FIG. 3.

Acid tolerance assay. (A) S. mutans strains UA159, TW26, and TW42 were grown in BHI medium adjusted to pH 7.0. Cultures with an OD₆₀₀ of ∼0.3 were harvested, washed with 0.1 M glycine buffer, pH 7.0, and subjected to acid killing by incubating the cells in 0.1 M glycine buffer, pH 2.8. For preadapted acid killing, cultures with an OD₆₀₀ of ∼0.2 were harvested and resuspended in fresh BHI medium adjusted to pH 5.0. Following two additional hours of incubation, cells with an OD₆₀₀ of ∼0.3 were prepared for acid killing as described above. Survival rate was determined by plating in triplicate on BHI agar plates, and results are expressed as percent survival rate versus time at pH 2.8. (B) Cell-free supernatants from UA159 (W) and TW26 (S) were reconstituted with concentrated TV medium to 0.5×, and their impact on acid tolerance was measured by incubating the cells in 0.1 M glycine buffer, pH 2.8, for 45 min as described above.