In vitro antimicrobial susceptibility assay (MIC determination)

Before analysis, compounds 5a–f were dissolved in 96% ethanol in a concentration of 2 mg/ml as stock solutions. Inoculums were prepared with fresh cultures of microbial strains, cultured on tryptic-soy agar or Sabouraud 2% (m/v) dextrose agar for 18 h (48–72 h for fungi) at 30 °C with physiological saline containing approximately 3 × 10⁶ CFUs/ml. Inoculum density (0.5 McFarland units) was adjusted with a bio Mériex (France) densitometer. Minimum inhibitory concentration (MIC) was determined by the twofold micro-dilution method in Mu¨ller–Hinton broth for bacterial strains and RPMI 1640 + 2% m/v dextrose for fungi according to the EUCAST recommendations in a sterile 96-well flat-bottom plastic tissue culture plate²⁰. MIC was determined using a linear regression curve after incubation of compounds and reading the optical density at 570 nm using an iEMS microplate reader. MIC was defined as the lowest concentration of extract that allows no more than 50% growth of microbes in comparison with intact microbial cells (negative control). All tests were performed in triplicate and expressed as the mean.