Viable Bacterial Cell Counting Methods Based on Electrochemical Properties of Tetrazolium Salts
Authors: Ikeda Hikaru , Tokonami Akira , Fujimura Hayato , Kawanaka Miya , Sadanaga Yasuhiro , Shiigi Hiroshi 
Published: December 2024

【Introduction】 Bacterial activity is an essential evaluation index for hygiene management and quality control of medicine and food because some bacteria such as Escherichia coli, Salmonella enterica, and Staphylococcus aureus are causative organisms that cause food poisoning and adversely affect the human body. Conventional bacterial testing has widely used methods that count colonies formed based on cell proliferation in culture. When the mother cell divides to produce her two daughter cells, the surviving cells multiply logarithmically. Most bacteria divide at least once within an hour, but some species take longer than 20 hours to divide, so visual confirmation of bacterial colonization may take at least 48 hours, and in some cases about 2 weeks. Hence, although this method reflects viability with accurate cell counts, it requires a long inspection time. A rapid and simple technique for bacterial detection based on dissolved oxygen, enzymatic reactions, and electron mediators was developed to circumvent this problem. This study focused on the electrochemical properties of tetrazolium salts to develop a simple method for evaluating viable bacterial counts, which are indicators of hygiene control at food and pharmaceutical manufacturing sites [1]. Given that the oxidized form of 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), which has excellent cell membrane permeability, changes to the insoluble reduced form of formazan inside the cells. The number of viable cells was estimated by focusing on the reduction current of MTT remaining in the suspension. Dissolved oxygen is an important substance for bacterial activity; however, it interferes with the electrochemical response of MTT. We investigated the electrochemical properties of MTT to obtain a potential-selective current response that was not affected by dissolved oxygen. Real-time observation of viable bacteria in suspension revealed that uptake of MTT into bacteria was completed within 10 min. In addition, we observed that the current response depends on viable cell density regardless of the bacterial species present. Our method enables a rapid estimation of the number of viable bacteria, making it possible to confirm the safety of food products before they are shipped from the factory and thereby prevent food poisoning.

【Experimental】MTT solution and 0.20% glucose-added nutrient broth medium were mixed and diluted with electrolytes, such as 0.10 M KCl aqueous solution and 0.10 M phosphate-buffered saline (PBS) solution (pH 7.4), to obtain 0.10 mM MTT solution. E. coli suspension (2.0 × 109 CFU mL－
1) was dropped into 0.10 mM MTT solution and incubated at 310 K for 1 h. Cyclic voltammetry (CV) was performed to measure MTT remaining in the suspension before and after incubation at a sweep rate of 0.10 Vs－
1 using a glassy carbon-disk electrode as the working electrode, a platinum coil as the counter electrode, and Ag|AgCl (3 M KCl) as the reference electrode.

【Results】Two sharp reduction peaks were observed at －0.1 V (II
c) and －0.6 V (I
c) at performing a cathode sweep from +0.2 V (Fig.1). These current responses are based on the reactions shown in the following equations.

MTT + e－ ⇄ MTT・（intermediate） [1]

MTT・ + H+ + e－ ⇄ formazan [2]

The MTT-based current responses remaining in these electrolytes gradually decreased with incubation time and almost disappeared after 60 min of incubation. This means that MTT was taken up into cells and the generated insoluble formazan was deposited inside the cells. Although dissolved oxygen is certainly an important substance for bacterial activity, it can interfere with the electrochemical response of MTT. The current response of formazan formation (I
c) was significantly different in the presence of dissolved oxygen (12%). However, the current response based on the intermediate formation reaction (II
c) was unaffected (0%). Quantification of viable bacterial counts based on the current response of II
c (103–107 CFU mL－
1) was achieved by measuring the amount of residual MTT in the electrolyte. Furthermore, the current response was confirmed to be dependent on viable cell density, independent of bacterial wall structure and metabolic processes.

【Reference】

[1] H. Ikeda, A. Tokonami, S. Nishii, Y. Yamamoto, X. Shan, Y. Sadanaga, H. Shiigi, Anal. Chem., 95(33), 12358-12364 (2023).

【Images】

Fig.1 CVs of 0.10 mM MTT in 0.10 M PBS solution (pH 7.4) containing E. coli (2.0×109 CFU mL−1) during incubation at 310 K.

Figure 1