2.6.2. Minimum Inhibitory Concentration (MIC) Assay

MIC was conducted to inspect the influence of different concentrations of the developed AMCS and its quaternized form on the growth of the studied bacteria. MIC experiment was performed according to the reported studies using the microtiter plate method [44]. In brief, bacterial strains (E. coli, P. aeruginosa, S. aureus and B. cereus) were overnight cultured by incubating them in LB broth under a shaking rate of 150 rpm at 37 °C. Next, bacterial cultures were diluted 100 times using the same LB medium to gain optical densities of 0.9 via measuring the bacterial turbidity at 600 nm. Various concentrations of tested samples (25, 50, 100, 200 and 250 µg/mL) were added into sterile 96-well microplates containing 20 µL of the bacterial culture suspensions. The wells were completed to 200 µL with LB broth free medium and followed by mixing for 2 min at 100 rpm using a bench-shaker. Finally, the wells were left overnight for aerobic incubation at 37 °C. Additionally, the negative and positive controls were prepared separately by mixing the diluted bacterial cultures and the examined samples with the free LB medium, respectively. The microtiter plates were shaken for 30 s using a microplate reader, and the turbidity of bacterial cultures was assayed at 600 nm. The test was conducted in triplicate and the inhibition (%) of microbial growth was calculated according to the following Equation (3):

where OD_a and OD_b represent the optical density of normal and inhibited microbial growth, respectively.