Confocal laser scanning microscopy observation of biofilms.

To assess the air-liquid interface biofilms, pellicles were grown in glass chambers with individual chamber dimensions of 1 × 1 × 4 cm (chambered no. 1.5 German cover glass system; Nunc, Inc.). To observe the structures of the pellicles, SYTO9 (Molecular Probes, Invitrogen) was used to stain the pellicles, and then buffers were removed from glass chambers to allow the pellicles to drop onto the coverslips. Fluorescent and differential interference contrast images were acquired using a FV1000 confocal laser scanning microscope (Olympus, Japan). The biofilm biomass and thickness were quantified by using COMSTAT software (45).