Figure9a-d_i.nd2

Figure 9. S. aureus MRSA biofilms grown without P. aeruginosa cell-free conditioned media (CFCM) (A – D) or with CFCM (E –H) for 24 hours. Wheat Germ Agglutinin Alexa Fluor 647 was used to identify S. aureus cells (A and E), while SYTO 84 was used to label both intracellular and extracellular DNA (B and F) including eDNA in the matrix of the biofilm. Depolarized membranes were stained using DiBAC4 voltage sensitive dye (C and G). All channels were overlayed, showing DiBAC4 staining of S. aureus cells (D and H) while volume rendering show all layers of S. aureus biofilms are depolarized upon treatment of CFCM (I – J). Membrane depolarization was quantified using corrected total cell fluorescence (K). Statistical analysis was performed by one-way ANOVA with Dunnett’s multiple comparison test with 95% (p<0.05) of significance. ****: p < 0.0001.