Minimum Inhibitory Concentration Assays

Inoculum were prepared following standard microdilution assay procedures^22–24 using Mueller-Hinton broth for all bacteria and YM broth for yeast. Each triazole and triazolium bromide compound was prepared as a 10 mM solution in DMSO. 10 μL of each DMSO stock solution was diluted into 190 μL broth and a 1:1 serial dilution was performed in a 96 well plate. Addition of 100 μL inoculum to each well resulted in a range of 250, 120, 62, 31, 16, 8, 4 and 2 μM concentrations for each MIC assay. Plates were incubated for 20 h (bacteria) and 24 h (yeast) at 37 °C then examined by eye for cloudiness indicating microbial growth. The most dilute member within a serial dilution that remained transparent after 24 h was defined as the minimum inhibitory concentration (MIC) value for that compound/organism combination. Assays were performed in triplicate. Any compounds displaying activity at the minimum 2 μM concentration of the original assay were tested again at a 10-fold dilution using the same microdilution procedure with a 1 mM DMSO stock solution.