Biofilm Quantification and Visualization

A crystal violet assay was performed to evaluate the biomass of biofilms, including codY^–ASyycF, codY^–, codY⁺ASyycF, codY⁺, and ATCC29213 (as a control). After 24 h of culture in TSB medium, the biofilm samples were stained with 0.1% (w/v) crystal violet for 15 min. The dye bound to the biofilms was transferred into a new plate, and the absorbance was measured with a microplate reader (ELX800, Gene) at 595 nm (Wu et al., 2021b (link)). In addition, the biofilms were labeled with SYTO9 and PI for epifluorescence observation. Live strains were stained green, while dead strains appeared red. Three random fields in each specimen were visualized using epifluorescence microscopy (Nikon Eclipse TE-2000S, Melville, NY).