To determine the optimal temperature for biofilm formation, the S. aureus attached to polypropylene was studied at different temperatures. We observed that this process was more efficient at 37 °C than at 30 or 25 °C (data not shown). Adhesion was also studied at different pH ranges (5.6–8.0). At a slightly acidic pH, the biofilm formation was 3.5-fold higher than at the basic

pH. However, at the physiological pH range, the biofilm formation was more stable (Fig. 2a). When different pH values were assayed, the extracellular metabolites (eROS and NO) increased significantly with a rise in pH. However, the increase in iROS was not as important at basic pH (Fig. 2b–d). The level of biofilm formation was inversely related to the extracellular metabolites acquired, and the increase of extracellular reactive species was also more significant PF-562271 chemical structure than iROS. We compared S. GSK2126458 aureus biofilm formation under aerobic and microaerobic growth

conditions in TSB and in thioglycolate medium, respectively. When assays were performed with thioglycolate medium in aerobiosis, an increase in biofilm formation was seen with respect to TSB (Fig. 3a). For this condition, the thioglycolate medium produced better biofilm formation, with lower ROS and ON occurring (Fig. 3b–d). The total production of biofilm with TSB medium was found to be approximately the same for both aerobic and microaerobic conditions at 37 °C. However, incubation under the microaerobic condition in thioglycolate medium resulted in significantly less biofilm formation for all the strains, compared with aerobic incubation (Fig. 4a). In contrast to the aerobic condition, for microaerobiosis, the biofilm formation in thioglicolate medium did not strongly stimulate biofilm formation, but produced eROS and NO (Fig. 4c

and d) (P vs. TSB <0.005). CSLM staining of the bacterial DNA and the glycopolysaccharide of the matrix was used to quantify structural biofilm changes with respect to differences in the culture conditions. Images were obtained using a CSLM microscope Racecadotril and two fluorescence stainings were used (propidium iodide and FITC–Con A). The panel in Fig. 4 shows laser scanning fluorescence images for XY (top) and XZ (bottom), of the glycocalyx matrix (green) and dead cells (red) of S. aureus ATCC 29213. Similar images were obtained with clinical strains (data not shown). Biofilm formation in the thioglycolate medium in aerobiosis was greater than in TSB (5.96 vs. 5.02 μm). In microaerophilia, in thioglycolate medium less biofilm was formed than for aerobiosis (5.96 vs. 5. 25 μm). The presence of microcolonies were observed with more dead cells (40%). The strains producing biofilm display greater adhesive abilities in comparison to nonproducing ones (Svensäter et al., 2001; Rollet et al., 2009).