0.22 μm pore

Phage was isolated from chicken stool in Shanghai, China, using the method described previously by Cao et al. [41 (link)]. Briefly, 25 g of sample was mixed homogeneously with 40 mL of LB broth, supplemented with 350 μL of 1 M CaCl₂, and inoculated with 1 mL of Salmonella suspension (10⁹ CFU/mL). After incubation at 37 °C overnight, the medium was centrifuged at 8000 rpm for 15 min (5424, Eppendorf AG 22331, Hamburg, Germany). The supernatant was filtered with a 0.22 μm pore (Millipore, Billerica, MA, USA) size syringe filter, and the presence of lytic phage in the sample was confirmed by spot testing. A district plaque was observed and suspended in 1 mL of salt magnesium (SM) buffer for purification. The phages were purified at least three times to create phage stock, using a double-layer agar technique [42 (link)].

For the imaging analyses of virus movements, the glass bottoms of culture dishes (Matsunami, Kishiwada, Japan) were coated with bovine mucin (1 mg/ml; Sigma-Aldrich, St. Louis, MO, USA), which was dissolved in phosphate-buffered saline (PBS) and filtered through a polyvinylidene fluoride membrane filter with a 0.22-μm pore (Millipore, Billerica, MA, USA) to remove insoluble mucin, and the dishes were then incubated for 1 h at room temperature. The glass surface of each dish was washed twice with PBS to remove any unbound mucin. This cycle of coating and washing was repeated twice. To examine virus movement, viruses diluted in PBS were added to the mucin-coated glass surfaces and observed by SRICM (Nikon, Tokyo, Japan) using a 100× lens objective (APO TIRF; numerical aperture, 1.49). Virus images were acquired every 1 s for a total duration of 15 min. Images of 50 filamentous AA, 30 spherical AA, and 30 spherical Taylor viruses were analyzed. Virions with lengths of more than 0.5 μm were regarded as filamentous, whereas virions with diameters of less than 0.3 μm were regarded as spherical.

The FBS was collected in sterile conical tubes (Corning) and centrifuged (GH3.8 rotor, Beckman GPR Centrifuge; Beckman Coulter, Inc., Brea, CA, USA) at 900× g for 10 min and filtered using a 0.22 μm pore (Millipore, Burlington, MA, USA). The samples were then centrifuged (SW28 rotor, Beckman XL-90 Ultracentrifuge, Beckman Coulter, Inc.) at 120,000× g for 18 h at 4 °C [22 (link)] and stored at 2 °C until use.