Lipid extraction and analysis.

About 125 ml of M. tuberculosis cultures was grown in a roller flask containing tyloxapol-free m7H9 medium containing 0.2% glucose. Lipids were extracted from whole bacterial cells by incubation in chloroform-methanol (1:2, vol/vol) for 2 days at room temperature and then in chloroform-methanol (2:1, vol/vol). Collected organic phases were pooled, washed twice with water, and dried to get crude lipid extracts. Extracted lipids were suspended in chloroform at a final concentration of 20 mg/ml and analyzed by high-performance thin-layer chromatography (HPTLC; Camag). Equal amounts of lipids were spotted on silica gel 60 plates (Merck) with a Camag ATS4 apparatus. The plates were developed using a Camag ADC2 device in various solvent systems (petroleum ether-diethyl ether, 9:1 [vol/vol], for PDIM; chloroform-methanol-water, 65:25:4, for glycolipids). PDIMs were visualized by spraying the plates with 10% phosphomolybdic acid in ethanol, followed by heating, and glycolipids were visualized by spraying the plates with a 0.2% anthrone solution in concentrated sulfuric acid, followed by heating. DIM A and B in enriched fractions were identified by nuclear magnetic resonance spectroscopy.