One - step growth experiments ( bacteriophages )
200 µL overnight culture is inoculated in 100 mL culture flask with 50 mL growth medium ( e . g . , LB ) .
Incubate on a shaking table until the density in the culture has reached cell density of ~ 5 × 108 CFU mL–1 ( corresponding to an OD525 of ~ 0.3 ) .
1 mL aliquots of the bacterial culture are mixed with subsamples of the phage stock in triplicate microfuge tubes at an multiplicity of infection ( MOI ) of approximately 0.01 .
Incubate for 10 min to allow the phages to adsorb to the host cells .
Centrifuge the cells ( 6000g , 10 min ) .
Remove the supernatant .
Resuspend the pellet in 1 mL growth medium ( e . g . , LB ) .
Repeat steps 5 - 7 to wash out any further unadsorbed phages .
Transfer 50 µL of the resuspended culture ( bacteria and adsorbed phages ) to 50 mL growth medium in a 100 mL culture flask and mix well .
Transfer 1 mL to a microfuge tube .
Incubate the triplicate 50 mL cultures on a shaking table .
Determine the number of PFU ( total infectious centers ) by plaque assay in the collected sample .
Continue to collect samples for PFU over time for 6–8 hours .
