One - step growth experiments ( cyanophages )
Set up triplicate adsorption tubes ( AT ) in 1.5 mL microtubes .
Add phages of a known titer to the cyanobacteria host in exponential growth ( cell concentration determined by microscopy ; Cyanoinput ) at an MOI of approximately 0.02 .
Allow the phages to adsorb to the hosts for 60 min .
at room temperature and an illumination of 25µmol quanta m–2s–1 .
Set up control tubes ( CT ) as above , except :
a . positive controls : replace host cells with media ( this gives input phage numbers ) .
b . negative controls : omit virus After 60 min , remove unadsorbed phages ( T = 0 ) .
Prepare nine 15 - mL centrifuge tubes containing 10 mL algal growth media .
Add 100 µL from AT to FGT ( 10–2 dilution ) .
Mix and remove 0.1 mL sample for T = 0 .
Perform plaque assay immediately to determine total infective centers ( TICT = 0 ) .
Add 100 µL from FGT to SGT ( 10–4 dilution ) .
Incubate FGT and SGT tubes at room temperature and ca .
25 µmol quanta m–2s–1 Remove 100 µL samples every 3 h from FGT ( at 3 , 6 , 9 , 12 , 16 h ) and SGT ( from 12 h onwards ) for ca . 30 h .
Determine the TIC via plaque assay .
Add 100 µL from positive control tube ( CT ) to FGT - control ( 10–2 dilution ) .
Determine the total phage input ( Phageinput ) .
