Fig. 7.

TLC-bioautography assay against Pseudomonas aeruginosa ATCC 27853. The active component in the crude extract responsible for antimicrobial activity was identified by the TLC-bioautography agar overlay method, which is a combination of contact and direct bioautography. a TLC profile of metabolites under long UV light. The crude extract was extracted from fermented broth using Potato dextrose broth (PDB) as a culture medium using ethyl acetate (EA). The EA crude extract ~ 10 µl was spotted onto TLC plates (Silica gel 60 F254, Merck) with at least 1 cm between the spots and placed in a solvent mixture of Chloroform: Methanol (9:1, v/v). Once the mobile phase reached the top, the TLC plate was removed, the solvent evaporated and observed under short and long UV. b Bioautography assay. The TLC plate was placed in a 90 mm Petri-plate and covered with molten Mueller–Hinton agar medium to a thickness of 2–3 mm. Once solid, the agar was overlaid with 10⁶ CFU/ml of P. aeruginosa ATCC 27853. The plates were incubated at 37 ± 2 °C for 18–24 h. c Sprayed with MTT solution, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, 0.5 mg ml⁻¹) and d Colorless inhibition zone (marked with a circle) against a purple background, showing the active component in the crude extract responsible for antimicrobial activity. Metabolically-active microorganisms convert the tetrazolium salt (MTT) into the corresponding intensely colored formazan (dehydrogenase activity of the microorganisms). As expected, dead bacteria cannot reduce the MTT, hence unable to produce the characteristic color change from yellowish to purple