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 .
