In this study, electrochemical detection of viable bacterial cells was performed using a tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), which was converted to an insoluble and redox active formazan compound in viable microbial cells. The insolubility of this formazan was effectively exploited as a surface-confined redox event. An indium-tin-oxide electrode was applied to a microbial suspension that had been incubated with MTT and was heated to dry for the extraction and adsorption of formazan. Drying led to the appearance of a distinctive voltammetric oxidation peak at +0.1 V vs Ag|AgCl, the magnitude of which was successfully correlated to the number of viable microbes in the suspension. Thus, the electrochemical detection of formazan was effectively coupled with the thermal lysis of microbes. It is also noteworthy that this lysis-adsorption technique was highly selective to the hydrophobic formazan molecule due to the removal of hydrophilic cell components during equilibration in a phosphate buffer before voltammetric measurement. This technique was capable of detecting microbes above 2.8 × 10 CFU mL and required only a 1 h incubation. The results of this study indicate that the sensitivity of the present technique is up to 10 000-fold higher than that of MTT colorimetry. The higher sensitivity was mainly ascribed to the concentration of the microbially produced formazan on the electrode by thorough desiccation of the bacterial suspension.

We have developed a novel bacterial detection technique by desiccating a bacterial suspension deposited on an electrode. It was also found that the use of an indium-tin-oxide (ITO) electrode dramatically improved the resolution of the voltammogram, allowing us to observe two pairs of redox peaks, each assigned to the adsorption of isoprenoid ubiquinone (UQn) and menaquinone (MKn), which were present in the bacterial cell envelopes, giving midpeak potentials of -0.015 and -0.25 V versus Ag|AgCl|saturated KCl| at pH 7.0, respectively. Most of the microorganisms classified in both the Gram-negative and -positive bacteria gave well-defined redox peaks, demonstrating that this procedure made the detection of the quinones possible without solvent extraction. It has been demonstrated that the present technique can be used not only for the detection of bacteria, but also for profiling of the isoprenoid quinones, which play important roles in electron and proton transfer in microorganisms. In this respect, the present technique provides a much more straightforward way than the solvent extraction in that one sample can be prepared in 1 min by heat evaporation of a suspension containing the targeted bacteria, which has been applied on the ITO electrode.

Ubiquinone and menaquinone play important roles in microbial respiratory chains. S. oneidensis is a facultative anaerobe, and it has been known that this microbe requires menaquinone (MK) and/or methylmenaquinone (MMK) to reduce inorganic and organic compounds, such as Fe(III), Mn(IV), fumarate, and nitrate, under anaerobic conditions. In this article, we show that voltammetry is useful to quantify ubiquinone (UQ), MK and MMK located in the cytoplasmic membrane simply by heating the microbe (thermal lysis) deposited on an indium-tin oxide electrode. It was found that the microbe predominantly produced UQ under aerobic conditions, while the production was switched to that of MMK under anaerobic conditions. This transition occurred at a dissolved oxygen concentration of ∼0.3 mg L −1 . It was also found in this study that the total quinone concentration (UQ + MK + MMK) in a single cell was constant at all stages of the culture in a nutrition broth, suggesting that the cytoplasmic membrane was saturated with the quinones. The reduction of Fe(III) citrate with S. oneidensis was also studied. It was found that this microbe decreased the UQ and MMK levels to 45% and 39% of the initial values, respectively, after finishing the reduction.