Effect of Thioflavin T on the Elongation Rate of Bacteria

The growing field of bacterial electrophysiology examines the relationship between bacterial membrane potential and cell division, growth, sporulation, and biofilm formation. These experiments require Nernstian fluorescent dyes to monitor membrane potential. Our research uses single cell imaging to determine if a common fluorescent dye, Thioflavin T (ThT), affects the growth of bacteria. Materials and

The increased cellular accumulation of ThT is a standard measure of hyperpolarization in bacterial electrophysiology. Growth curves, a bulk measurement, are typically used to determine suitable concentrations of ThT for use in experiments. Single cell measurements show that cells incubated with ThT have decreased elongation rates. This creates a potential experimental artifact that could lead to misinterpretation of data. Hyperpolarized cells internalize more ThT. This increased intracellular concentration of ThT, rather than the change in membrane potential, could lead to decreased growth. These experiments point toward the importance of single cell measurements to detect subtle changes in cell growth. We hope this research will be useful for other researchers in their choice of dye for the detection of membrane potential.

We use a combination of standard growth curve measurements and single cell imaging, both brightfield and fluorescence microscopy, to monitor the growth of Bacillus subtilis and Escherichia coli as a function of ThT concentration. Increased membrane potential (hyperpolarization) leads to increased intracellular accumulation of ThT: High fluorescence intensity is an indicator of hyperpolarization. Blue light is used to hyperpolarize a subpopulation of cells to monitor cellular elongation in response to increased cellular internalization of ThT.

Single cell imaging shows that the elongation rates of B. subtilis and E. coli are decreased when these cells are incubated with ThT. At micromolar concentrations of ThT, this effect may be masked in standard growth curves, but is visible with single cell measurements on agarose pads. Conclusions: The increased cellular accumulation of ThT is a standard measure of hyperpolarization in bacterial electrophysiology. Growth curves, a bulk measurement, are typically used to determine suitable concentrations of ThT for use in experiments. Single cell measurements show that cells incubated with ThT have decreased elongation rates. This creates a potential experimental artifact that could lead to misinterpretation of data. Hyperpolarized cells internalize more ThT. This increased intracellular concentration of ThT, rather than the change in membrane potential, could lead to decreased growth. These experiments point toward the importance of single cell measurements to detect subtle changes in cell growth. We hope this research will be useful for other researchers in their choice of dye for the detection of membrane potential.