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
