Extraction and Purification of Mycobacterial Cell Wall Fatty Acid Methyl Esters (FAMEs) and Mycolic Acid Methyl Esters (MAMEs)

Mycobacterial cell wall fatty acid and mycolic acids was purified following protocols as described (Dupont and Kremer, 2014). The liquid mycobacterial cultures of M. smegmatis were inoculated and grown, in Middlebrook 7H9 broth and incubated at 37°C with slow shaking till the OD₆₀₀ reached (OD₆₀₀ = 1) of the culture. Exponentially growing mycobacteria were harvested for mycolic acid extraction. The mycobacterial pellet was resuspended in 4 ml of tetrabutylammonium hydroxide (TBAH) and incubated overnight at 100°C for hydrolysis of complete fatty/mycolic acid. To the 4 ml of TBAH mixture, 8 ml CH₂Cl₂, 600μl CH₃I and 4 ml water were added and mixed well for 1.5 h at room temperature for methyl esterification of the mycolic acids. It was centrifuged at 3,500 rpm for 10 min at room temperature to separate the mixture in two phases: a lower organic phase containing the lipids and an upper aqueous phase that was discarded. The tube was mixed for 25 min after adding 5 ml of water and centrifuged at 3,500 rpm for 10 min at room temperature. The wash step was repeated three more times and the upper phase was removed. The lower organic phase was dried, 4 ml diethyl ether was added, and sonication was performed in a water bath for 5–10 min at room temperature. It was further centrifuged at 3,500 rpm for 10 min at room temperature and methyl esters were transferred in a new glass tube. The diethyl ether was evaporated under a stream of nitrogen and the residue was resuspended in 100–200 µl CH₂Cl₂. The extracted fatty acid methyl esters (FAMEs) and mycolic acid methyl esters (MAMEs) were resolved by thin layer chromatography using hexane/ethyl acetate (19:1, v/v). The TLC plate was sprayed with 5% ethanolic molybdophosphoric acid, lipid bands corresponding to the different mycolic acids revealed following charring using a heat gun.