Determination of Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC), and Minimum Fungicidal Concentration (MFC) of Endophytic Fungal Extracts

The minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) were determined using broth micro-dilution method in a sterile 96 well micro plate, as described by Gulluce et al. (2007). Serial dilution of each fungal extract was prepared to get final concentrations ranging from 0.781 to 100 μg/μL. Each well was supplemented with 10 μL of bacterial or fungal suspension, 90 μL of liquid culture broth and 100 μL of fungal extract. The last well containing the above-cited components without addition of the fungal extract was considered as positive control. The one containing DMSO without extract was the negative control. Plates were incubated at 37°C for 24 h for bacterial strains and for 3 days at 30°C for fungal strains. Afterward, 25 μL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was added to each well for evaluation of the microorganisms viability. After incubation of plates for 30 min at 37°C, the clear wells indicated the inhibition of cell growth. The MIC is defined as the lowest concentration of extract that inhibit the growth of microorganisms. The MBC values were determined after incubation of plates for 48 h at 37°C as the highest dilution of extract that completely inhibit the growth of bacteria. MFC was considered as the first well with no visible growth of the test fungi. It was determined by inoculating the PDA plates with 10 μL of the well content followed by incubation for 3–5 days at 30°C. The MFC values were interpreted as the lowest concentration of endophytic fungal extract that inhibits fungal growth.