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Applied and Environmental Microbiology, December 2005, p. 8157-8164, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8157-8164.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

High-Temperature Fluorescent In Situ Hybridization for Detecting Escherichia coli in Seawater Samples, Using rRNA-Targeted Oligonucleotide Probes and Flow Cytometry

Ying Zhong Tang,1 Karina Yew Hoong Gin,2* and Tok Hoon Lim2

Department of Ocean, Earth and Atmospheric Sciences, Old Dominion University, Norfolk, Virginia 23529,1 School of Civil and Environmental Engineering, Nanyang Technological University, Block N1, 50 Nanyang Avenue, Singapore 6397982

Received 1 June 2005/ Accepted 4 August 2005

Fluorescence in situ hybridization (FISH) is a widely used method to detect environmental microorganisms. The standard protocol is typically conducted at a temperature of 46°C and a hybridization time of 2 or 3 h, using the fluorescence signal intensity as the sole parameter to evaluate the performance of FISH. This paper reports our results for optimizing the conditions of FISH using rRNA-targeted oligonucleotide probes and flow cytometry and the application of these protocols to the detection of Escherichia coli in seawater spiked with E.coli culture. We obtained two types of optimized protocols for FISH, which showed rapid results with a hybridization time of less than 30 min, with performance equivalent to or better than the standard protocol in terms of the fluorescence signal intensity and the FISH hybridization efficiency (i.e., the percentage of hybridized cells giving satisfactory fluorescence intensity): (i) one-step FISH (hybridization is conducted at 60 to 75°C for 30 min) and (ii) two-step FISH (pretreatment in a 90°C water bath for 5 min and a hybridizing step at 50 to 55°C for 15 to 20 min). We also found that satisfactory fluorescence signal intensity does not necessarily guarantee satisfactory hybridization efficiency and the tightness of the targeted population when analyzed with a flow cytometer. We subsequently successfully applied the optimized protocols to E. coli-spiked seawater samples, i.e., obtained flow cytometric signatures where the E. coli population was well separated from other particles carrying fluorescence from nonspecific binding to probes or from autofluorescence, and had a good recovery rate of the spiked E. coli cells (90%).

* Corresponding author. Mailing address: School of Civil and Environmental Engineering, Nanyang Technological University, Block N1, 50 Nanyang Avenue, Singapore 639798. Phone: 65 6790 4775. Fax: 65 6791 0676. E-mail: cyhgin{at}ntu.edu.sg.

Applied and Environmental Microbiology, December 2005, p. 8157-8164, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8157-8164.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.





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