2.2.3. Minimal Inhibitory Concentration (MIC)

The resazurin microtiter-plate assay [19] was used to evaluate the MIC. Samples were dissolved in sterile DMSO; then, they were distributed in a multiwell plate with different volumes of sterile Muller-Hinton broth (Sigma Aldrich Italy) previously prepared. Two-fold serial dilutions were performed to have 50 μL of the test material in serially descending concentrations in each well. A 35 μL amount of 3.3 × strength iso-sensitized broth and 5 μL of resazurin, used as indicator solution, were added to achieve a final volume/well of 240 μL. Finally, 10 μL of bacterial suspension was added to each well to reach a concentration of about 5 <sup>×</sup> 105 cfu/mL. Sterile DMSO and ciprofloxacin (Sigma Aldrich Italy, prepared dissolving 1 mg/mL in DMSO) were used as negative and positive control, respectively. Multiwell plates were prepared in triplicate and incubated at 37 ◦C for 24 h. The lowest concentration at which a color change occurred (from dark purple to colorless) revealed the MIC value.
