14c acetic acid

For lipid analysis, M. chelonae biofilms and planktonic cultures were grown as described before, but Sauton’s media was supplemented with ¹⁴[C]-acetic acid (1 μCi/mL, Perkin Elmer). Different lipid fractions were extracted and resolved by thin layer chromatography as described previously (Besra, 1998 (link)). Lipid species were visualised by autoradiography by exposing X-ray films Kodak Carestream) to the resolved TLC plates for 48 h.

The composition of sebocytes in major neutral lipids was investigated by separating crude lipid extracts by TLC. A total of 2 μCi of [¹⁴C]-acetic acid (Perkin-Elmer Corp., Norwalk, CT, USA) was added to the medium for 6 h. After removing the medium, cells were detached and transferred into 1.5-mL tubes containing 200 μL of a chloroform/methanol (2:1) mixture. After cell debris was removed by centrifugation at 13,000 rpm for 5 min, the tubes were dried using a vacuum evaporator. The lipid components extracted from sebocytes were analyzed by chromatographic separation on 20-cm silica gel TLC plates that had been previously charged with chloroform. After spotting the lipids (40 μL) dissolved in chloroform, the plates were developed three times as follows: (1) hexane to the top, (2) toluene to the top, and (3) hexane/ether/acetic acid (70:30:1) 10 cm from the top. A 30-min drying period in a standard fume hood was performed between each mobile phase to ensure complete evaporation of the solvent.

Radiolabeling with [¹⁴C]-acetic acid (PerkinElmer) (1 mCi/mL) was performed on cultures at mid-log phase (optical density at 600 nm (OD₆₀₀) = 0.5) for 24 h before harvesting or after 30 min of treatment with or without 0.025% SDS and harvested after a 6-h incubation at 37 °C. Mycolic acids were extracted using a modified method based on the method described in ref. 48 (link). Dried whole-cell pellets were subjected to alkaline hydrolysis by 10% tetrabutylammonium hydroxide overnight at 100 °C and methylation by addition of 4 mL of CH₂Cl₂, 500 µL of CH₃I, and 2 mL of water for 30 min. The resultant lower organic phase was collected and washed with water to recover fatty acid and MAMEs. For analysis by 2D argentation TLC, samples were loaded at 15,000 counts per minute (cpm) onto Silica Gel 60 F254 plates and resolved, unless otherwise stated, using petroleum ether 60–80/acetone (19:1 [vol/vol]) twice in the first direction and three times in petroleum ether 60–80/ethyl acetate (18:2 [vol/vol]) in the second direction. Autoradiograms (Carestream Kodak BioMax MR) were exposed for 3 d.