Adsorption of phage to cyanobacteria
Have everything ready to perform plaque assay:
a. Plating cells, aliquoted, and set aside.
b. Bottom plates labeled (24 + plates).
c. Top agar aliquoted and temperature equilibrated.
d. Dilution tubes—these contain 1.5 mL media, labeled and kept on ice.
e. Cyanophage stock diluted into 1–5 mL media Set up a table to record times such as Table 1 (guidelines).
Set up adsorption cultures (e.g., 250 mL polycarbonate Erlenmeyer flasks with screw cap).
Fill flask with 100 mL host cells.
Add cyanophage stock of known titer to host at an MOI of ca. 0.01 and quickly mix to disperse the virus.
Immediately remove a subsample and dilute 100× for time zero: Transfer 15 µL to a tube containing 1.5 mL of ice cold media.
Vortex to mix.
Pellet host for 5 min at ca.
16,000g and 4°C; note the time.
Carefully remove a small aliquot (50 µL) of the supernatant to a new tube and keep cold for plaque assay; note time.
Place adsorption cultures under usual conditions (e.g., light and temp).
Repeat sampling at 15 min intervals for 1 to 1.5 h Determine the concentration of viruses remaining in the supernatant for each time point by plaque assay.
