Reviving and Culturing Bacterial Isolates

Colonies of this isolate were revived from a glycerol stock (stored at −80°C) by streaking them onto chocolate II agar (Becton, Dickinson Co. Sparks, MD, USA) in 5% CO₂ at 37°C for 48 h (14 (link)). Multiple isolated colonies from a single plate were used to inoculate 6 ml of brain heart infusion broth (Hardy Diagnositics, Santa Maria, CA, USA) supplemented with 0.1% Trizma base (Sigma-Aldrich, St. Louis, MO, USA) and 0.01% thiamine monophosphate (Sigma-Aldrich; this medium is referred to as BHI-TT [15 (link)]) in a 50-ml conical tube. The inoculated BHI-TT was vortexed, and a target optical density at 600 nm (OD₆₀₀) of 0.2 was verified. The total volume of the suspension was then brought to 45 ml with fresh BHI-TT (resulting in a new OD₆₀₀ of 0.02) and incubated at 37°C with shaking at ∼190 rpm for 6 to 8 hours until an OD₆₀₀ of 0.3 (mid-log-phase growth) was reached (VCN measured as ∼1 × 10⁹ CFU/ml) where the cultures reach a maximum OD₆₀₀ of 0.6 in 24 hours.