Development of Fluorescence In Situ Hybridization as a Rapid, Accurate Method for Detecting Coliforms in Water Samples

Abstract

Coliform bacteria are indicators of water quality; however, most detection methods for coliform bacteria are time-consuming and nonspecific. Here, we developed a fluorescence in situ hybridization (FISH) approach to detect four types of coliform bacteria, including Escherichia coli, Klebsiella pneumoniae, Enterobacter aerogenes, and Citrobacter freundii, simultaneously in water samples using specific probes for 16S rRNA. This FISH method was applied to detect coliform bacteria in simulated water and domestic wastewater samples and compared with traditional detection methods (e.g., plate counting, multiple-tube fermentation (MTF) technique, and membrane filter (MF) technique). Optimal FISH conditions for detecting the four types of coliforms were found to be fixation in 3% paraformaldehyde at 4 °C for 2 h and hybridization at 50 °C for 1.5 h. By comparing FISH with plate counting, MTF, MF, and a commercial detection kit, we found that FISH had the shortest detection time and highest accuracy for the identification of coliform bacteria in simulated water and domestic wastewater samples. Moreover, the developed method could simultaneously detect individual species and concentrations of coliform bacteria. Overall, our findings indicated that FISH could be used as a rapid, accurate biosensor system for simultaneously detecting four types of coliform bacteria to ensure water safety.

Keywords: biosensor; coliform bacteria; coliform detection method; fluorescence in situ hybridization; water safety.

Figure 1

Establishment of the optimal fixation conditions for detecting E. coli using fluorescence in situ hybridization (FISH). (a–c) E. coli cells were fixed using 3% paraformaldehyde at 4 °C for different times (1, 1.5, or 2 h) and then stained with Cy3. (d–f) E. coli cells were fixed using 3% paraformaldehyde at 25 °C for different times (1, 1.5, or 2 h).

Figure 2

Identification of the optimal hybridization temperature for detecting K. pneumoniae using FISH. (a–d) K. pneumoniae cells were detected by FISH with specific probes at different hybridization temperatures (a: 42 °C, b: 46 °C, c: 50 °C, and d: 62 °C) for 1.5 h.

Figure 3

Identification of the optimal hybridization time for detecting coliform bacteria using FISH. E. coli (a) and E. aerogenes (b) cells were detected by FISH using specific probes for hybridization for different times at 50 °C.

Figure 3

Identification of the optimal hybridization time for detecting coliform bacteria using FISH. E. coli (a) and E. aerogenes (b) cells were detected by FISH using specific probes for hybridization for different times at 50 °C.

Figure 4

Detection of four coliform strains using the established FISH method under optimal conditions.

Figure 5

Simultaneous detection of E. coli and K. pneumoniae to demonstrate the specificity of the probes in FISH.

Figure 6

Detection of coliform bacteria in domestic wastewater using FISH.