Fig. 1

Bacillus subtilis cell-free supernatant (B. subtilis CFS) inhibits the growth of planktonic and biofilm Staphylococcus aureus (S. aureus). a The growth curve of planktonic S. aureus. S. aureus (5 × 10⁸ CFU/mL, 100 µL) was grown in TSB/PBS (control), TSB/B. subtilis CFS, TSB/PBS with gentamicin (0.75 µg/mL), or TSB/PBS with penicillin (32 µg/mL). Samples were taken out for OD₆₀₀ evaluation at indicated time points. Data are shown as mean ± SE (n = 4 biologically independent samples per time points). b Representative images of crystal violet staining for S. aureus biofilm. Experiments were repeated independently from 4 different colonies of S. aureus. c Quantitative analysis of biofilm formation. Crystal violet-staining was dissolved and measured at 600 nm in a microplate reader. N = 4/group, **P < 0.01, Mann-Whitney U test. d Representative images of SYTO9-PI staining for biofilm S. aureus. S. aureus (5 × 10⁷ CFU/mL) was grown at 37℃ for 24 h, and then treated with TSB/PBS (control), TSB/B. subtilis CFS, TSB/PBS with gentamicin (0.75 µg/mL), or TSB/PBS with penicillin (32 µg/mL) for 8 h. After being washing with PBS, biofilm S. aureus was examined with SYTO9-PI, followed by analysis using a fluorescence microscope. Both live and dead bacteria were stained green from SYTO9, and dead ones red from PI. Scale bar 100 μm