Determination of MIC and MBC values

Stationary phase cultures were diluted to a final cell concentration of 10⁷ cfu/ml in a 96-well plate with wells holding a final reaction volume of 200 μl of fresh medium containing different concentrations of the investigated antimicrobial substance. The tested concentrations of BAC (Sigma-Aldrich, 234427), benzyl-dimethyl-dodecylammonium chloride (BAC₁₂; Sigma-Aldrich, 13380) and NBD-DDA were 10, 20, 30, 40, 60, 100, and 150 μM. The plates were incubated at 37°C with agitation for 24 h and growth was assessed visually. To determine the MBC (minimum bactericidal concentration), 10 μl from each well without visible growth were spotted onto LB agar medium and incubated at 37°C for 24 h. The first concentration that reduced the initial cell density by a factor of 10³ or more was designated the MBC. NBD-DDA was dissolved in DMSO. To assess the effect of DMSO on growth, a MIC (minimum inhibitory concentration) assay with the solvent DMSO alone was conducted. Growth was not inhibited by DMSO in the concentration range tested here. All experiments were conducted with n = 3 biological replicates and yielded the same MIC and MBC.