Phage infection and timed harvest of E. coli and B. subtilis cells

Prepare 30 mL subcultures of E. coli and B. subtilis at 1:100 dilution (300 µL in 30 mL) and grow to a target OD₆₀₀ of 0.4 in a shaking incubator (180 rpm). For both E. coli and B. subtilis this is ~2 h at 37 °C. Supplement the subculture media (LB) with 1 mM CaCl₂ and 1 mM MgCl₂.

Aliquot 500 µL of cell culture into several 1.5 mL microcentrifuge tubes and inoculate with 50 µL of phage at the correct dilution to reach your target MOI (see our protocol on MOI calculations, linked below).

Calculating multiplicity of infection (MOI)

Here, we are infecting E. coli with T4 and and B. subtilis with SPO1. Be sure to simultaneously carry out a control experiment where you mock-infect 500 µL E. coli and B. subtilis cultures with 50 µL sterile SM buffer. Once inoculated, incubate the microcentrifuge tubes at 37 °C on a heated shaker block, shaking at 300 rpm.

Plate out phage and bacteria to calculate the actual MOI used in the experiment.

Pre-cool a bench-top centrifuge to 4 °C and at desired time points, harvest cells by centrifuging at 5000 × g for 3 min. For example, cells can be harvested at 5 min and 15 min in the hope of capturing both earlier and later infection stages. This timing will be highly phage-dependent!

Remove the supernatant from the pellet and immediately flash-freeze the pellets in liquid nitrogen. You must be speedy during this process to effectively halt phage infection. You can store bacterial pellets at –80 °C and use later for downstream applications such as RNA extraction.