3.6.3. Determination of Minimum Inhibitory Concentration (MIC)

Microdilution experiments in 96-well microplates were performed to determine MICs [43]. EOBD was diluted in 0.2% agar, whereas the positive control was suspended in MHB and SDA medium with 5% DMSO. Afterward, 100 µL of matter solution was used to produce a range of concentrations using factor 2. All wells except the first, which acted as negative growth control, were inoculated with 50.00 µL of microbial solution. At the end of the experiment, the microplates were incubated at 36 °C for bacteria and 30 °C for yeast. 2,3,5-triphenyl tetrazolium chloride (TTC) was applied to each well before assessing the absorbance after incubation. Wells with bacterial growth became pink owing to dehydrogenases, but wells without growth remained colorless. MIC is the lowest concentration without pink color. Microdilution was also employed to investigate antifungal activity (mold strains) [42]. Briefly, samples were diluted in a PDB medium using tubes with 5 mL, which were inoculated with 100.00 µL of fresh fungal conidia formerly adjusted to 10⁶ conidia/mL. Next, tubes were incubated for 5 days at 27 °C. After incubation, the MIC was known as the lowest concentration of EOBD that kill bacterial growth in tubes.