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Bioassay guidance was used along the whole process including method development, isolation and identification of antibacterial neem ( Azadirachta indica ) oil compounds. The biomonitoring was performed by direct bioautography (DB), a combination of thin-layer chromatography (TLC) and antimicrobial detection. DB of neem oil showed one antibacterial zone that was not UV-active; therefore, the TLC separation was improved under DB control. The chromatographic zone that exhibited activity against  Bacillus subtilis ,  Xanthomonas euvesicatoria ,  Aliivibrio fischeri ,  Staphylococcus aureus  and methicillin-resistant  Staphylococcus aureus  was characterized by TLC reagents, indicating a lipophilic, fatty acid-like chemical feature. Two compounds were found and identified in the active zone by high-performance liquid chromatography–electrospray ionization mass spectrometry as linoleic and oleic acids. Both fatty acids inhibited  B. subtilis , but  A. fischeri  was sensitive only against linoleic acid.

Rapid, Bioassay-Guided Process for the Detection and Identification of Antibacterial Neem Oil Compounds