Isolation of Phages from Pig Fecal Samples

Fecal samples were obtained from a pig farm in Hongseong-gun, Chungcheongnam-do,
Korea, suspended in 50 mL SM buffer (50 mM Tris-HCl [pH 7.5], 0.1 M NaCl, and 8
mM MgSO₄·7H₂O), and centrifuged at 4,000×g
for 10 min. The supernatant was filtered using a 0.45 μm membrane filter
(Dismic^®-25CS, Advantec, Japan) to prepare a sample
solution. Next, 18 mL supernatant and 2 mL ETEC FC02 bacterial cell culture
(OD₅₄₀=0.4) were mixed and incubated at 37°C for 18
h. After centrifugation at 4,000×g for 10 min, the supernatant was
filtered using a 0.45 μm membrane filter. Then, 100 μL filtered
supernatant was mixed with the host bacteria E. coli FC02
bacterial cell culture and 5 mL Luria-Bertani (LB) broth containing 0.7%
soft agar. The mixture was poured into 1.5% LB agar and incubated
overnight at 37°C. Individual single plaques were harvested from the agar
plate using sterile pipette tips, resuspended in 500 μL SM buffer, and
the double-layer agar technique was repeated (Šimoliūnas et al., 2014 (link)).