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.
