Bacterial Microcolony Formation and Dispersal

The induction assays have been described previously (8 (link)). Briefly, bacteria were resuspended in prewarmed DMEM containing 1% FBS, filtered through 5-μm filters, and diluted to 10⁷ CFU/ml. The bacterial solutions were transferred to 24-well glass-bottom plates (MatTek) and allowed to grow and form microcolonies for 3 h at 32°C or 37°C in a 5% CO₂ environment in a live-cell observer (Axiovert Z1; Zeiss). Prewarmed DMEM supplemented with sodium l-lactate (L7022; Sigma-Aldrich) was added to microcolonies at a 1:1 ratio at final concentrations of 10 mM. During treatment with spectinomycin, the antibiotic was added at a final concentration of 100 μg/ml 30 min before the induction. Three positions were chosen per well, and images were acquired every 5 to 10 min for 4 or 5 h using a 40× objective. The aggregation phase is described as the start of incubation until the dispersal of bacteria starts. The dispersal phase is described as the initiation of bacterial dispersal until no aggregates are observed. The planktonic phase is described as dispersed single bacteria.