2.7. Determination of minimal inhibitory concentration
Minimum inhibitory concentration of all the fungal extract and plant extract were done by using microtiter broth dilution technique of Keskin et al. [23] with minor modification. A sterile 96 round-bottom well plate was used and was labeled properly. Crude extract of volume 100 μL was added into the first row of the plate. To rest of the wells, 50 μL of potato dextrose broth was added and then serial dilutions were performed using a micropipette (A1–A10). Then, 50 μL of broth containing bacterial suspension (106 cfu/mL) was added to each well. Single antimicrobial extract in progressive dilutions was added in each column of wells. Each plate had a set of both growth (A11) and sterility control (A12). Plates were sealed with parafilm and placed in an incubator at 37 °C for 24 h. After incubation, 10 μL of 0.2% 2,3,5-triphenyl tetrazolium chloride (TTC) solution was added to each well. The plates having TTC were then incubated at 37 °C for 1 h. A visible color changes from purple to pink, indicated growth of microorganism and were recorded as negative. The MIC value of the extract was taken as the lowest concentration that showed no microbial growth.