Effect of temperature, cholesterol and nerve tissue on multiplication of armadillo M. leprae.

The effect of temperature, nerve tissue and certain constituents of the medium on multiplication of armadillo M. leprae was studied using Hanks BSS. An equal or better growth was seen at 30 degrees C and 10 degrees C compared to 37 degrees C. Multiplication was also seen at -20 degrees C. Adding cholesterol, foetal calf serum, cystine-HCl, sodium thioglycollate or nerve suspension and covering medium with liquid paraffin each showed beneficial effect. Hanks containing foetal calf serum, cholesterol with sodium thioglycollate or cystine-hydrochloride showed maximum multiplication. These combinations may be used for testing additional factors for further improvement of the medium.

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.