2.2. Phage Propagation and Purification

SU7 was previously isolated from the Käppala wastewater treatment plant located 15 km East of Stockholm, Sweden, in November 2016. For phage enrichments, fresh LB media was inoculated with 100 µL of overnight bacterial cultures and allowed to grow to the mid-log phase at 37 °C with shaking or until the optical density at 600 nm (OD₆₀₀) was 0.6. SU7 was enumerated using the agar overlay (OA) method with 65% w/v (22.75 g/L) TYA as previously described [13,14]. Concentrated stocks of SU7 were produced using a modified polyethylene glycol (PEG) precipitation protocol [15]. In brief, crude phage lysate suspensions were centrifuged at 3864× g for 10 min and the supernatant passed through a sterile 0.45 µm syringe filter (Sarstedt Filtropur, Nümbrecht, Germany). Phages were precipitated by adding solid NaCl and PEG8000 (Acros Organics, Fisher Scientific, Schwerte, Germany) to the partially purified suspension to final concentrations of 1 M and 10% w/v, respectively, and then stored at 4 °C for two weeks. Following refrigeration, phages were centrifuged at 11,000× g for one hour at 4 °C, the pellet re-suspended in 50 mL phosphate-buffered saline (PBS), pH 7.4, and the phage content determined using the OA method.