Micro-dilution culture to determine minimal inhibition and minimal bactericidal concentrations
In a sterile 96-well plate, serial dilutions were made with the test substance to final volume of 50 μl. Inoculum was then prepared in Mueller-Hinton broth to 0.5 McFarland and diluted before adding 50 μl to the well to make ∼1x105 CFU/ml. The culture plate was incubated at 35 °C for 20-24 hrs. A positive growth control and sterility wells were also prepared along with the culture wells. Absorbance at OD600 was taken using BMG Labtech SPECTROstar Omega spectrophotometer. MIC is determined by the lowest concentration without growth. IC50 is determined from plotting a dose-response curve, see Data Analysis below. To determine the MBC, using a multi-channel pipette, 1 μl of culture broth was taken from the same 96-well microdilution growth plate and carefully inoculated on surface of MH agar plate. The plate is then incubated for additional 20 hrs. at 35 °C. The MBC value is the lowest concentration without observed growth on the agar.