Roseobacter Screening Of Surface Waters For Viruses
Pull Roseobacter cultures from freeze and grow in 5 ml broth cultures. 
Perform virus screening in sterile 96 well flat-bottom tissue culture treated microtiter plates. 
Store seawater filtrates at 4°C. 
Absorb the viruses to the bacteria. 
Place 15 µl of log phase cells and 15 µl of seawater filtrate (containing virus) into wells of a 96 well plate. 
Allow the viruses to absorb to the bacteria for 1 hour. 
Add 200 µl growth media (1/2 or 1/10 YTSS) to the wells. 
Seal the plate with parafilm and incubate with shaking. 
Look for infected wells that show lysis with respect to uninoculated controls Enrich for viruses by transferring from initial 96 well plate to another 96-well plate containing fresh liquid media (“frogging” into new wells).  
Once enriched sufficiently, filter out cells with 0.2um filtration into 1.5ml microfuge tubes Spin for 5 mins. at max to pellet cells. 
Transfer supernatant to fresh tube for storage.  
Store at 4°C.  
Plaque-purify wells containing virus by growing cells plus virus on solid media.   
Prepare solid agar media and 0.5% overlay agar. 
Absorb virus to cells using as a starting point, 25 µl virus from 96 well plate enrichments and 200 µl log-phase host bacteria for 1hr. 
Mix the virus-absorbed cells in the 5 ml overlay agar. 
Pour over the solid media. 
Incubate and look for plaque formation. 
Pick representatives of all plaque types present and record appearance of the plaques. 
Store the broth that contains the agar plugs at 4°C and allow the viruses to diffuse out of the agar into the broth. 
Once the viruses are plaque-purified they can be scaled-up in 5 ml broth cultures for DNA purification Take 25 µl of the plaque-purified virus (agar plug in ~100 µl broth) and absorb to 200 µl log-phase bacteria for 1 hr. 
Add to 5 ml broth and grow with shaking. 
Centrifuge out cells and filter culture through 0.2 µm filter SYBR stain a portion of the filtrate to determine how much virus is present.  
Purify DNA using the Promega Wizard Lambda DNA kit
