Field-scale evaluation of CFDA/SE staining coupled with multiple detection methods for assessing the transport of bacteria in situ

Abstract

Abstract This research was undertaken to evaluate staining with the fluorescent compound CFDA/SE (5-(and-6-)-carboxyfluorescein diacetate, succinimidyl ester) coupled with multiple cell detection methodologies as a means to monitor bacterial transport during field-scale experiments. Stained cells of Comamonas sp. strain DA001 were injected into a shallow, aerobic aquifer in Oyster, VA, USA. Groundwater samples analyzed in the laboratory using epifluorescence microscopy, flow cytometry, ferrographic separation, and microplate spectrofluorometry yielded comparable concentrations of CFDA/SE-stained DA001 cells, although each method had a different effective lower limit of detection. Determination of cell concentrations in the field using microplate spectrofluorometry allowed the track of the bacterial plume to be monitored in near-real time, but produced results that were not as accurate as laboratory analyses. The CFDA/SE stain was well retained in the cells over a 5-month period. Normal handling of samples under fluorescent and incandescent lighting did not significantly affect sample integrity, but exposure to sunlight resulted in rapid loss of total and per cell fluorescence. The combination of CFDA/SE staining and multiple detection methods was demonstrated as an effective means to study bacterial transport in groundwater.