Fig. 5

Biofilm biomass estimation using CV staining and fluorescent imaging. Staphylococcal strains were allowed to form biofilm in 96-well plates for 24 h in 200 µl of LB medium, which was supplemented with 0.25×, 0.5×, and 1× MIC of APLss or 1× MIC of Van. Percentage inhibition of biofilm grown in the presence of APLss or Van in comparison to the untreated control using CV staining is shown for strains including (A) MSSA 25923 and (B) MRSA 33591. Subsequently, fluorescent images were captured after staining with FITC and PI to visually assess the extent to which biofilm biomass was inhibited for included strains. FITC staining revealed green fluorescence from biofilm extracellular polysaccharides, and PI staining revealed red fluorescence from bacterial DNA. The data are presented as mean ± SEM. Differences between groups were evaluated using one-way ANOVA followed by Tukey’s multiple comparison test, and bars labeled with a (*) or (****) indicate statistical significance at the p < 0.05 and p < 0.0001 levels, respectively. Abbreviations: APLss, antimicrobial peptidase lysostaphin; FITC, fluorescein isothiocyanate; MIC, minimum inhibitory concentration; ns, not significant; PI, propidium iodide; Van, vancomycin