Rapid Quantification of Escherichia coli in Potable Water by Fluorescence In Situ Hybridization Performed in Liquid (liq-FISH) and a Microfluidic System

Abstract

Specific and sensitive detection of fecal microbes in potable water is essential for ensuring the safety of water supplies. To this end, because conventional culture-based methods typically require at least 24 h to detect fecal bacteria, rapid and simple microbiological detection methods are considered necessary. Fluorescence in situ hybridization (FISH) is a useful culture-independent technique for selectively and rapidly detecting target bacteria using fluorescently labeled probes that hybridize with intracellular ribosomal RNA. However, typical FISH assays are relatively complicated to perform, making FISH unsuitable for routine tests. In this study, we developed an “in liquid-fluorescence in situ hybridization” assay (liq-FISH) to enumerate Escherichia coli cells, indicator of fecal contamination, rapidly. The assay performs the entire in situ hybridization procedure in liquid and requires only two simple steps—addition of fixative followed by the addition of fluorescent probe. Important processes in FISH, fixation and hybridization, were optimized, and then specificity of the optimized liq-FISH procedure was confirmed by E. coli and other eight gammaproteobacterial species. The findings showed that only E. coli cells fluoresced under a fluorescence microscope; however, filtration process is required to observe and count hybridized cells by fluorescence microscopy. For simple and semi-automated counting following liq-FISH, our developed microscope-based microfluidic counting system was applied. Hybridized cells were injected into a microfluidic device, which permitted the detection and enumeration of E. coli cells flowing through the microchannel (width: 100 μm, depth: 15 μm). The obtained results were compared with those obtained by conventional fluorescence microscopy, and results showed the similarity (r = 0.908). E. coli cells could be counted within 5 h (filtration for concentration of low numbers of E. coli cells (if necessary): 0.5 h, fixation of cells: 2 h, in situ hybridization: 2 h, counting: 0.5 h), and this method would be useful for rapidly quantifying E. coli cells in potable water.