Virions were purified using a two-layer CsCl gradient, as in reference (28 (link)). A sample of phage concentrate from D5 bioreactor effluent (collected following PEG precipitation) was brought up to a final volume of 14 mL with SM buffer. Sample density was adjusted to 1.39 g/mL and loaded above 2 mL 1.7 g/mL CsCl. Samples were then ultracentrifuged at 150,000 × g for 4.5 h at 4°C. The density fractions between 1.41 and 1.50 g/mL were recovered and stored at 4°C. Before TEM imaging, 1 mL of the recovered phage suspension was centrifuged at 25,000 × g for 1 h at 4°C, after which 0.9 mL of supernatant was removed, and pelleted phages resuspended by gentle pipetting. A 5 μL droplet of the phage suspension was placed onto a glow-discharged copper grid with carbon-coated Formvar film and incubated for 30 s at room temperature. The excess solution was drained away on filter paper. Grids were incubated with 1% uranyl acetate for 10 s. Phage particles were viewed on an FEI Tecnai G2 F20 transmission electron microscope (FEI Company, Eindhoven, the Netherlands) and a Gatan Ultrascan 4k CCD camera (Gatan, Pleasanton, CA, USA).
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