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

Fuller, M.E., B.J. Mailloux, S.H. Streger, J.A. Hall, P. Zhang, W.P. Kovacik, S. Vainberg, W.P. Johnson, T.C. Onstott, and M.F. de Flaun, 2004, "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, "Appl. Environ. Microbiol. 70: 1680-1687.

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.

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