Crystal Violet Biofilm Quantification

A crystal violet staining assay was conducted using 96-well plates, as previously reported (Lee et al., 2021 (link)). S. aureus cells (~10⁷ CFU/mL) were inoculated into LB medium and retinoic acids were added at 0, 2, 5, 10, 20, 50, or 100 μg/mL to the wells of 96-well plates and cultivated for 24 h at 37°C without agitation. Biofilm formation was measured by discarding planktonic cells and washing the plates three times with distilled water. Biofilm cells were then stained with 0.1% crystal violet (300 μL) for 20 min and washed three times with water to remove crystal violet. Crystal violet stained cells were then extracted with 95% ethanol (300 μL) by shaking vigorously. Absorbances were measured at 570 nm (OD₅₇₀) using a Multiskan plate reader (Thermo Fisher Scientific, Waltham, MA, United States). Biofilm formation results are obtained from three independent cultures of six replicate wells.