Fig. 6.

Microscopic analyses of the impact of cationic proteins and eDNA on S. aureus cell aggregation and biofilm stability. A, Freshly cultivated biofilm cells were harvested, washed, pelleted, and resuspended in the following solutions, which were adjusted to either pH 5.5 or pH 8, and investigated by fluorescence microscopy after staining with the eDNA stain Toto-1 and Syto62 as a counterstain: planktonic or biofilm ECM supernatant, fresh growth medium supplemented with BSA or CytC as control proteins, planktonic or biofilm ECM supernatant supplemented with cDNA isolated from S. aureus, medium control, medium control plus cDNA. Representative images of 15 randomly selected areas of duplicate experiments are shown. The scale bar indicates 10 μm. B, Biofilms were cultivated in flow cells for 12 h followed by a treatment with a growth medium control, Proteinase K, and alkalized growth medium of pH 12, respectively. Biofilms were subsequently challenged by elevated shear forces to test biofilm stability and analyzed by confocal laser scanning microscopy (CLSM). For each biofilm, 3 CLSM images of randomly selected areas (spanning 100 μm × 100 μm) were acquired after 0, 2, and 5 min of elevated shear stress in duplicate experiments. A representative image of each treatment and time point is shown. C, Biofilms were cultivated in flow cells for 12 h, stained with the eDNA stain Toto-1 (green) and Syto62 (red) as a counterstain, washed and analyzed by CLSM. A representative image of 5 randomly selected areas of duplicate experiments is shown. The scale bar indicates 10 μm.