Further Studies on the Cultivation of Mycobacterium Leprae

Abstract

We 1 have previously reported the cultivation of Mycobacterium leprae in minced chick embryo suspended in Tyrode's solution direct from leprous nodules containing acid-fast organisms. Evidence of growth and multiplication was obtained in as few as 5 days although subsequent experience has shown that better growth occurs in from 10 days to 2 weeks. The leprosy nodules were digested with 3% sodium hydroxide to free the organisms from the tissue and to destroy contaminants and, following neutralization, the tissue medium was inoculated. Human embryonic tissue (spleen, liver, lung) has also been employed and more experience with this tissue leads us to believe that it is a better medium than chick embryo, contrary to our previous report, although it is, of course, more difficult to obtain.

Leprosy nodules which have been treated as described above, the acid-fast organisms from which have been seeded in minced embryonic tissue suspended in Tyrode's solution, have been found to contain acid-fast organisms which are unquestionably alive. After carrying such cultures in the tissue medium through several generations we wished to test the viability of these acid-fast organisms by placing them on a solid medium, such as the hormone glycerol agar, under a gaseous tension of CO₂ and oxygen. Such cultures were prepared and were incubated under these conditions for 3 months. In one series of 10 cultures of this type we have found 6 to be positive as judged by definite micro-colonies of acid-fast organisms (quite similar to those which have been described for B. tuberculosis obtained from the blood stream) present in smears from the cultures when such smears are taken from suspicious areas which can be identified grossly. These micro-colonies are somewhat difficult to demonstrate and require a perseverance not usually required in the ordinary casual examination of a bacteriological smear, but their identification is definite and, we believe, represents evidence that the acid-fast organisms in the tissue culture are viable after several generations in such mediums. The demonstration of these micro-colonies may prove to be significant in offering an explanation of the frequent failure to demonstrate acid-fast organisms in the very early lesions of leprosy.