Plan apochromat 63 1.40 oil m27 objective lens

Overnight cultures of S. aureus ATCC25923 and N315, S. epidermidis ATCC35984, and P. aeruginosa PAO1 were diluted 1: 100 in TSB or LB media in the presence of 10 mM NAC, or 20% serum, or a combination of NAC and serum. Bacteria were cultured in 15 mm glass-bottom cell culture dishes (polystyrene) on a rocker platform at 37 °C. After incubation for 24 h, the dishes were washed with water three times and fixed with 4% formaldehyde. Two ml of a 1: 1000 dilution of the nucleic acid stain SYTO 61 (Invitrogen) was added to the dishes, followed by incubation in the dark at room temperature for 30 min. The dishes were then washed and 2 ml of 50 µg/ml FITClabelled concanavalin A type IV (Sigma-Aldrich) was added. After incubation in the dark for 5 min at 37 °C, the dishes were rinsed with water three times and air-dried. Visualization of biofilms was performed using a laser scanning confocal microscope (LSM780, Carl Zeiss, Plan-Apochromat 63×/1.40 Oil M27 objective lens) with 561 nm excitation and 640 nm emission wavelengths for SYTO61, and 488 nm and 537 nm for FITC-ConA. Images were acquired from at least three distinct regions of each cell culture dish and processed using the ZEN image analysis package (Carl Zeiss). Biofilm thickness was measured by Z-stack images [15] . Three independent replicas were conducted for each experiment.