Minimum inhibitory concentration (MIC)

To determine the minimum inhibitory concentration (MIC), the strains were inoculated in TSB medium for 18 h at 37 °C. Bacterial cultures were centrifuged at 1721 × g, and the supernatant was removed, P. aeruginosa was normalised to an optical density of 0.05 at OD _{600 nm} in double-strength TSB, which equated to ca. 0.5 × 10⁸ CFU mL⁻¹. The antibiotics were dissolved to a final concentration of 2048 μg mL⁻¹. In a 96 well plate, the outer wells were filled with 200 µL sterile TSB. In the second column of wells, 200 µL of the antimicrobials at the maximum concentration and 100 μL of sterile distilled water was added to the sequential columns. A twofold dilution was carried out, 100 μL of the solution was transferred into the next column (column three); this process was repeated until column ten, where 100 µL of the solution was discarded ensuring all wells had a 100 µL total volume. Normalised P. aeruginosa  solutions were combined with 0.15% triphenyl blue chloride (TBC) (this was included as a metabolic activity indicator). Aliquots of 100 μL of the bacterial suspension was then added to each well, giving a final volume of 200 μL. To avoid evaporation, the plate was closed and sealed with Parafilm^®. The outer wells that contained sterile TSB served as the negative control. Column eleven contained P. aeruginosa but no antimicrobial (positive control). The plates were incubated at 37 °C for 18 h without agitation. This study was also conducted with the solvents the antimicrobials were dissolved in to ensure that the antimicrobial activity was not produced solely by the solvents. Following incubation, the MIC of the antimicrobial was defined as the minimum concentration that inhibited visible growth in that well, compared with the positive and negative controls utilised in this study (n = 3).