Development of a Vital Fluorescent Staining Method for Monitoring Bacterial Transport in Subsurface Environments

Abstract

Previous bacterial transport studies have utilized fluorophores which have been shown to adversely affect the physiology of stained cells. This research was undertaken to identify alternative fluorescent stains that do not adversely affect the transport or viability of bacteria. Initial work was performed with a groundwater isolate, Comamonas sp. strain DA001. Potential compounds 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 cell adhesion or viability. Members of many other gram-negative and gram-positive bacterial genera were also effectively stained with CFDA/SE. More than 95% of CFDA/SEstained Comamonas sp. strain DA001 cells incubated in artificial groundwater (under no-growth conditions) remained fluorescent for at least 28 days as determined by epifluorescent microscopy and flow cytometry. 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 autofluorescing sediment particles by epifluorescence microscopy. A high throughput 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 laboratory-scale bacterial transport experiments performed with intact sediment cores and nondividing DA001 cells revealed good agreement between the aqueous cell concentrations determined by the microplate assay and those determined by other enumeration methods. This research indicates that CFDA/SE is very efficient for labeling cells for bacterial transport experiments and that it may be useful for other microbial ecology research as well.

Simultaneous Transport of Two Bacterial Strains in Intact Cores from Oyster, Virginia: Biological Effects and Numerical Modeling

“…Isolation and identification of adhesion-deficient variants of these strains have been described elsewhere (12,17). The inocula were grown and radiolabeled as described previously (17,18). The DA001 cells injected into cores HS54 and SG11 were labeled with 35 S. The DA001 and OYS2-A cells injected into SG48 were labeled with 35 S and 14 C, respectively.…”

Bacteriocin-producing strains of Lactobacillus plantarum inhibit adhesion of Staphylococcus aureus to extracellular matrix: quantitative insight and implications in antibacterial therapy

“…Prior to the binding assays, the L. plantarum strains were labelled with the fluorescent dye cFDA-SE, based on previous investigations, which established that cFDA labelling of LAB strains retained the viability of the cells and was also suitable for performing in vitro adhesion assays (Fuller et al, 2000;Mukherjee et al, 2013). For the adhesion assays, L. rhamnosus GG was selected as a standard probiotic strain (Doron et al, 2005) for comparative evaluation.…”

dksA Is Required for Intercellular Spread of Shigella flexneri via an RpoS-Independent Mechanism

“…5). The 5-(and 6)-carboxytetramethylrhodamine succinimidyl ester probe used in this study forms peptide bonds with cellular proteins located on the outer surfaces of eukaryotic cell plasma membranes (10,32). The probe does not cross the plasma membrane and thus does not label intracellular proteins.…”