Biofilm formation inhibition assay and the determination of inhibitory concentration (IC50) value

The biofilm formation inhibition capabilities were evaluated according to the previously described protocol [27, 38, 39] with slight modifications. Escherichia coli was cultured on TSB and incubated at 37 °C till the culture turbidity matched (0.5 McFarland), and diluted on fresh and sterile TSB as 1:100 dilutions. Then, 100 μL of bacterial culture was added to each well of a 96-well microtiter plate and incubated for 4 h at 37 °C to allow cell attachment. Following incubation, 100 μL of each plant extracts at a final concentration of 500 μg/mL was added to each well. An equal volume of ciprofloxacin with a final concentration of 1.25 μg/mL was added as a positive control and MHB as negative control instead of plant extracts. In blank wells, 200 μL of MHB was used without a bacteria culture to ensure the sterility of the experiment. The plates were covered with a lid and incubated at 37 °C for 48 h. The concentration of plant extracts and ciprofloxacin was maintained below their MIC value.

After incubation, the cultures were decanted on a paper towel, rinsed two times with 200 μL sterile phosphate buffer saline (PBS) of pH 7.2. Then, the plates were heat-fixed by incubating at 60 °C for 1 h. Then, the plates were stained with 0.1% crystal violet (CV) solution for 20 min at room temperature. After CV staining, plates were washed three times with PBS of pH 7.2 to free the stain from the microtiter plates. Then, the plates were air-dried and de-stained with 200 μL of 95% ethanol (v/v) for about 30 min. Finally, the absorbance was taken at 590 nm.

The percentage of inhibition of biofilm formation was calculated by using the following formula.

Finally, IC₅₀ was calculated based on percentage inhibition with the different concentrations of plant extract (500–100 μg/mL) [20, 40, 41]. Each inhibition assay was performed in triplicate and done twice to check the reproducibility of the result.