After incubation, the supernatant was separated for extracellular oxidative and nitrosative stress assay and the plate was rinsed with phosphate-buffered solution (PBS, pH 7.2). After drying, staining for adherent biofilms was performed using CV (1%). Then, the CV was removed and cells were rinsed three times with 300 μL PBS (pH 7.2) before drying for 24 h at room temperature. A quantitative assessment of the biofilm formation was obtained

by extracting the CV with 200 μL per well of the bleaching solution: ethanol/glacial acetone (70 : 30). The intensity of the coloration was determined at 595 nm using a microplate reader (Model 680 BioRad, Hercules, CA). All strains were tested in three independent experiments on different days. The average OD595 nm value was determined by three replicates and was interpreted by the following scale: positive (>0.24), weak (>0.12 and <0.24) or negative (<0.12) (Deighton et NSC 683864 manufacturer al., 2001). The biofilm biomass unit (BBU) was arbitrarily defined with 0.1 OD595 nm=1 BBU. Biofilm formation BVD-523 datasheet was investigated at 12, 24 and 48 h, and the effect of temperature was evaluated at 25, 30 or 37 °C. Static conditions were used at 37 °C for 24 h at different pH values (5–8). The influence of the reduction conditions was assayed in thioglycolate broth and microaerobic conditions with TSB or thioglycolate broth were

also studied. Three wells with 200 μL TSB or thioglycolate were added to serve as negative controls and to obtain a background value, which was then subtracted from the values obtained from the cells in the wells. The intra- (iROS) and extracellular (eROS) production of ROS was detected by the reduction of nitro-blue

tetrazolium (NBT, Sigma) to nitroblue diformazan. The below supernatant was separated by measuring the eROS. Then the biofilms in individual wells of sterile 96-well polystyrene microtiter plates were treated with 0.05 mL dimethyl sulfoxide (Merck) to extract the reduced NBT using 0.1 mL NBT (1 mg mL−1) and 0.1 mL TSB (for the final volume) at 37 °C for 30 min, followed by the addition of 0.02 mL hydrochloric acid (0.1 M) to stop the reaction and measure iROS. The reaction is detectable by the byproducts of the assay, which are proportional to the ROS generated in biofilm and were measured by OD at 540 nm (Paraje et al., 2009; Aiassa et al., 2010; Páez et al., 2010). The supernatant under different conditions was separated for extracellular nitrosative stress assay and incubated for NO measurement. The NO was evaluated as nitrite by a microplate assay method using Griess reagent (Paraje et al., 2009). One hundred microliter aliquots of supernatant were mixed with 200 μL Griess reagent [sulfanilamide 1.5% in 1 N HCL and N-1-naphthyl ethylenediamide dihidrochloride 0.13% in sterile distilled water (dH2O)].