Continuous Culture of S. mutans Strains

S. mutans strains UA159 and ΔspxA2 were grown under continuous culture conditions using a BioFlo 2000 fermenter (New Brunswick Scientific, Edison, NJ) in TY medium (3% tryptone, 0.1% yeast extract, 0.5% KOH, and 1 mM H₃PO₄) containing 1% glucose, as described previously (Faustoferri et al., 2015 (link)). Cultures were grown at a constant dilution rate of 0.24 h⁻¹ under glucose-limiting (2.3 mM) conditions. Culture pH was continually maintained throughout the experiment via the addition of 2 N KOH and verified by a pH probe (Mettler Toledo, Billerica, MA). Cultures were harvested after 10 generations at steady-state pH 7, followed by addition of excess glucose (20 mM), thereby lowering culture pH. Cultures were similarly harvested at pH 5 after 10 generations at steady-state and stored at −80°C.