Water soluble complex of palmitic acid in media for cultivation of leprosy-derived psychrophilic mycobacteria from Mycobacterium leprae infected tissues.

Abstract

Palmitic acid and palmitates were transformed into water soluble complexes with crystalline heptakis-2,6-di-0-methyl-beta-cyclodextrin. This formulation was incorporated into liquid and solid chemically well-defined media. The fatty acid served as C and energy source, ammonium thioglycolate as the sole source of N with the SH group as further source of energy. Minute amount of dimethyl-sulfoxide added was used for its known effect on cell membrane permeability. The media were inoculated with host grown Mycobacterium leprae cells isolated from human, armadillo and Nu mice foot pad lepromata. No growth occurred in the liquid medium at 22 or 32 degrees C, but cultures and subcultures of acid fast rods were grown at 10 degrees C. Bacilli in the cultures were solid, strongly acid fast rods, growing in clumps like globi. Growth on the semisolid media was visible as smooth round colonies, of white to ivory in colour, slowly expanding flatly at the periphery of the colony on the agar surface. Colonies developed within 2-3 weeks and reached maximum size at 50-80 days depending on the size of inoculum. Subcultures grow faster and more abundantly with adaptation to the media. No growth was seen without the water soluble complexes of palmitic acid or palmitates in the media. The free fatty acid or its salts had an equal growth supporting effect. Identical psychrophilic cultures were obtained from 7 out of 9 armadillo, 12 out of 12 Nu mice and 1 out of 2 human lepromata. None of the cultures grow on Loewenstein, Dubos or 7H9 media at 10 degrees C, 20 degrees C or 32 degrees C, respectively. The tested 4th to 7th subcultures of the strains were strongly positive for phenolic glycolipid-1. Heat killed suspensions of up to 7th subcultures gave negative late skin reaction in all of 16 LL cases. In 19 I, B and T cases the late skin reactions were all similar to that obtained with authentic human lepromin.