Biofilm imaging on confocal microscopy.

Bacteria were cultured on glass coverslips in 6-well polystyrene plates at 37°C under 5% CO₂ in RPMI 1640 medium. The coverslips were washed five times with phosphate-buffered saline (PBS) and stained with 5 μM Syto9 (Life Technologies, Carlsbad, CA) for 10 min. The coverslips were washed twice and fixed with 4% paraformaldehyde in PBS before mounting on glass slides with Fluoromount/Plus (Diagnostic BioSystems, Pleasanton, CA). Fluorescent images were acquired in the Alexa Fluor 488 laser unit on an Olympus FV1200 IX81 microscope using a 60× objective and captured with a charge-coupled-device (CCD) camera.