Dimethyl sulfoxide (dmso)

The determination of MIC and MBC values was performed by the microdilution technique. MBC was determined by sub-culturing the samples having a value of lesser or equal to MIC value on the appropriate media namely Mueller Hinton (MH) for bacteria and Sabouraud Chloramphenicol agar (SC) for Candida strains. The highest dilution (lesser concentration) that yielded no single bacterial colony was taken as MBC value. Each extract was diluted in 5% dimethyl sulfoxide (DMSO, Kemika, Zagreb, Croatia) and subjected to serial dilution starting from 150 mg/ml. The 96-well plates (round bottom) were prepared in the same way that each well contains 95 μl of Sabouraud or MH broth, 5 μl of microbial suspension and 100 μl of dilution of the extract (300 mg/ml) to be tested. The negative control well contains 195 μl of enrichment broth without extract and 5 μl of suspension of the microorganism to be tested. After incubation at 37°C for 18 to 24 h, the lowest concentration, at which there was no turbidity, was also regarded as MIC value of the extract. MBC/MFC values are estimated by the concentration of the extract which totally inhibits the growth of bacteria or yeast, and this concentration was subsequently confirmed on MH or SC agar after incubation 24 h at 37°C.