Background

From fundamental microbiology research to antimicrobial susceptibility testing (AST), quantifying the number of live bacteria is an important task. When it comes to detecting live bacteria, the colony forming unit (CFU) assay, dating from the 19^th century, is still the gold standard due to its reliability and sensitivity. However, the CFU assay has a major drawback; it requires 1-3 days of incubation. To overcome this limitation, developments in rapid bacterial detection technology has flourished in recent years (Iqbal et al., 2000; Ahmed et al., 2014). Examples of recently developed technologies are based on impedance spectroscopy, Raman spectroscopy or detection of biomolecules (e.g., ATP, DNA). Nevertheless, overcoming the tradeoff between speed, accuracy and robustness remains a challenge.

Membrane potential indicators are proven useful for distinguishing live and dead bacteria by flow cytometry and fluorescence microscopy (Sträuber and Müller, 2010). However, this method is technically challenging because it requires a careful calibration for bacterial species/strains, media conditions, indicator concentrations, light sources and detectors. In a recent study, we showed that such limitations can be overcome using optical measurements of the electrical response dynamics of membrane potential under stimulation by an external electrical field (Stratford et al., 2019). The responses of proliferative and inhibited cells were distinguishable within a minute; hence, it substantially shortens the time required for a live bacterial assay. Simulations of a phenomenological mathematical model and biophysical understanding suggested that this technology is applicable for different microbial species and antimicrobial treatments. This means that by comparing the electrical dynamics of unperturbed and antimicrobial-treated cells, it may be possible to accelerate AST. An important next step is examining how widely this technology can be used with environmental and pathogenic microbes. This protocol will assist conducting electrical stimulation experiments for AST and live bacteria detection assays with various bacterial species and strains as well as with various antimicrobial treatments.

In addition to its fundamental role in cell proliferation, membrane potential mediates bacterial electrical signaling in biofilms and during the processes of sporulation, mechano-sensation and cell division (Strahl and Hamoen, 2010; Prindle et al., 2015; Bruni et al., 2017; Sirec et al., 2019, Benarroch and Asally, 2020). Membrane potential is also associated with antibiotic resistance (Damper and Epstein, 2010). While these recent studies are beginning to garner attention, membrane potential remains largely overlooked in the field of microbiology. We suspect this oversight is partially due to the technical difficulties associated with experimentally controlling membrane potential. To this end, this protocol will also assist biophysical and microbiological investigations into bacterial electrophysiology.