2.2. Phage Propagation and Purification

Before propagating the phage, Nocturne116 was plaque-purified by subculturing individual plaques twice as described elsewhere [13] and subsequently propagated using double-agar overlays with 20 Petri dishes to achieve the confluent lysis of the bacterial lawn. Top soft agar layers containing lysed bacterial lawns and phage progeny were collected, vortexed for ~30 s after addition of 5 mL LB media per soft agar from one plate, incubated at RT for ~ 30 min, and then centrifuged at 8228× g on an Eppendorf 5810R centrifuge (Eppendorf) for 30 min before carefully decanting supernatant which was subsequently filtered through a 0.45 μm pore size syringe filter (Sarstedt). These steps yielded about 100 mL of the phage-containing filtrate with ~2 × 10⁸ pfu/mL. Purification of the propagated phage was carried out by gel-filtration on 4 Fast Flow Sepharose (Cytiva, Marlborough, MA, USA) followed by Q Sepharose High Performance (Cytiva) ion exchange chromatography.

Phage presence in peak fractions (PBS buffer of ~7.3 pH as medium) was analyzed by titration and TEM. Finally, the purified phage was concentrated on Amicon Ultra-15, 100 K MWCO filters (Merck, Kenilworth, NJ, USA) to 0.5 mL at 3214× g on an Eppendorf 5810R centrifuge (Eppendorf).