Microbial Metabolite Fractionation and TLC

All bacterial isolates were cultured individually in TSB (1 L) at 28 °C with shaking at 180 rpm. After 48 h, bacterial pellets were separated from supernatant after centrifuging cultured broth at 10,000×g for 20 min at 4 °C. The resulting supernatant was subjected to fractionation of secondary metabolites by successively adding four different organic solvents (hexane, ethyl acetate, chloroform, and butanol) as described previously by Mollah et al. [35 (link)]. Resulting extracts were resuspended in methanol (0.4 mg/mL) and further diluted with DMSO or methanol to desired concentrations based on experimental purposes. During each fractionation step, the metabolite extraction was subjected to thin layer chromatography (TLC) by spotting it on a silica gel plate (20 × 20 cm: Merck, Darmstadt, Germany). A mixture of chloroform, methanol, and acetic acid (7:2.5:0.5, v/v) was used as an eluent. Spots in the silica gel plate were visualized and marked under a fluorescence analysis cabinet (Spectroline, CM-10, Westbury, NY, USA).