After sterilization by autoclaving the entire setup was placed in an incubator at 37°C. The inoculum was prepared as YH25448 chemical structure follows. 10 ml of broth was inoculated with a single colony from a YPD agar plate. Cultures were incubated on a shaker at 280 rpm and 30°C to an OD600 of 0.4–0.5. This was used to inoculate a fresh 10 ml broth culture at 0.05 OD600 which was grown overnight under
the same conditions. From this culture 20 ml of cells at 108 cells/ml in phosphate selleck compound buffered saline (PBS) (0.1 M, pH 7.0) was prepared. The tubular reactor was clamped downstream of the air trap and, using a 20 ml syringe, the reactor was filled by drawing the cell suspension into the tubing from the effluent end. The inoculated reactor was incubated for 1 h at 37°C before starting the
medium flow at 1 ml/min. Planktonic cultures Batch cultures were grown at 37°C on a shaker at 280 rpm. Preparation of the inoculum for planktonic cultures was the same as for biofilm cultures. The medium volume of batch cultures grown for different periods of time was adjusted so that the cells would be exposed to the same volume of medium as the biofilm for each time point. Accordingly, batch cultures were all inoculated with 1 × 108 cells and cultured in final volumes of 30, 60, 90, 120 and 180 ml for the 30, 60, 90, 120 and 180 min time points, respectively. selleck chemicals For 90, 120 and 180 min time
points the initial medium volume was 60 ml, and 30 ml aliquots of medium were added at appropriate times. Biofilm sectioning Biofilms were sectioned using two methods. For embedding in Spurr’s resin  biofilm samples were fixed in situ at 4°C in 3% gluteraldehyde in PBS. The fixed samples were washed at room temperature for 10 min in 20, 50 and 100% ethanol solutions successively. Samples were incubated in a series of Spurr’s: 1:2 Spurr’s: propylene oxide (overnight at 4°C); 1:1 Spurr’s: propylene oxide (8–10 h at room temperature), 2:1 Spurr’s: propylene oxide (overnight at 4°C) and full strength Spurr’s (6–8 h these at room temperature). The Spurr’s solution of the last incubation was replaced by a fresh one and samples were baked for 10–12 h in an oven at 70°C. After cooling to room temperature, the silicone tube was removed from each sample and the hardened Spurr’s column containing the biofilm was sectioned using a Reichert OM-U2 ultramicrotome. Sections were mounted on slides and imaged using a Nikon Eclipse E600 in epi-fluorescence mode. Samples for cryosectioning were prepared by excising a section of the silicone elastomer tube used to grow the biofilm with a fresh razor blade without disturbing the biofilm. Excess medium in the tube was carefully removed using a 10 ml syringe and needle. The tubing was cut lengthwise and the upper half was removed.