Cholesterol, a factor which is required for growth of mycobacteria from leprous tissues

In a yeast extract, glycerol and sheep serum containing medium, slow but abundant growth of mycobacteria occurred when media were inoculated with M. leprae isolated from leprous tissues of armadillos (Dasypus novemcinctus Linn.). The lipid fraction of the serum was the essential factor for growth. Cholesterol not only replaced, but surpassed the growth promoting effect of the lipid fraction. However, growth of mycobacteria was observed only when media were enriched with serum. The relationship of the obtained strains of mycobacteria to leprosy is not yet clear. The following cholesterol medium, stabilized with lecithin, is proposed for primary cultivation of mycobacteria from leprous tissues: KH2PO4-8.2 gm, Na2HPO4-0.5 gm, yeast extract (Difco)-4 gm, and glycerol 30 gm, dissolved to make one liter basal medium in distilled water. Cholesterol (200 mg) dissolved in 4 ml warm acetone is injected with a syringe into the basal medium. The solution is autoclaved for ten minutes to evaporate the acetone. Lecithin, 200 mg dissolved in 20 ml of the basal medium is mixed to the medium cooled to room temperature. Nine milliliter aliquots are distributed into each of a series of 50 ml screw cap tubes and autoclaved for 25 minutes. One milliliter of filter sterilized sheep serum is added to each of the tubes containing 9 ml of the cholesterol-lecithin medium. Semisolid media are prepared the same way but 1.5% agar w/v is added to the cholesterol-lecithin medium before autoclaving. When cooled to 56 degrees C, 10% w/v sheep serum is mixed to the liquid. The medium is distributed into screw cap tubes and agar slants are poured and allowed to solidify in the inclined tubes at room temperature. Macrophages contain considerable amounts of cholesterol. Cholesterol is proposed as a possible growth factor for host grown M. leprae in the macrophages of the susceptible host and the same sterol as a growth factor for primary cultivation of mycobacteria from leprous tissues.