Kinetics of P. aeruginosa Killing by Ethyl Acetate Extract

The rate of kill of P. aeruginosa by the ethyl acetate extract was determined using a modified method from Sim et al. [28 (link)]. Extracts and bacteria used were prepared as described previously for determination of antibacterial activity [29 (link)]. Extract concentrations in the range of 12.5–100 µg/ml were separately added to the wells of a 96-well microplate with an equal volume of the standardised bacterial inoculum. Preincubation absorbance readings of the plate were measured at 590 nm using a microplate reader (Tecan GENios Pro microplate reader, Grödig, Austria). The cell density was then measured at 590 nm at 30-minute intervals for the first two hours, followed by two-hour intervals for 8 hours using a microplate reader (Tecan GENios Pro microplate reader, Grödig, Austria). The profiles of killing and regrowth of bacteria were assessed as a function of both time and extract concentrations.