Minimum Inhibitory Concentration

The minimum inhibitory concentration (MIC) of the organic extract was determined by micro-broth dilution assay in a sterile 96-well plate (Padhi and Tayung, 2013) against P. aeruginosa, S. aureus, E. coli, and C. albicans. A two-fold dilution of the extracts with the concentration ranging from 1,000 to 62.5 μg/ml was made. The wells were filled with 90 μl of each test bacterial and fungal suspension (approx. 10⁶ CFU/ml). Test extracts (10 μl) of different concentrations were added into each well to make upto a final volume of 100 μl. Medium containing 10% DMSO was used as the negative control. After incubation at 37°C for 24 h for bacteria and 48 h for fungal pathogens, a solution (10 μl) of triphenyltetrazolium chloride (TTC) was added to each well as microbial growth indicator, and the microplates were incubated for an additional 30 min. MIC was determined as the lowest concentration of the test extract at which no pink color appeared.