Vibrio harveyi cultures (Vh01, Vh51, Vh102, and Vh105) used as bacterial hosts for the phages in this study were isolated from shrimp hatcheries (Chrisolite et al., 2008). Bacteria were grown at 30 °C in peptone yeast extract sea salt (PYSS) broth (Oakey & Owens, 2000) containing 5 g L−1 peptone and 3 g L−1 yeast extract dissolved in Macleod’s artificial sea salt water (HiMedia, Mumbai, India). Phages designated as φVh1, φVh2, φVh3, and φVh4, selected from a pool of 76 phages isolated from shrimp hatcheries (Chrisolite et al., 2008), were used in this study. Phage lysates were prepared (Carlson, 2005) and

concentrated by ultracentrifugation at 200 000 g for 2 h at 4 °C, using SW-41 swinging-bucket rotor (Beckman, Palo Alto, CA). Phage pellet was resuspended in sterile phage buffer and treated with DNase Dabrafenib order I (1 μg mL−1) and RNase A (100 μg mL−1) (Genei, Bangalore, India) to degrade the nucleic acid residues of host bacteria. PD-0332991 mw Phage concentrate was further purified by cesium chloride (SRL, Mumbai, India) gradient ultra-centrifugation at 490 000 g for 18 h at 20 °C. The band containing phage particles was drawn from the centrifuge tube using sterile needle and dialyzed against phage buffer overnight at 4 °C and stored at 4 °C

for subsequent studies. Titer of the purified phage suspension was determined by agar overlay method (Carlson, 2005). Purified phages (with a titer of oxyclozanide about 108 PFU mL−1) were tested by spot assay (Carlson, 2005) to test the spectrum of bactericidal activity against 125 isolates of V. harveyi and an isolate each of Vibrio species such as Vibrio logei, Vibrio fischeri, Vibrio splendidus, Vibrio alginolyticus, Vibrio paraheamolyticus, Vibrio anguillarum, Vibrio cholerae (Non-O1), Vibrio fluvialis, Vibrio mimicus, Vibrio ordalii, Vibrio vulnificus, and Vibrio metschnikovii. A 10-μL suspension of purified phages was placed on 200 mesh carbon-coated copper grids and stained with potassium

2% phosphotungstate (pH-7.2) for 20 s. Excess stain was removed immediately by placing the grids on blotting paper. The grids were examined in a Tecnai G2 Spirit Bio-Twin Transmission Electron Microscope (Eindhoven, The Netherlands). Total nucleic acid of the bacteriophages was extracted using the protocol described earlier (Santos, 1991) with some modifications, dried, and resuspended in 50 μL of sterile Milli-Q water. The phage nucleic acid was treated with DNase I, RNase A (Genei, Bangalore, India), and S1 nuclease (New England Biolabs, MA) according to the manufacturer’s instructions to confirm the nature of the nucleic acid of the bacteriophages (Sambrook et al., 1989).