2.6. Antimicrobial activities of the fungal extract

Ten milligrams of ethyl acetate crude extract were dissolved in 1 ml of dimethyl sulfoxide (DMSO) to make a stock solution of fungal crude extract. Using Muller Hinton agar (HiMedia, India) for bacteria and Sabouraud Dextrose Agar (HiMedia, India) for Candida albicans, the antimicrobial activity of the prepared stock solution was evaluated using the agar diffusion technique [29,30]. The prepared agar plates were inoculated with 100 μl of overnight grown culture (10⁶ CFU/ml) of Staphylococcus aureus ATCC 6538 (S. aureus), Bacillus cereus ATCC 10,987 (B. cereus), Escherichia coli ATCC 8739 (E. coli), Salmonella typhimurium ATCC14028 (S. typhimurium), Klebsiella pneumonia ATCC 13,883 (K. pneumonia) and 48 h old culture of Candida albicans ATCC 10,231 (C. albicans). Using a sterile cork borer, wells (8 mm in diameter) were cut in the inoculated agar media and 100 µl of crude extract stock solutions were transferred into each well. Ciprofloxacin (30 μg) was used as antibiotic control for bacterial strains while fluconazole  (25 μg) was used as antifungal agent control for Candida albicans as well as DMSO which was used as a negative control. All plates were left for 2 h at 4 °C until the extract was diffused then incubated for 24 h at 37 °C in case of bacteria and 48 h at 28 °C for Candida albicans. The experiment was performed three times. After incubation, the inhibition zones were measured, and the mean values were calculated.

Minimum inhibitory concentration (MIC) of an antibacterial agent is stated in (µg/ml) and is defined as the lowest concentration of an antimicrobial agent that, under tightly regulated in vitro conditions, totally inhibits observable growth of the test strain [31]. The MIC values of crude extract of the fungal isolate were evaluated using a microdilution test in 96-well plates according to Sharaf et al. [32]. The above-mentioned standard bacterial and yeast strains were inoculated, with a cell suspension concentration of 10⁶ CFU/ml, in Muller Hinton broth and Sabouraud dextrose broth media, respectively, and then 200 µl was loaded into wells. The fungal crude extract was diluted in twofold concentrations (7.8- 1000 μg/ml) and tested against bacterial and yeast strains. The differences in optical density were evaluated using the wells that contained a negative control (medium + tested stock solutions at the tested concentrations). The absorbance was measured at 630 nm after an 18-h incubation period at 37 °C.