Crystal Violet Biofilm Assay

The biofilm formation ability assay was performed according to the methods by O’Toole with some minor modifications (Niemirowicz et al., 2016). An overnight culture of each isolate was incubated at 37°C/180 rpm up to the logarithmic phase with an OD₆₀₀ value of 0.6, and the turbidity was adjusted to 0.5 McFarland standard, further diluted to 1:100, and CDB at 0, 32, 64, 128, 256, and 512 μg/mL concentrations were added, respectively. Then, 100 μL of the dilution was added to the 96-well polystyrene micro-test plate (Flat bottom with lid, Sterile; Corning, United States), and 3 replicate wells were set up. Following 24-h incubation at 37°C with shaking at 75 rpm, the upper planktonic bacteria was decanted, and biofilms attached to the well surfaces were stained with 100 μL of 1% (w/v) crystal violet solution (lot number: NO.20190324, Beijing Solarbio Biotechnology Co., Ltd., China) for 15 min. The bound dye was solubilized for 30 min with 100 μL of the eluent (95% absolute ethanol and 5% glacial acetic acid) and subsequently quantified by measuring the OD₅₉₅ value by the Multiskan FC Microplate Reader.