AME 25:65-74 (2001)  -  doi:10.3354/ame025065

Double-stranded DNA measurement in lakes with the fluorescent stain PicoGreen and the application to bacterial bioassays

James B. Cotner*, Megan L. Ogdahl, Bopaiah A. Biddanda

Department of Ecology, Evolution and Behavior, University of Minnesota, 1987 Upper Buford Circle, St. Paul, Minnesota 55108, USA

ABSTRACT: We used the double-stranded DNA (dsDNA) stain PicoGreen with a microplate fluorometer to measure bacterial abundance, biomass, and growth rates in lake water. PicoGreen fluorescence units (PFU) correlated closely with bacterial abundance measured with acridine orange direct counts (R2 = 0.95 to 0.98) as well as bacterial biomass inferred from image analysis (R2 = 0.95 to 0.98) in eutrophic waters. PicoGreen fluorescence increased proportionally with bacterial size, indicating that it was a good indicator of biomass as well as abundance. In oligotrophic Lake Superior, there was a weaker, but significant (p < 0.05) correlation between PFU and abundance (R2 = 0.52) as well as PFU and biomass (R2 = 0.54). Growth rate measurements in bottle cultures showed a similar relationship, with PFU, abundance, and biomass being more tightly correlated in productive waters than in oligotrophic waters. Parallel dilution cultures were performed in microplate wells (nanocosms) and 1 l bottles. The slope of nanocosm fluorescence to bottle fluorescence was ca 1, indicating that nanocosms mimicked bacterial abundance and growth in bottle cultures. Preservation of Escherichia coli with formaldehyde showed that there was an initial loss of dsDNA of about 10 to 15% with little subsequent loss for 2 wk, indicating that bacteria from growth experiments conducted in the field could be preserved for subsequent analysis in the laboratory. Bacterial cellular dsDNA content in 3 Minnesota lakes varied between 0.6 and 6.2 fg cell-1 and was highest in the most eutrophic and most rapidly growing bacterial community, i.e., the more eutrophic lakes. These results suggest that the PicoGreen method is effective for growth bioassays in systems with moderate to high levels of bacterial productivity. PicoGreen coupled with a microplate fluorescence reader is a promising method for determining bacterial biomass and growth rates, especially in meso- to eutrophic systems.

KEY WORDS: dsDNA · Bioassay · Bacterial growth rate · PicoGreen · Biomass · Abundance

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