Biofilm imaging

Biofilms grown on glass slides for 48 h were imaged by confocal laser scanning microscopy (CLSM) (Olympus; FV3000). 20 μL of the overnight grown culture of PA14 was added into a fresh 20 mL LB broth, in a 50 mL falcon along with a microscope glass slide and grown for 48 h. The biofilm was then washed with PBS followed by staining with FilmTracer LIVE/DEAD Biofilm Viability kit (ThermoFisher). Next, biofilm was incubated with SYTO™ 9 and propidium iodide stains for 20 min in the dark at room temperature as per the manufacturer’s protocol. After that, the biofilm was washed with PBS and the slides were imaged using a OlympusFV3000 microscope with an objective lens of ×60 (oil). Each experiment included three independent biological replicates and three images were taken for each replicate. For each glass slide, five image stacks were taken with a z-step size of 0.7 μm. ImageJ software⁶¹ was used to calculate the number of live (SYTO9; green) and dead (propidium iodide; red) cells.