Crystal Violet Staining Assay

A crystal violet staining assay (Li et al., 2014) was used to quantify the biomass of the biofilms with or without NaF treatment. The pegs with biofilms were first inserted into a 0.01% crystal violet solution (200 μL/well) for 5 min, washed twice with distilled water, and then inserted into 2% sodium deoxycholate to destain for 5 min. The absorbance of the used destaining solution was measured at 608 nm using a spectrophotometer (Spectramax Plus, Molecular Device, Sunnyvale, CA, United States). The change in biomass during the 16-h overnight incubation was calculated as: the OD₆₀₈ of 48-h biofilms minus the OD₆₀₈ of 32-h biofilms. The data are presented as the percentage of biofilm formation in a NaF group relative to that in the corresponding non-NaF treated control group.