Membrane-potential dynamics mediate bacterial electrical signaling at both intra- and intercellular levels. Membrane potential is also central to cellular proliferation. It is unclear whether the cellular response to external electrical stimuli is influenced by the cellular proliferative capacity. A new strategy enabling electrical stimulation of bacteria with simultaneous monitoring of single-cell membrane-potential dynamics would allow bridging this knowledge gap and further extend electrophysiological studies into the field of microbiology. Here we report that an identical electrical stimulus can cause opposite polarization dynamics depending on cellular proliferation capacity. This was demonstrated using two model organisms, namely Bacillus subtilis and Escherichia coli, and by developing an apparatus enabling exogenous electrical stimulation and single-cell time-lapse microscopy. Using this bespoke apparatus, we show that a 2.5-second electrical stimulation causes hyperpolarization in unperturbed cells. Measurements of intracellular K+ and the deletion of the K+ channel suggested that the hyperpolarization response is caused by the K+ efflux through the channel. When cells are preexposed to 400 ± 8 nm wavelength light, the same electrical stimulation depolarizes cells instead of causing hyperpolarization. A mathematical model extended from the FitzHugh–Nagumo neuron model suggested that the opposite response dynamics are due to the shift in resting membrane potential. As predicted by the model, electrical stimulation only induced depolarization when cells are treated with antibiotics, protonophore, or alcohol. Therefore, electrically induced membrane-potential dynamics offer a reliable approach for rapid detection of proliferative bacteria and determination of their sensitivity to antimicrobial agents at the single-cell level.

11Membrane-potential dynamics mediate bacterial electrical signaling at both intra-and inter-cellular 12 levels. Membrane potential is also central to cellular proliferation. It is unclear whether the cellular 13 response to external electrical stimuli is influenced by the cell's proliferative capacity. A new strategy 14 enabling electrical stimulation of bacteria with simultaneous monitoring of single-cell membrane 15 potential dynamics would allow bridging this knowledge gap and further extend electrophysiological 16 studies into the field of microbiology. Here we report that an identical electrical stimulus can cause 17 opposite polarization dynamics depending on cellular proliferation capacity. This was demonstrated 18using two model organisms, namely B. subtilis and E. coli, and by developing an apparatus enabling 19exogenous electrical stimulation and single-cell time-lapse microscopy. Using this bespoke apparatus, we 20show that a 2.5 sec electrical stimulation causes hyperpolarization in unperturbed cells. Measurements 21 of intracellular K + and the deletion of the K + channel suggested that the hyperpolarization response is 22caused by the K + efflux through the channel. When cells are pre-exposed to UV-violet light, the same 23 electrical stimulation depolarizes cells instead of causing hyperpolarization. A mathematical model 24 extended from the FitzHugh-Nagumo neuron model suggested that the opposite response dynamics are 25 due to the shift in resting membrane potential. As predicted by the model, electrical stimulation only 26induced depolarization when cells are treated with antibiotics, protonophore or alcohol. Therefore, 27electrically induced membrane potential dynamics offer a novel and reliable approach for rapid detection 28 of proliferative bacteria and determination of their sensitivity to antimicrobial agents at the single-cell 29 level. 30 31

Detecting live bacteria is an important task for antimicrobial susceptibility testing (AST) in the medical sector and for quality-monitoring in biological industries. Current methods for live-bacteria detection suffer limitations in speed or sensitivity. In a recent paper, we reported that electrical response dynamics in membrane potential enable single-cell rapid detection of live bacteria. The electrical response can be observed within a minute after electrical stimulation. Thus, it has potential in accelerating AST and the monitoring of biological samples. This method also enables experiments for biophysical and microbiological investigations into bacterial electrophysiology. With the hope that more researchers, scientists and engineers will use electrical stimulation for their assays, here we detail each step of the electrical stimulation experiment.