Columbia blood agar base cba

Fresh fecal samples from 20 chickens randomly picked were subjected to culture recovery from D20. Fecal microbiota was recovered on Columbia Blood Agar base (CBA, Becton Dickinson and Company, Franklin Lakes, NJ, United States) supplemented with 5% defibrinated sheep blood (Fisher Scientific, Hampton, NH, United States) and 100 μg/mL cycloheximide (Sigma-Aldrich). CBA plates supplemented with 32 μg/mL of ampicillin sodium (Life Technologies, Grand Island, NY, United States) were used to recover Amp^r bacteria. The dry mass of chicken feces was spun down by centrifugation at 4°C at full speed (Eppendorf 5415R, Germany) and homogenized in stomacher bags by a stomacher (Seward Stomacher 80 Lab System, United Kingdom). Homogenized samples were serially diluted in sterile saline and plated on corresponding agar plates. The plates were incubated at 37°C for 48 h in a GasPak 150 anaerobic system with GasPak EZ anaerobe container system sachets (Becton Dickinson and Company). The upper and lower detection limits of the plate counting enumeration method are 10¹⁰ CFU/g and 10² CFU/g, respectively.

Standard chicken diet P10109 prepared by Ohio Agricultural Research and Development Center (OARDC) Poultry Facility was used in the study. Feed composition was illustrated in Supplementary Table S1. Poultry feed, two pounds each in autoclaving-safe box (30 cm × 30 cm × 10 cm) or cylinder jar (20 cm in diameter, 20 cm in height), was processed in a sterilizer (AMSCO Renaissance series 3021, Mentor, OH, United States) under gravity mode at 121°C, 103.4 kPa for 15 min, cooled down to room temperature, and heated with the same parameters again to minimize bacterial population including spore-forming cells. The bacterial population of the resulting feed was less than 5 × 10² CFU/g, assessed by plate counting on Plate Count Agar (Becton, 100 Dickinson and Company, Franklin Lakes, NJ, United States) supplemented with 100 μg/mL cycloheximide (Sigma-Aldrich) and Columbia Blood Agar base (CBA, Becton Dickinson and Company, Franklin Lakes, NJ, United States), supplemented with 5% defibrinated sheep blood (Fisher Scientific, Hampton, NH, United States) and 100 μg/mL cycloheximide (Sigma-Aldrich).