2.3. Minimum Inhibitory Concentration (MIC) Assay

The antimicrobial activities of the single compounds, including Tob, Cipro and IA, as well as the combinations of Tob:IA and Cipro:IA (molar [antibiotic]:[IA] of 1:5) were investigated by standard microbroth dilution assays with planktonic PA14 wt in 96 well-plates. A suspension of PA14 wt prepared from mid-log cultures in PPGAS medium was first centrifuged and diluted to OD600 (absorption at 600 nm) 0.02, which corresponds to approximately 2 × 10⁷ cfu/mL (cfu, colony-forming units). Bacteria-containing wells were then treated with the respective samples. Concentration was serially diluted over a range of 0.315–300 μg/mL. Bacteria incubated with PBS served as control. After incubation for 16h at 37 °C, inhibitory concentration (IC) IC90 values were determined by sigmoidal curve fitting of absorption values (600 nm) that were measured on a Tecan microplate reader. The IC90 values are defined as concentrations at which the growth of bacteria is inhibited by 90%. Three independent experiments were conducted in triplicate.