Figure 8. Sensitivity of V. anguillarum strains to antimicrobial products. Cultures of V. anguillarum grown to an OD600 of 0.2 (108 cells ml21) were diluted 10-fold in PBS containing either (A) 10 mg ml21 of lysozyme or (B) 10 ng ml21 of polymyxin B sulfate. Bacterial survival was measured at room temperature by determining the cfu in each sample at 0 min and 20 min for lysozyme and 0 min and 1 h for polymyxin B sulfate. Percent survival is the average of three experiments. Asterisks indicate a p-value of ,0.05 as determined by the Student’s t-test. Strains are designated by their mutation. Mutation designations followed by a C are complemented. doi:10.1371/journal.pone.0037678.g008 sandwiching them between the two coverslips. Additional drops of HBSS with PEST were added to the edges of the coverslips to prevent drying out. The fish scales were then incubated overnight at 12uC to allow the epithelial cells to migrate from the fish scale surface. For live-cell microscopy, glass bottom microwell dishes (MatTek) were pretreated with poly-L-lysine, which provided a sticky surface for highly motile V. anguillarum and enhanced O-Antigen Prevents Phagocytosis by Fish Skin Cells visualization of bacterial-host interactions using the live-cell microscope. For poly-L-lysine pretreatment, 300 ml of a 0.01% poly-L-lysine solution (70,000–150,000 mol. wt.) was placed on the glass bottom of a microwell dish. The microwell dish was incubated overnight at room temperature followed by 3 washings with phosphate buffered saline (PBS). Following the PBS washings, 100 ml HBSS with PEST were spotted into the poly-L-lysine treated glass bottom and 5 scales were equally distributed on the glass surface. A coverslip larger than the microwell was placed on top of the scales to apply pressure that forces the scales towards the glass bottom. An additional 1.4 ml of HBSS and PEST was added to the microwell dish, which was then incubated overnight at 12uC to allow the epithelial cells to migrate from the fish scale surface. These epithelial cell preparations were then used in live-cell microscopy or confocal 3D-imaging microscopy. Live-cell microscopy Live cell microscopy was used to visualize motility and phagocytic activity of the rainbow trout skin epithelial cells. Cells isolated in the glass-bottom microwell dishes were used. The top coverslip was removed and the epithelial cells were gently washed 3 times in HBSS to remove PEST prior to infection with bacteria. After washing, the epithelial cells were overlaid with 1 ml of HBSS and infected with bacteria. Prior to infection, bacterial strains were grown overnight at 24uC in TSB-1% with aeration, sub-inoculated as a 1/10 dilution in TSB-1%, and incubated for an additional 3 hours in TSB-1%. From each bacterial culture, an equivalent of 108 cells were removed (OD600 of 0.2 equals 16108 cells ml21), pelleted in a microfuge, washed, and resuspended in 1 ml of HBSS. To infect the epithelial cell culture, 1 ml of each bacterial sample in HSBB was added to a microwell containing epithelial cells giving a final concentration of 16105 bacteria ml21. Following the addition of bacteria, single, motile epithelial cells were visualized using an inverted Nikon Eclipse Ti live-cell microscope equipped with a Plan Apo VC 606/1.40 oil objective. Motility and phagocytic activity of the epithelial cells were recorded for 20 min taking 30 frames per second. The bacteria- host cell association was then analyzed frame by frame of a recorded movie. All images were processed with real-time deconvolution using the NIS-Elements AR 3.2 software. For the initial experiment to demonstrate phagocytic activity of the rainbow trout epithelial cells, 2 mm-latex beads (,104 beads ml21, Chemika) were used in the place of bacteria. For mannose inhibition of phagocytosis, the epithelial cells were pretreated before bacterial addition with HBSS containing 1 mM mannose for 20 min at room temperature followed by 3 washings with HBSS without mannose. All live-cell microscopy analyses were repeated in at least three-independent experiments and videos and images presented are representative of these experiments. Confocal microscopy For 3D-imaging of bacteria within the epithelial cells, the epithelial cells isolated on collagen I coated coverslips were used in confocal 3D-imaging microscopy. The top coverslip was removed and the bottom coverslip with attached epithelial cells was washed 3 times in HBSS to remove PEST before infecting with bacteria. The coverslip was placed in a small petri dish containing 1 ml of HBSS. To infect the epithelial cells, bacterial cells were prepared as described above for live-cell microscopy and were added to the epithelial cells to a final concentration of 16105 bacteria ml21. After 3 hours infection at room temperature, the coverslip was washed 3 times in PBS to remove unattached epithelial cells and bacteria. The cells were then fixed with 4% paraformaldehyde (pH 7.4) in PBS for 10 minutes at room temperature. After PLoS ONE | www.plosone.org 10 May 2012 | Volume 7 | Issue 5 | e37678