A culture medium for cultivation of mycobacteria, probably Mycobacterium leprae from Mycobacterium leprae infected tissues.

Mycobacterium leprae suspensions were prepared from infected armadillos. The M. leprae cells were inoculated into culture media containing KH2PO4 4.7. g. Na2HPO4 2 g, sodium thioglycolate 1 g, (NH4)2SO4 2 g, MgSO4 0.1 g, ferric ammonium citrate 0.05 g, and lipoic acid (thioctic acid) 0.1 g in one liter distilled water. The solution was enriched with heat killed, sonicated leprosy derived Mycobacterium X or crude mycobactin extract from M. phlei to contain + 0.2 micrograms mycobactin per 1 ml in the final medium. Twenty ml media was distributed into each of 25 ml screw cap tubes and autoclaved for 30 minutes. Positive growth was obtained from seven out of ten specimens when incubated at 34 degrees C. The cultures developed as a sediment in the liquid media, suggesting preference for microaerophylic conditions. No growth was seen on the surface of the semi-solid agar media containing the same ingredients. Latency period of growth was estimated as 10-16 days and time of division as 6 days. Subcultures were obtained. Cells were long, acid fast, arranged side by side or end to end, with a tendency to form long spiral cords or clumps when sedimented on siliconized slides. Pyridine extraction eliminated acid fastness, but not gram positivity. Cultures did not grow on Dubos, Lowenstein or 7H10 media. They produce the disease in the foot pads of mice characteristic of M. leprae. Subcultures remain dependent on the heat killed sonicated mycobacteria, or crude mycobactin extract, and reduced oxygen tension in the media. Results suggest that cultures might be identical to M. leprae.

Absence of mycobactin in Mycobacterium leprae; probably a microbe dependent microorganism implications.

Ferric mycobactins were prepared from Mycobacterium phlei. Mycobacterium avium--intracellulare A and H, isolated respectively from armadillo and human leprosy specimens. Attempts were made to extract mycobactin from host grown M. leprae cells. The crude ferric mycobactin extracts were tested for growth supporting effect on the mycobactin dependent M. paratuberculosis strain ATCC 19698. Mycobactins prepared from M. phlei and the two M. avium--intracellulare strains had growth promoting effect on M. paratuberculosis. The same test organism did not grow in media supplemented with the extract prepared from M. leprae. Results indicate the absence of mycobactin from host grown M. leprae. Since M. leprae cells contain cytochrome c and since mycobactin is essential to growth of all mycobacteria, M. leprae might be considered as a microbe dependent microbe. It is proposed that secondary mycobacteria present in M. leprae infected humans and armadillos might provide mycobactin for in vivo multiplication of M. leprae.