estimate the viability of M. leprae and can be used effectively as a surrogate for the conventional MFP technique in many studies.