Quantification of Bacterial Biofilms

A crystal violet assay was utilized to evaluate bacterial biofilms as previously described (Gaddy et al., 2009; Lu et al., 2021). Briefly, overnight GBS cultures were diluted 1:10 in THB-CP medium in 96-well plates. To analyze the effect of lactoferrin on biofilm inhibition, lactoferrin was applied in increasing concentrations (0, 250, 500, 750, or 1000 µg/mL) at the time of inoculation. Biofilms were allowed to form at 37°C in ambient air overnight. OD₆₀₀ was determined using a spectrophotometer and supernatant was removed and replaced with 0.1% crystal violet stain for thirty minutes. Wells were washed with deionized water three times and dried. The retained crystal violet was resolubilized with a solution of 80% ethanol and 20% acetone. Plates were incubated for at least 30 minutes and optical density was determined at 560 nm (OD₅₆₀). Quantification was determined by using a ratio of OD₅₆₀/OD₆₀₀.