Nvt65 rotor

Virulent phages 53, 73, and 128 were isolated from failed mozzarella productions in Uruguay. S. thermophilus host strains Uy01, Uy02, and Uy03 were obtained from a local starter culture supplier and were grown in LM17 medium at 42 °C. Reference phages DT1 (cos-type) and 2972 (pac-type), including their respective S. thermophilus hosts SMQ-30110 (link) and DGCC771023 (link), were obtained from the Félix d’Hérelle Center (www.phage.ulaval.ca). Phages and bacterial strains were stored, as frozen stocks, in LM17 supplemented with 15% (v/v) glycerol. Phages were amplified in LM17 supplemented with 10 mM CaCl₂ (LM17-CaCl₂). Phage titers were determined as described elsewhere16 . Each phage was plaque purified three times and concentrated through CsCl gradient41 . Briefly, one liter of phage lysate wasemented with 10% polyethylene glycol (PEG 8000). PEG-concentrated phages were recovered by centrifugation and subjected to ultracentrifugation using a discontinuous CsCl gradient in a Beckman SW41 Ti rotor (35,000 rpm, 3 h at 20 °C). The phage band was retrieved and a second ultracentrifugation was performed with a continuous gradient of CsCl using a Beckman NVT65 rotor (60,000 rpm, 18 h at 20 °C). The purified phages were dialyzed in buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 8 mM MgSO₄) and stored at 4 °C.

Viral particles were purified from 30 15-cm Petri dishes of iD98HR1 cells that had been treated with OHT for 3 days. The extracellular virions were collected from the culture supernatant. Cell debris was removed by centrifuging the supernatant at 6,000 g for 15 min. The intracellular viral particles were first released from cell pellets by three cycles of freeze and thaw. Viral particles in extracellular or intracellular fractions were concentrated by centrifugation at 130,000 g for 1 h on a 50% OptiPrep (Axis-Shield) cushion. The virions at the interface were collected, and the OptiPrep was adjusted to 25%. Subsequently, a gradient was generated by centrifugation at 350,000 g for 3 h with an NVT65 rotor (Beckman). Fractions of 1 ml were collected from the bottom of the tube. Proteins in each fraction were analyzed by immunoblotting with antibodies. Viral particles in the fractions were absorbed onto a formvar/carbon-coated grid (Ted Pella, Inc.), blotted dry, and negatively stained with 1% uranyl acetate for 15 min at room temperature. The morphology of the viral particles was examined, and images were obtained using a JEOL JEM-1200 transmission electron microscope.