protocol_571.txt

Isolation of cyanophages by liquid enrichment assay
Prefilter at least 3 L water sample through a glass fiber filter.
Filter sample through 0.2 µm or 0.45 µm low protein binding PVDF filter.
Dispense the filtered water samples (e.g., 0.5 L or more) into culture vessels, e.g., 1 L or larger Erlenmeyer flasks.
Add nutrients to the filtered water to support growth of the target cells.
Seed the filtered water with 1% to 10% v/v of host culture.
As a control, replace the filtered environmental sample with virus-free (0.02 µm filtered or heat-killed) water sample.
Incubate the flasks at the temperature and light conditions appropriate for the cyanobacteria and look for signs of lysis.
Remove an aliquot of the enrichment culture, and pellet remaining cells by centrifugation (e.g., 20 minutes at 6000g).
Filter the supernatant through a 0.22 µm or 0.45 µm PVDF filter.
Store the lysate at 4°C until further analysis.
Verify the presence of lytic phages by liquid assay (or plaque assay).
To propagate/amplify the lytic agent, the liquid bioassay is repeated using the putative lytic agent as the test sample.