Fig. 2.

Effect of benzyl alcohol on M. smegmatis lipids. A: Live and heat-killed cells were treated with benzyl alcohol for 60 min, serially diluted, and plated onto Middlebrook 7H10 agar, confirming that rapid heating is effective in killing M. smegmatis. B: HPTLC analysis of PIMs after heat killing followed by benzyl alcohol treatment. PIMs were chromatographed using a solvent containing chloroform, methanol, 13 M ammonia, 1 M ammonium acetate, and water (180:140:9:9:23, v/v/v/v/v) and visualized by orcinol staining. A region of the HPTLC plate corresponding to Rf of 0.22–0.48 is shown. C–E: HPTLC analysis of lipids: (C) PIMs separated and visualized as in panel B (Rf = 0.20–0.48); (D) phospholipids separated as in panel B and visualized by molybdenum blue (Rf = 0.35–0.61); (E) GPLs and TDM separated by chloroform/methanol/water (9:1:0.1, v/v/v) and visualized by orcinol. DMSO, vehicle control. CL, cardiolipin; and PE, phosphatidylethanolamine. All experiments were repeated at least twice, and representative results are shown. DMSO, dimethyl sulfoxide; GPL, glycopeptidolipid; HPTLC, high-performance thin layer chromatography; PIM, phosphatidylinositol mannoside; TDM, trehalose dimycolate.