Media was pumped AZD2014 mw into the chambers at a flow rate of 60 ml h-1, dripping onto the stainless steel slides (8.5 cm × 1.3 cm) placed in the chambers. The reactors were placed on a stand inclined at 10° from horizontal and PBM would flow the length of the coupon and drain from the reactor. The reactors were inoculated by adding 1 ml of an overnight culture to 15 ml of fresh PBM used to cover the slides (inoculum OD600 ≈ 0.3) in PBM (1 g l-1 glucose). The reactor was sealed by clamping the effluent tubes and the inoculum was allowed to

sit in the reactor for 18-24 h on a level surface. After the inoculation period, the reactor was inclined and flow was initiated. The entire drip-flow reactor was kept in a 37°C incubator. Medium flowing from outside the incubator was warmed by passing the silicone tubing through a grooved aluminum block kept in the incubator. MX69 purchase The biofilms were grown in the drip flow reactors for 72 hours after the static inoculation phase. Biofilm protein synthetic activity patterns P. aeruginosa PAO1 (pAB1) biofilms were grown

for 72 hours in drip flow reactors. The medium was then supplemented with 1 mM IPTG and flow continued for 4 h. After this ARS-1620 datasheet induction period, biofilm-covered slides were removed from the reactor and cryo-embedded in Tissue-Tek O.C.T. (VWR Scientific). Cryo-embedded biofilms were cryo-sectioned, and examined by confocal laser scanning microscopy with a Leica TCS NT with excitation at 488 nm and emission filter of 500 – 530 nm. Dimensions of the biofilm and the GFP-expressing zone were determined by image analysis using Scion Image software (Scion). Some specimens were counterstained with rhodamine B following IPTG induction of the GFP. In these cases, rhodamine B was introduced into the medium at a concentration of 5 μg ml-1

for 30 min. The biofilms were others then rinsed with fresh medium for 30 min before cryo-embedding. Oxygen concentrations in biofilms Oxygen concentration profiles in biofilms were measured with microelectrode technology described in detail elsewhere [90, 91]. The microelectrode manipulator was placed inside the incubator so that the measurements could be made at 37°C. Antibiotic susceptibility of biofilms After 72 hours of growth in the absence of antibiotic, the desired antibiotic was added to the growth medium, and the flow continued for an additional 12 hours. Tobramycin was applied at 10 μg ml-1 and ciprofloxacin at 1.0 μg ml-1. After treatment the stainless steel coupons were removed from the reactor and the number of viable cells was determined by scraping the biofilms into 9 ml of phosphate buffer (pH 7.2, 1.4 mM) and homogenizing for 1 min. The resulting cell suspensions were serially diluted and plated on TSA. Killing was reported as a log reduction. The log reduction was calculated relative to the cell count at time zero.