Breath easy membrane

To draw growth curves, starter cultures were grown in 5 ml LB or supplemented M9 medium and the OD₆₀₀ values were measured. The bacteria were then diluted to an OD₆₀₀ value of 0.01 and 5 μl of this suspension was added to 200 μl of medium (LB, LB-Miller, or supplemented M9) in a sterile microtiter plate. For blanks, no bacteria were added. The plates were sealed with a BreathEasy® membrane (from Sigma-Aldrich). The plates were incubated in a Biotek Synergy plate reader with controlled temperature and orbital rotation at the “slow” setting; absorbance at 600 nm was recorded at 20-min intervals. For plotting, values from four biological replicates were averaged.

Minimum inhibitory concentrations (MICs) were determined by the broth microdilution method (Wiegand et al., 2008 (link)). Exponentially growing precultures were seeded in clear 96-well flat-bottom plates (Greiner Bio-One) at OD₆₀₀ = 0.05 in the presence of two-fold serial dilutions of assay compounds in a volume of 200 μl/well. Assay plates were sealed (Breath-Easy membrane, Sigma-Aldrich) and incubated for 7 days at 37°C and 80 rpm prior turbidity determination (OD₆₀₀, Tecan M200Pro plate reader). MIC curves were plotted using the Graph Pad Prism 5 software and the concentration that inhibits 50 and 90% of growth compared to the drug-free control were defined as MIC₅₀ and MIC₉₀, respectively. To establish the Minimum bactericidal concentration (MBC), the colony forming units (CFUs) per assay well were determined by plating serial dilutions of samples onto agar (Murugasu-Oei and Dick, 2000 (link)). Colonies were counted after 3–4 weeks of incubation at 37°C. The MBC was defined as the lowest concentration of test compound that killed 90% of the initial inoculum.

Minimum inhibitory concentrations (MIC) were determined by the broth microdilution method (Wiegand et al., 2008 (link)). Exponentially growing precultures were seeded in clear 96-well flat-bottom plates (Greiner Bio-One) at OD₆₀₀ = 0.05 in the presence of two-fold serial dilutions of assay compounds in a volume of 200 μl/well. Assay plates were sealed (Breath-Easy membrane, Sigma-Aldrich) and incubated for 7 days at 37°C and 80 rpm prior to turbidity determination (OD₆₀₀, Tecan M200Pro plate reader). Percentage growth was determined compared to untreated control and plotted as a function of drug concentration (Graph Pad Prism 5 software). The concentrations that inhibit 90% of growth were recorded as MIC.