Plaque Purification and Phage Propagation

A single plaque was picked and resuspended in 500 μl of SM buffer (5.8 g NaCl, 2.0 g MgSO₄⋅7H₂O, 50 ml 1 M Tris–HCl, pH 7.4, in 1 L of dH₂O). After incubating for 60 min at 37°C, the solution was centrifuged, and the supernatant was filtered through 0.22 μm pore size filters. To produce a homogenous phage stock, seven rounds of single plaque purification were performed. Thereafter, the propagation of the phage was performed by preparing a plate lysate. The lysate was centrifuged, and the supernatant was filtered through a 0.22 μm pore size filter to remove bacterial cells (Bonilla et al., 2016). The titer of propagated phage was measured by the spot test as described in Mazzocco et al. (2009) with modifications. Briefly, phage lysate was serially diluted (12-fold) in SM buffer, and 10 μl of each dilution was spotted on a double-layer LB plate. The bottom layer contains 1% (w/v) LB agar and the top layer is composed of 0.5% (w/v) LB agar mixed with 100 μl of exponentially growing P. aeruginosa culture. The plates were all dried for 25 min in a laminar flow hood (ASTEC, MicroFlow) before it was incubated at 37°C for 18 h. The plaque forming unit (pfu/ml) was measured by counting the plaques in the dilution that have 3–30 countable plaques using the following calculation: Average number of plaque × 100 × reciprocal of dilution = pfu/ml.