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Applied and Environmental Microbiology, March 2004, p. 1680-1687, Vol. 70, No. 3
0099-2240/04/$08.00+0 DOI: 10.1128/AEM.70.3.1680-1687.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Application of a Vital Fluorescent Staining Method for Simultaneous, Near-Real-Time Concentration Monitoring of Two Bacterial Strains in an Atlantic Coastal Plain Aquifer in Oyster, Virginia
Mark E. Fuller,1* Brian J. Mailloux,2,
Sheryl H. Streger,1 James A. Hall,2,
Pengfei Zhang,3,
William P. Kovacik,4,|| Simon Vainberg,1 William P. Johnson,3 Tullis C. Onstott,2 and Mary F. DeFlaun1,#
Envirogen, Inc., Princeton Research Center, Lawrenceville, New Jersey 08648,1 Department of Geosciences, Princeton University, Princeton, New Jersey 08544,2 Department of Geology and Geophysics, University of Utah, Salt Lake City, Utah 84112,3 Division of Biological Sciences, University of Montana, Missoula, Montana 598124
Received 26 June 2003/ Accepted 25 November 2003
Two differentially labeled bacterial strains were monitored in near-real time during two field-scale bacterial transport experiments in a shallow aquifer in July 2000 and July 2001. Comamonas sp. strain DA001 and Acidovorax sp. strain OY-107 were grown and labeled with the vital fluorescent stain TAMRA/SE (5 [and -6]-carboxytetramethylrhodamine, succinimidyl ester) or CFDA/SE (5 [and -6]-carboxyfluorescein diacetate, succinimidyl ester). Fluorescently labeled cells and a conservative bromide tracer were introduced into a suboxic superficial aquifer, followed by groundwater collection from down-gradient multilevel samplers. Cells were enumerated in the field by microplate spectrofluorometry, with confirmatory analyses for selected samples done in the laboratory by epifluorescence microscopy, flow cytometry, and ferrographic capture. There was general agreement in the results from all of the vital-stain-based enumeration methods, with differences ranging from <10% up to 40% for the analysis of identical samples between different tracking methods. Field analysis by microplate spectrofluorometry was robust and efficient, allowing thousands of samples to be analyzed in quadruplicate for both of the injected strains. The near-real-time data acquisition allowed adjustments to the predetermined sampling schedule to be made. The microplate spectrofluorometry data sets for the July 2000 and July 2001 experiments allowed the transport of the injected cells to be related to the site hydrogeology and injection conditions and enabled the assessment of differences in the transport of the two strains. This near-real-time method should prove effective for a number of microbial ecology applications.
* Corresponding author. Mailing address: Shaw Environmental, Inc., Princeton Research Center, 4100 Quakerbridge Rd., Lawrenceville, NJ 08648. Phone: (609) 936-9300. Fax: (609) 936-9221. E-mail: Mark.Fuller{at}shawgrp.com.
Present address: Columbia Earth Institute, Columbia University, New York, NY 10115.
Present address: Carnegie Institution of Washington, Washington, DC 20015.
Present address: Department of Earth and Atmospheric Sciences, City College of New York, New York, NY 10031.
|| Present address: Pacific Northwest National Laboratory, Richland, WA 99352.
# Present address: GeoSyntec Consultants, Princeton, NJ 08540.
Applied and Environmental Microbiology, March 2004, p. 1680-1687, Vol. 70, No. 3
0099-2240/04/$08.00+0 DOI: 10.1128/AEM.70.3.1680-1687.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
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