TITLE: No Title
AUTHORS: 

[DESCRIPTION]
The following continued adaptation of Hennes et al. (1995) is a result of FLVP assay optimizations for the <em>Vibrio alginolyticus</em> PWH3a-phage P1 phage-host system (PHS) for use with SYBR Green I stain. Novices to microscopy would benefit from reading Wen et al. (2004) for details on preparing and storing slides using SYBR Green I stain. This protocol is a modification of the standard slide preparation techniques for bacteria and viruses in aquatic samples (see Suttle and Fuhrman 2010, <a href="http://www.aslo.org/books/mave/" target="_blank">this volume</a>).

[GUIDELINES]
<strong>Materials and Equipment:<br /></strong><br />• FLVP stock or 1:100 working stock (diluted in 0.02-µm filtered water or seawater medium) <br />• 1.5-mL microcentrifuge tubes <br />• appropriate host diluent (e.g., sterile seawater medium) <br />• 0.2-µm Anodisc (Whatman) and 0.45-µm cellulose (backing) filters <br />• microscope slides and coverslips (25 × 25 mm) <br />• mounting medium containing antifade (50:50 phosphatebuffered saline:glycerol with 0.1% p-phenylenediamine) <br />• epifluorescence microscope capable of 1000× magnification and equipped with a blue-light excitation filter (such as Olympus U-MWB2/U-MWIB2 filters).<br /><br /><strong><em>Additional Experiments</em><br /><br /></strong><strong>FLVP microcosm experiments:</strong> <br /><br />Seawater samples for the microcosm experiments were obtained from either a seawater holding tank at the University of British Columbia (49°16’N, 123°15′W) or a station in Vancouver Harbor (49°18’N, 123°06’W). All samples, before use in experiments, were screened for the endogenous presence of PWH3a using the FLVP assay.<br /><br />In the first experiment, where the added (exogenous) bacterium did not dominate the system, PWH3a was added to a final concentration of 6.3 × 10<sup>6</sup> cells mL<sup>–1</sup> in 500 mL of a natural background of prokaryotes at a final concentration of 2.1 × 10<sup>6</sup> cells mL<sup>–1</sup> (~3:1 final ratio PWH3a:natural). The microcosm was enriched with MLB (10% final concentration) and incubated at 20°C with a 14:10 h light:dark cycle. Subsamples were taken at ~24-h intervals for prokaryotic cell counts (see Suttle and Fuhrman 2010, <a href="http://www.aslo.org/books/mave/" target="_blank">this volume</a>) and FLVP assays. Shortly after the 72-h time point, PWH3a-P1 virus was added to the microcosm at a final concentration of ~10<sup>5</sup>–10<sup>6</sup> viruses mL<sup>–1</sup>. <br /><br />In the second microcosm, where the added (exogenous) bacterium did dominate the system, the PWH3a final concentration was increased to 1.9 × 10<sup>7</sup> cells mL<sup>–1</sup> in 50 mL of a further reduced natural prokaryotic background of 4.4 × 10<sup>5</sup> cells mL<sup>–1</sup> (~40:1 final ratio PWH3a:natural). The microcosm was enriched as before and maintained at the same light level as the previous microcosm, yet the incubation was at the higher temperature of 30°C with aeration. Subsamples were taken as before, and infection of the microcosm was performed shortly after the 48-h time point with the same concentration of PWH3a-P1 virus as above. <br /><br /><strong>FLVP receptor titration experiment:</strong> <br /><br />Four flasks of 10 mL sterile MLB were inoculated with PWH3a at a final concentration of 1.4 × 10<sup>6</sup> cells mL<sup>–1</sup>. FLVPs were added to three of the flasks at the following MOIs: 0 (uncoated control), 1000, and 10, to attempt to coat the PWH3a bacterium’s cell surface receptors and render it resistant to subsequent infection. PWH3a-P1 virus was then added to the three flasks at an MOI of 0.01–0.1. The fourth flask did not contain any FLVPs or PWH3a-P1 and served as the control. Subsamples were taken from the four flasks at 2-h intervals for FLVP assays.<br /><br /><br />

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.