After washing, 20 ml 0·9% NaCl containing CaCl2 were added. To determine the number of bacteria in the alginate beads the beads were dissolved to release the bacteria using 0·1 M citric acid buffer pH 5. Serial dilutions were made and cultured on a modified Conradi-Drigalski medium (SSI), selective for Gram-negative rods. After overnight incubation at 37°C PS-341 datasheet the number of colony-forming units (CFU) was determined. The concentrations of P. aeruginosa in both the small beads (SB) and large beads (LB) varied from 0·2 to 0·7 CFU/ml; in no experiment did the concentration of bacteria in the beads differ more than 19%, and the bacterial concentration was lowest in the SB in all experiments.

In the present work we made beads in two different sizes. For the SB we used the 0·250 mm nozzle, an alginate flow rate 20 ml/h and the airflow 105 mBar. For the LB the 0·500 nozzle, alginate flow rate 60 ml/h and airflow 35 mBar were used. The diameter of the beads were measured using a light microscope (Olympus, Tokyo, Japan) and a picture-analysing program (Visiopharm Image Analysis and Stereology, Alleroed, Denmark). Two diameters at right

angles were determined for each bead and presented as the mean. Female 11-week-old BALB/c mice were purchased from Taconic Europe A/S (Lille Skensved, Denmark) and allowed to acclimatize for 1 week before use. A total of 207 mice were used in the experiments. Mice had free access to chow and water, and were under the observation of trained personnel. All experiments were authorized by the National Animal Ethics Committee, Denmark. Mice were anaesthetized subcutaneously PRKD3 Aurora Kinase inhibitor (s.c.) with a 1:1 mixture of etomidate (Janssen, Birkeroed, Denmark) and midazolam (Roche, Basel, Switzerland) (10 ml/kg body weight) and tracheotomized. SB or LB seaweed alginate beads embedded with PAO579 were installed into the left lung of BALB/c mice using a bead-tipped needle. All mice received the same amount of alginate and number of P. aeruginosa (0·66 × 109 CFU/ml for the SB group versus 0·71 × 109 CFU/ml for the LB group). An additional 32 mice were challenged with

beads prepared as described but without adding P. aeruginosa to the alginate. Mice were killed using an overdose of barbiturate at days 1, 2, 3, 5 or 6 after challenge. Peripheral blood was collected by cardiac puncture and serum isolated after centrifugation of coagulated blood. Serum was kept at −70°C until analysis. Half the number of lungs were collected aseptically and transferred to 5 ml of sterile phosphate-buffered saline (PBS) and kept on ice until further analysis. The left lungs from the remaining number of mice were fixed in a 4% w/v formaldehyde solution (VWR, Copenhagen, Denmark). Evaluation of pulmonary histopathology was performed as described previously [8]. The fixed lungs were embedded in paraffin wax and cut into 5-µm-thick sections, followed by haematoxylin and eosin or Alcian blue staining.