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

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.

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Evaluation of detection of Legionella spp. in water samples by fluorescence in situ hybridization, PCR amplification and bacterial culture

International Journal of Medical Microbiology ◽

10.1078/1438-4221-00213 ◽

2002 ◽

Vol 292 (3-4) ◽

pp. 241-245 ◽

Cited By ~ 22

Author(s):

Susanne Buchbinder ◽

Karlheinz Trebesius ◽

Jürgen Heesemann

Keyword(s):

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Water Samples ◽

Bacterial Culture ◽

Pcr Amplification

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Processing Deep-Sea Particle-Rich Water Samples for Fluorescence In Situ Hybridization: Consideration of Storage Effects, Preservation, and Sonication

Applied and Environmental Microbiology ◽

10.1128/aem.70.1.25-33.2004 ◽

2004 ◽

Vol 70 (1) ◽

pp. 25-33 ◽

Cited By ~ 6

Author(s):

Phyllis Lam ◽

James P. Cowen

Keyword(s):

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Water Samples ◽

Deep Sea ◽

Cell Counting ◽

Epifluorescence Microscopy ◽

Size Fractionation ◽

Cell Abundance ◽

Abundance Estimates

ABSTRACT Particles are often regarded as microniches of enhanced microbial production and activities in the pelagic ocean and are vehicles of vertical material transport from the euphotic zone to the deep sea. Fluorescence in situ hybridization (FISH) can be a useful tool to study the microbial community structures associated with these particles, and thus their ecological significance, yet an appropriate protocol for processing deep-sea particle-rich water samples is lacking. Some sample processing considerations are discussed in the present study, and different combinations of existing procedures for preservation, size fractionation sequential filtration, and sonication were tested in conjunction with FISH. Results from this study show that water samples should be filtered and processed within no more than 10 to 12 h after collection, or else preservation is necessary. The commonly used prefiltration formaldehyde fixation was shown to be inadequate for the rRNA targeted by FISH. However, prefiltration formaldehyde fixation followed by immediate freezing and postfiltration paraformaldehyde fixation yielded highly consistent cell abundance estimates even after 96 days or potentially longer storage. Size fractionation sequential filtration and sonication together enhanced cell abundance estimates by severalfold. Size fractionation sequential filtration effectively separated particle-associated microbial communities from their free-living counterparts, while sonication detached cells from particles or aggregates for more-accurate cell counting using epifluorescence microscopy. Optimization in sonication time is recommended for different specific types of samples. These tested and optimized procedures can be incorporated into a FISH protocol for sampling in deep-sea particle-rich waters.

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Detection and Differentiation of Chlamydiae by Fluorescence In Situ Hybridization

Applied and Environmental Microbiology ◽

10.1128/aem.68.8.4081-4089.2002 ◽

2002 ◽

Vol 68 (8) ◽

pp. 4081-4089 ◽

Cited By ~ 52

Author(s):

Sven Poppert ◽

Andreas Essig ◽

Reinhard Marre ◽

Michael Wagner ◽

Matthias Horn

Keyword(s):

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Single Step ◽

Detection Methods ◽

The Novel ◽

Antibody Staining ◽

Uncultured Bacteria ◽

Direct Fluorescence ◽

Probe Set

ABSTRACT Chlamydiae are important pathogens of humans and animals but diagnosis of chlamydial infections is still hampered by inadequate detection methods. Fluorescence in situ hybridization (FISH) using rRNA-targeted oligonucleotide probes is widely used for the investigation of uncultured bacteria in complex microbial communities and has recently also been shown to be a valuable tool for the rapid detection of various bacterial pathogens in clinical specimens. Here we report on the development and evaluation of a hierarchic probe set for the specific detection and differentiation of chlamydiae, particularly C. pneumoniae, C. trachomatis, C. psittaci, and the recently described chlamydia-like bacteria comprising the novel genera Neochlamydia and Parachlamydia. The specificity of the nine newly developed probes was successfully demonstrated by in situ hybridization of experimentally infected amoebae and HeLa 229 cells, including HeLa 229 cells coinfected with C. pneumoniae and C. trachomatis. FISH reliably stained chlamydial inclusions as early as 12 h postinfection. The sensitivity of FISH was further confirmed by combination with direct fluorescence antibody staining. In contrast to previously established detection methods for chlamydiae, FISH was not susceptible to false-positive results and allows the detection of all recognized chlamydiae in one single step.

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