Then, 50 μl of a bacterial suspension (1.5 × 108 CFU/ml) was inoculated into the well and incubated aerobically at 37°C for 6 days. The biofilm deposited on a silicone sheet was gently rinsed with sterile saline to remove bacterial cells, except for those included in the biofilm. After rinsing, the biofilm was soaked in 1 of the following treatment solutions for 24 h at 37°C: NAC (0,

0.5 mg/ml, 1 mg/ml, 2.5 mg/ml) and ciprofloxacin (0, 1/2MIC, 1MIC, 2MIC, 4MIC, 8MIC) combinations according to a checkerboard design. Then, the sheets were rinsed 3 times with PBS to remove planktonic bacteria, individually sonicated for 10 min and vortexed for 3 min in 1 ml of MHB. The number of viable cells was counted by the method described above. The lg (CFU) per cubic Cilengitide centimeter values of sheets for different groups were calculated. Measurement of extracellular polysaccharides (EPS) The amount of carbohydrates produced by each bacterial strain was determined using a modified version of the acid hydrolysis method of Dall and Herndon [27].

Briefly, polysaccharides were precipitated with ethanol and then dehydrated with concentrated acid to a furfural. When tryptophan reacted with furfural, a condensation product was formed, which developed a brownish violet Selleckchem KPT-8602 color. The amounts of EPS were determined spectrophotometrically by measuring the INK1197 purchase absorbance at 490 nm. Six-day old bacterial biofilms on silicone sheets, grown as described above, were gently rinsed with PBS. After rinsing, the biofilm was placed in alginate producing (AP) medium described by Terry et al. [28] for 48 h, then soaked in AP medium containing Tryptophan synthase NAC (0, 0.5 mg/ml, 1 mg/ml) for an additional 24 h, gently rinsed with PBS to remove bacterial cells, except for those included in the biofilm,

individually sonicated for 10 min and vortexed for 3 min in 1 ml of PBS. The suspension was adjusted to 90% light transmittance at 400 nm (about 2.1 × 107 CFU/ml), then centrifuged at 950 × g for 10 min, and the supernatant was filtered through a sterile 0.22-μm membrane filter. Then, 0.5 ml of the supernatant fluid, which contained the polysaccharide, was precipitated by adding it in drops to 4 ml of cold absolute ethanol. Polysaccharides were pelleted by centrifugation (2,400 × g, 15 min) and resuspended in 200 μl of distilled water, after which they were digested with 700 μl of sulfuric acid (77%) to form monosaccharides. Samples were cooled for 10 min in an ice bath, then 0.1 ml of cold tryptophan (1%, wt/wt) was added to each tube and mixed. After heating in a boiling bath for 20 min, the tubes were cooled on ice, and the absorbance at 490 nm was read with a spectrophotometer (Pulang New Technology Corporation, China) using a PBS blank subjected to the same procedure. The amount of EPS was expressed in μg/μl. The assay was calibrated using a dextran standard (Dextran T500, Pharmacia) subjected to the same procedure.