Comparison of polyvinyl chloride membrane electrodes sensitive to alkylphosphonium ions for the determination of the electrical difference (ΔΨ) of Streptococcus mutans and Lactobacillus casei

Abstract

Polyvinyl chloride membrane electrodes sensitive to tetraphenyl phosphonium (TPP +), butyltriphenyl phosphonium (bTPP +), and methyltriphenyl phosphonium (mTPP +) ions have been compared for the determination of the electrical potential difference (ΔΨ) of the oral bacteria, Streptococcus mutans DR0001 6 and Lactobacillus casei RB1014. All three types of electrode proved suitable for determining Δ,gY, although the TPP +-sensitive electrode was particularly susceptible to interference by protonmotive force (Δp) dissipators known to inhibit sugar uptake by the bacteria. The mTPP +-sensitive electrode was the least affected. Similarly, both strains had high nonspecific binding capacity for TPP + and bTPP + ions, and this increased for all three ions when the bacteria were heated to 80°C for 1 h to abolish glucose uptake and metabolism. This heat-treatment procedure is therefore not a suitable control for determination of nonspecific binding to cells. However, 1% ( v v ) toluene, 20 μ m gramicidin, or 10 μ m valinomycin effectively depolarized the bacteria without interfering with nonspecific binding. The ionophores were therefore used subsequently for the determination of nonspecific binding of the lipid-soluble cations. The mTPP + ion and corresponding electrode proved the most effective system, and ΔΨ values of −89 and −107 mV were obtained for S. mutans and L. casei, respectively, harvested from glucose-limited continuous cultures and incubated in 100 m m Hepes-KOH buffer (pH 7.0), containing 1 m m dithiothreitol and 10 m m glucose. Although the ΔΨ of S. mutans decreased significantly in the presence of Mes-KOH and potassium phosphate buffers at pH 7.0, it increased to −119 mV in Tris-HCl buffer (pH 7.0). Addition of 100 m m KCl to the Tris buffer showed that the inhibiting effects of the former buffers resulted from their high K + content. Glucose uptake and acid production by both strains was also markedly inhibited by high concentrations of Na +. The measuring system revealed that the ΔΨ of both strains decreased by up to 50% in the presence of 100 m m Na +, supporting the proposed role for Na + in the deenergization of Δp in oral bacteria.