Isolation of cyanophages by plaque assays
Prepare base plates Prepare top agar / agarose .
Prepare target indicator cells .
Prepare the sample : Environmental samples should be prefiltered as described earlier .
Adsorb 50 to 100 µL sample ( as is , and 10 - fold serial dilutions , up to ca .
3 levels ) to 0.5 mL target cells under the usual culturing conditions ( e . g . , for Synechococcus sp .
strain DC2 , constant 5–25 µmol quanta m–2s–1 , at 25°C ) , agitate occasionally to encourage adsorption of phage to host .
After 1 h , transfer virus : host mixture to 2.5 mL soft agar .
Quickly and gently vortex the mixture and pour the entire tube contents onto the surface of the agar plate .
Working rapidly , gently rock and swirl the plate to spread the mixture evenly onto the plate surface before the agar starts to gel .
Set aside on a flat surface to harden ( about 1 h ) .
Prepare a control plate containing only cells ; this plate will allow you to monitor cell growth .
Seal plate with parafilm , flip plates upside down .
Incubation of plates under constant low light conditions ( 5 to 25 µmol quanta m–2s–1 ) will produce darker lawns thus allowing for easier detection of plaques .
Note the number of plaque forming units ( PFUs ) , plaque size , and morphology .
Choose a well - isolated plaque on a plate that contains less than 100 PFUs .
Harvest the plaque using a Pasteur pipette : gently press the tip of the pipette into the plaque to the bottom agar ; using gentle suction , remove the plug .
Transfer the plug to 1 mL media and vortex briefly to break it up .
Place the tube at 4°C and allow the phages particle to elute from the plug overnight to form a plaque lysate .
Vortex and centrifuge the sample ( ca .
12,000g for 10 min ) to pellet cyanobacteria and agar .
Transfer the supernatant to a new tube ; typical titer of the plaque lysate can be 104 to 105 PFU mL–1 .
Repeat steps 14 to 19 for a minimum of 3 plaques .
Choosing plaques with different morphologies may result in the isolation of different phages .
