5 ml of molten 0.6% LB agar. The surface of the plates were overlaid with the soft agar and allowed to set. Luria Bertani (LB) broth (1.0 ml) containing 0.5% NaCl was added to the vial containing freeze-dried phage and 0.1 ml of the rehydrated phage was learn more spotted onto the overlay. The plate was tilted to spread the rehydrated phage over as much of the surface as possible. This was allowed

to dry and incubated at 37 °C overnight. After 24 h incubation, the soft agar was scraped from the surface of the agar plates using a sterile cell scraper. The soft agar was centrifuged at 1000 rpm for 25 min to sediment the cellular debris and agar. The supernatant was passed through a 0.22 μm Millipore filter and the filtrate was stored at 4–8 °C. The double layer plaque assay method was adapted from a method devised by Adams (1959). An actively growing broth culture of E. coli 11303 was prepared 18–24 h before performing the plaque assay. Plates of 1.2% LB agar were pre-warmed Pomalidomide purchase in an incubator at 37 °C. Plates were prepared as previously described. Serial dilutions of the samples obtained from the in vitro release study were prepared from 10−1 to 10−8. The agar overlay was prepared by adding 60 μl of the E. coli innoculum into 3 ml of 0.6% top agar and poured immediately onto the 1.2% agar plates and agitated to ensure even distribution. Samples of each serial dilution (20 μl) were spotted onto the overlay, with 4–5 dilutions per plate.

Each sample was spotted in triplicate

to ensure reproducibility. The plates were incubated at 37 °C overnight. Plaques were subsequently enumerated on plates at each dilution. Plaques appear as defined, circular zones of clearance within the bacterial these lawn, due to bacteriophage-mediated bacterial cell lysis. The concentration of bacteriophage present in each sample was calculated from the dilution in which plaques were most countable, and using the following equation: equation(1) Number of plaques×dilution factor×50=Concentration in PFU/mlNumber of plaques×dilution factor×50=Concentration in PFU/mlwhere 20 μl is plated, ×50 to calculate PFU/ml. An average of the three results was taken as the phage concentration. The delivery of a stock solution (5 × 108 PFU/ml) of T4 bacteriophage across neonatal porcine skin, using the hollow MN system was carried out using Franz diffusion cells (FDC-400 flat flange, 15 mm orifice diameter, mounted on an FDCD diffusion drive console providing synchronous stirring at 600 rpm and thermostated at 37 ± 1 °C, Crown Glass Co. Inc., Sommerville, NJ, USA). The orifice diameter in the receptor compartment was 15 mm. No donor compartment was used, to allow ease of use of the hollow MN device. The receptor compartment volume was calculated to be 12 ml. PBS pH 7.4 (11 ml) was accurately dispensed into the receptor compartment using a 5 ml Pipetteman®, assuming that the full 1000 μl would be delivered via the hollow MN device. The PBS was degassed prior to use by sonication.