A semi-static biofilm model was used to assess biofilm formation of S1 and S2, as described before[43 (link)]. Overnight bacterial cultures were diluted to an OD660 of 0.01 in 6 ml LB medium with 1%(wt/vol) glucose and added to a coverslip coated with poly-L-lysine (0.45 μm; diameter, 12 mm; Becton Dickinson) inside a well from a six-well polystyrene plate (Corning Inc.). Biofilms were grown at 30°C for 48 hat 120 rpm. After 48 h, the coverslips were washed with 0.85% (wt/vol) NaCl and the biofilms were chemically fixed with 8% (vol/vol) glutaraldehyde (Merck) for 20 min. Subsequently, the biofilms were stained with 15 μg/ml propidium iodide (PI) in 0.85% (wt/vol) NaCl that was removed after 15 min. The coverslips were transferred to glass microscope slides and analyzed by a confocal laser scanning microscope (CLSM) (Leica SP5), equipped with an oil plan-Neofluar ×63/1.4 objective. PI was excited at 488 nm. Z stacks were taken with an interval of 0.42 μm. Pictures were analyzed with LAS AF software (Leica), and biofilm thickness and biomass were quantified using Comstat[44 (link)]/Matlab R2013b software (the MathWorks). The average thickness and biomass of the biofilms were measured at ten randomly chosen positions and statistical significance determined by unpaired two-tailed Student's t-test. Experiments were performed twice in duplicate.
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