2.10 Antimicrobial activity
The ethyl acetate extracts from of the three isolates were subjected to determination of the minimum inhibitory concentration (MIC) using the broth microdilution method in 96-well microtiter plates, as described by the Clinical and Laboratory Standards Institute - CLSI (2012) [23], USA: standard M7-A9 (methodology of the antimicrobial dilution sensitivity tests for aerobic growth bacteria), with the modifications, and M27-A4 (susceptibility test for yeasts). Extracts were prepared at the initial concentration of 3.2 mg/mL and dissolved in 10% DMSO. Tests were performed against Escherichia coli ATCC 25922,Staphylococcus aureus ATCC 25923 and Candida albicans ATCC 60193. Each microtiter plate was inoculated with a previously prepared standard bacterial (107 CFU/mL) and fungal suspension (2.5 x 103 CFU/mL). The microtiter plates were incubated at 35-37 °C for 16 to 20 hours for E. coli ATCC 25922 and S. aureus ATCC 25923 and 35 °C for 24 h for C.albicans ATCC 60193. The lowest concentration of extract that inhibited visible growth was recorded as the MIC. Amoxicillin and oxacillin were used as standard antibacterial agents and fluconazole was used as the antifungal standard for positive inhibitory controls.