Phage Amplification and Purification

The DLA method was used to amplify the phages, based on a protocol described in a previous publication [62 (link)]. The top agar layer was collected in an SM buffer (50 mM Tris [pH 7.5], 100 mM NaCl, and 10 mM MgSO₄) and mixed for 1 h. The mixture was centrifuged, and the supernatant was filtered through a 0.45 μm syringe filter to eliminate contaminants. Then, a polyethylene glycol/NaCl solution was added to the sample to precipitate the phage particles. The cesium chloride (CsCl) density gradient centrifugation method was used to purify the phage particles [45 (link)]. Phage samples with gradient layers of CsCl solution were ultracentrifuged for 3 h at 50,000× g using a Type 70 Ti fixed-angle titanium rotor (Beckman, Brea, CA, USA). The sedimentation bands were collected and dialyzed using a 7000 MWCO Slide-A-Lyzer^® Dialysis Cassette (Thermo Scientific, Waltham, MA, USA). The purified samples (>10¹⁰ PFU/mL) were stored at 4 °C for further analysis.