Influence of pH and fluoride on properties of an oral strain of Lactobacillus casei grown in continuous culture.

A freshly isolated oral strain, Lactobacillus casei RB1014, was grown in continuous culture to compare the effects of pH and fluoride on growth and metabolism. The cells were grown at pH 7.0 to 3.2 in the absence of fluoride and from pH 7.0 to 5.4 with 20 mM NaF. Cell numbers varied from 3 X 10(9) to 30 X 10(9)/ml on blood agar during alterations in the growth pH from 7.0 to 4.27. Only when the culture was stressed by lowering the pH to 3.2 were cell numbers drastically reduced. Cells growing at pH 7.0 without fluoride were unable to grow when plated on fluoride agar (10.5 mM) at pH 5.5; however, when the growth pH was allowed to decrease to 4.94, cells grew on the fluoride plates in numbers equal to those growing on blood agar. This fluoride tolerance trait appeared rapidly once pH control was removed and was lost when the culture was returned to pH 7.0. The addition of 20 mM NaF to the culture medium did not adversely affect growth, provided that the pH was maintained at 6.0 or above; cells tolerant to 10.5 and 16 mM NaF appeared on pH 5.5 plates during this phase. In cells removed from the chemostat throughout the experiment and incubated at the pH of growth in a pH stat, glycolytic activity was optimum at pH 5.5 in the absence of NaF. Fluoride stimulated glycolytic activity by cells incubated at pH 7.0 and by cells growing with 20 mM NaF, provided that the pH of growth remained at or above 6.0. A more detailed examination of the adaptation to fluoride tolerance during shifts to acidic pH values revealed that cells capable of growth on acidic fluoride agar plates appeared within 2 h of the start of the fall in pH of the chemostat culture. Estimation of the intracellular pH during the period of the initial pH fall revealed that the intracellular pH was identical to the extracellular pH (i.e., no pH gradient [delta pH]), indicating that fluoride would not be transported into the cells to inhibit metabolism. However, once the pH of the medium was stabilized, delta pHs were generated, with the delta pH increasing as the pH declined. The inhibition of glycolysis by fluoride increased in proportion to the delta pH. Cells grown at pH 5.5 generated larger delta pHs than did cells grown at pH 7.0, although the values were normally small (approximately 0.9 U). The data suggest that the inherent fluoride tolerance of L. casei RB1014 was associated with relatively small delta pHs.