Bbl brain heart infusion broth modified

F. nucleatum (ATCC 10953) was obtained from the American Type Culture Collection (Manassas, VA, USA). F. nucleatum was cultivated under anaerobic conditions (80% N2, 10% CO2, 10% H2) in a Whitley A35 Anaerobic Station (Don Whitley Scientific, West Yorkshire, UK) at 37°C. For maintained growth on solid media, tryptic soy agar plates with 5% sheep blood (BD biosciences, San Jose, CA, USA) were used. For the base liquid medium, BBL brain heart infusion broth (modified) (BD biosciences, San Jose, CA, USA) was used. Filter sterilized succinate was added to the liquid base medium to a final concentration of 0, 25, 50, or 100 μM. All treatments were performed in triplicate, with 7 mL of medium in 15 mL Falcon tubes with caps loosely fashioned. An inoculation volume of 2% (vol/vol) of overnight liquid culture was used to inoculate all treatments. All liquid media was fully reduced under anaerobic conditions prior to inoculation. Colony-forming unit (CFU) measurements were performed on tryptic soy agar plates with 5% sheep blood.