Determination of minimum inhibitory concentration (MIC)

MICs against microbial strains were determined by broth microdilution using two-fold serial dilutions in Muller Hinton Broth for bacteria and RPMI 1640/MOPS for C. albicans as described by the Clinical and Laboratory Standards Institute (CLSI) method. The test was carried out in 96-well U-bottom microdilution plates. Microbial inocula were prepared by subculturing bacteria into Muller Hinton Broth (MHB) and Candida cells in Sabouraud Broth at 37 °C for 18 h and then diluted to approximately 10⁵–10⁶ CFU/ml in MHB or RPMI/MOPS. One hundred μl of test compounds were diluted 1:2 in appropriate medium and placed in a 96-well tissue culture plate. The initial concentrations of the compounds used was 250 mg/L. One hundred μl aliquots of test microorganisms were added to each well. Microplates were then incubated at 37 °C for 24 h. Each experiment was repeated at least three times. As positive growth control, wells inoculated with microorganisms in the absence of the tested compound were carried out. MIC value was defined as the lowest concentration of compound that inhibits microbial growth. The positive control for Gram-positive and Gram-negative bacteria was gentamicin, and fluconazole for C. albicans.