Development of a vital fluorescent staining method for monitoring bacterial transport in subsurface environments

Previous bacterial transport studies have utilized fluorophores which have been shown to adversely affect the physiology of stained cells. This resear ch was undertaken to identify alternative fluorescent stains that do not ad versely affect the transport or viability of bacteria. Initial work was per formed with a groundwater isolate, Comamonas sp. strain DA001, Potential co mpounds were first screened to determine staining efficiencies and adverse side effects. 5-(And 6-)-carboxyfluorescein diacetate, succinimidyl ester ( CFDA/SE) efficiently stained DA001 without causing undesirable effects on c ell adhesion or viability. Members of many other gram-negative and gram-pos itive bacterial genera were also effectively stained with CFDA/SE. More tha n 95% of CFDA/SE-stained Comamonas sp. strain DA001 cells incubated in arti ficial groundwater (under no-growth conditions) remained fluorescent for at least 28 days as determined by epifluorescent microscopy and flow cytometr y. No differences in the survival and culturability of CFDA/SE-stained and unstained DA001 cells in groundwater or saturated sediment microcosms were detected. The bright, yellow-green cells were readily distinguished from au tofluorescing sediment particles try epifluorescence microscopy, A high thr oughput method using microplate spectrofluorometry was developed, which had a detection limit of mid-10(5) CFDA-stained cells/ml; the detection limit for flow cytometry was on the order of 1,000 cells/ml. The results of labor atory-scale bacterial transport experiments performed with intact sediment cores and nondividing DA001 cells revealed good agreement between the aqueo us cell concentrations determined by the microplate assay and those determi ned by other enumeration methods. This research indicates that CFDA/SE is v ery efficient for labeling cells for bacterial transport experiments and th at it may be useful for other microbial ecology research as well.