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.
