Determination of Active Marine Bacterioplankton: a Comparison of Universal 16S rRNA Probes, Autoradiography, and Nucleoid Staining.

Applied and Environmental Microbiology
M Karner, J A Fuhrman

Abstract

We compared several currently discussed methods for the assessment of bacterial numbers and activity in marine waters, using samples from a variety of marine environments, from aged offshore seawater to rich harbor water. Samples were simultaneously tested for binding to a fluorescently labeled universal 16S rRNA probe; (sup3)H-labeled amino acid uptake via autoradiography; nucleoid-containing bacterial numbers by modified DAPI (4(prm1),6-diamidino-2-phenylindole) staining; staining with 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), a compound supposed to indicate oxidative cell metabolism; and total bacterial counts (classical DAPI staining), taken as a reference. For the universal-probe counts, we used an image intensifying and processing system coupled to the epifluorescence microscope. All of the above-mentioned methods yielded lower cell counts than DAPI total counts. Universal-probe counts averaged about half of the corresponding DAPI count and were highly correlated to autoradiography counts (r(sup2) = 0.943; n = 7). Nucleoid-containing cell counts could be lower than DAPI counts by as much as 1 order of magnitude but sometimes matched autoradiography or probe counts. CTC counts were 2 orders of magnitude below DAPI co...Continue Reading

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