5.2. Antibacterial Activity Testing—Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)

Samples of each of these chemical compounds were reconstituted into an appropriate volume of DMSO to achieve a final concentration of 10 mg/mL. MIC values for these compounds was determined by two-fold broth microdilution in 96-well microtiter plates. Briefly, overnight cultures of Escherichia coli ATCC 25922, Escherichia coli 4, MRSA ATCC 43300 and MRSA 06/04 (see Table S1 for further information about the isolates) were diluted in sterilised PBS to approximately 10⁵ CFU/mL. Aliquots of 5 µL were then transferred to separate wells in a 96-well plate that contained 100 µL of each compound at varying concentrations (ranging from 100–0.195 μg/mL) prepared from two-fold serial dilutions in Mueller-Hinton (MH) broth. Plates were incubated at 37 °C for 18 h using an Omnilog^® automated incubator (Biolog Inc.;21124 Cabot Boulevard, Hayward, CA 94545, USA) and MIC values recorded.

Determination of the MBC values for all compounds tested above was performed in MH broth media. Again, 5µL were collected from the MICs 96-well plates (above) and re-inoculated into fresh sterile 96-well plates containing fresh MH. Plates were incubated under the same conditions mentioned above. The assay was performed in triplicate for each compound. (see Table S1 for UCD Centre for Food Safety strains used for determination of antibacterial activity and Table S2 for Antibacterial activity of compounds tested – MIC and MBC results (triplicates)).