Monitoring the Metabolic Activity of a Single Bacterial Cell Based on Scattering Intensity

Abstract

Cell activity is evaluated using the number of colonies formed on a medium or the number of live cells in a suspension or by staining nuclei with fluorescent dyes to determine whether cells are dead. However, the culture methods generally require extended culturing times, and damage to the cell membranes observed using fluorescent dyes is not necessarily related to cell survival or activity. Hence, accurately determining the activities of individual cells is impossible. Therefore, we developed a method for quantitatively evaluating the metabolic activities of single cells by focusing on the optical and chemical properties of formazan dye, i.e., 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The oxidized form of MTT is soluble and highly permeable to cell membranes, but it is reduced to insoluble MTT formazan upon reaction with intracellular metabolic products. Single-cell observation using dark-field microscopy revealed that insoluble formazan aggregates within the cells formed particles that emitted characteristic scattered light. The formazan-derived scattered light component extracted via peak fitting was related to metabolic activity, demonstrating its usefulness as a parameter indicating the activity of an individual cell. This method enables the real-time evaluation of the activities of single cells, which should lead to not only the acceleration of bacterial screening and microbial control but also the development of antibiotics and suppression of drug-resistant bacteria.