Determination of MIC of colistin and gentamicin alone by micro broth dilution method

The MIC of colistin and gentamicin against the bacterial and yeast cultures was determined by micro broth dilution method as outlined by Clinical and Laboratory Standards Institute (CLSI) guidelines, 2012. Briefly, the drugs were serially diluted in Mueller Hinton broth to obtain their final working concentrations. The concentration of colistin ranging from 0.25–16 µg/mL, 8–1024 µg/mL, and 2–512 µg/mL was used against P. aeruginosa, S. aureus, and C. albicans, respectively. The concentration of gentamicin ranging from 0.5–512 µg/mL was used against P. aeruginosa and C. albicans, and 0.25–16 µg/mL was used against S. aureus. The micro broth dilution method was performed on a 96-well plate. A 100 µL of antimicrobial agent of varying concentrations was added to the plate along with 100 µL of the prepared bacterial or yeast inoculum having a density of 5 × 10⁵ CFU/mL and 0.5–2.5 × 10³ CFU/mL, respectively. Sterility and a growth control were added to the 96-well plates to validate the experiment. The plate was then incubated at 37°C for 24 h. The MIC, which is the lowest concentration of the drug that completely inhibits the growth of the organism, was determined for each drug individually against their respective organism. The quality control strains used were, ATCC 27853, ATCC 29213 for P. aeruginosa and S. aureus, respectively, to monitor the test performance.