Biofilm Inhibition Assay

The selected formula and MOX solution were tested at concentrations below the MIC (MIC/2, MIC/4, MIC/8, MIC/16, MIC/32, MIC/64, MIC/128, and MIC/256). The biofilm inhibition assessment was done as mentioned before.²⁵ Precisely, the MRSA USA300 suspension (10⁸ CFU/mL) in tryptic soy broth (TSB) was added in a flat-bottom 96-well ELISA plate (200 μL/well). Different concentrations of the selected formula/solution were added to the loaded wells (20 μL/well). Nothing was added to the control wells (untreated, 100% reference measurements). The plates were then incubated at 37°C for 24 h at static aerobic conditions. After incubation, the grown cultures’ optical density (OD600) was assessed by a spectrophotometric plate reader (Biotek, Synergy 2, USA). The formed biofilms were quantified as follows; wells were washed with saline then dried. The dried biofilm was then stained with crystal violet (0.1% w/v, 200 μL/well) for 30 minutes at room temperature. The wells were then washed three times with distilled water and dried. The crystal violet in the stained biofilm was liquified by adding absolute ethanol (200 μL/well) and incubating for 20 minutes at 4°C (to minimize evaporation). The OD₅₅₀ of the crystal violet solutions was assessed by spectrophotometric plate reader and divided by OD₆₀₀ of the grown cultures for normalization. The biofilm inhibition % was calculated by the following equation:²⁵