TRITC (tetramethyl rhodamine isothiocyanate)-labeled wheat germ agglutinin (Molecular Probes, Eugene, OR) was used at a concentration of 0.1 mg/mL to stain the PIA in biofilms [17]. Hemoglobin was purchased from Sigma and used as indicated concentrations. The Ethics Committee of the Zhongshan Hospital of Fudan MCC950 purchase University and the East Hospital of Tongji University both exempted this study from review because the current study only focused on bacteria. Cultivation of bacterial biofilms Biofilm cultivation in polystyrene microtitre plates was carried out as described previously [11]. Briefly, overnight cultures of Se strains grown in TSB (0.25% glucose) medium were diluted 1:200.
The diluted cultures were transferred
to wells of polystyrene microtitre plates (200 μL per well) and incubated at 37 °C for 24 h. After washing, the wells find more were stained with 2% crystal violet for 5 min. Then, the plate was rinsed, air-dried, redissolved in ethanol and the absorbance was determined at 590 nm. For cultivation of Se biofilms in the flow-chamber system, the flow-chamber system was first assembled and C188-9 order prepared as described previously [18]. Briefly, the flow chambers were inoculated by injecting 350 μL overnight culture diluted to OD600 = 0.001 into each flow channel with a small syringe. After inoculation, flow channels were left without flow for 1 h, after which medium flow (0.2 mm/s) was started using a Watson-Marlow 205 S peristaltic pump. Microscopy All microscopic observations and image acquisition were performed 3-oxoacyl-(acyl-carrier-protein) reductase using a Zeiss LSM 510 confocal laser scanning microscope (Carl Zeiss, Jena) equipped with detectors and filter sets for monitoring SYTO 9, PI, DDAO and TRITC fluorescence. Images were obtained using an x63/1.4i objective or an x40/1.3i objective. Simulated 3D images and sections were generated using the IMARIS software
package (Bitplane). Bacterial attachment assays Initial cell attachment was tested as described previously [11]. Briefly, cell suspensions from the mid-exponential phase of bacterial growth were diluted to OD600 = 0.1 in PBS, and then incubated in wells (1 mL per well) of cover-glass cell culture chambers (Nunc) for 30 min at 37°C, after which attached cells were calculated by microscopy. Quantification of extracellular DNA Extracellular DNA was quantified as described previously [11]. Overnight cultures were diluted to OD600 = 0.001 in AB medium supplemented with 0.5% glucose, 0.05 mM PI and 10% TSB. The diluted cultures were transferred to wells of polystyrene microtitre plates (150 μL per well) and incubated for 24 h at 37°C, upon which PI absorbance was measured at 480 nm and cell density was measured by OD600 using a Wallac microtitre plate reader. Relative amounts of extracellular DNA per OD600 unit were calculated.