Determination of active marine bacterioplankton: A comparison of universal 16S rRNA probes, autoradiography, and nucleoid staining

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; H-3-labeled amino acid uptake via autoradiography; nucleoid-containing bacterial numbers by modified DAPI (4',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(2) = 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 counts, Universal 16S rRNA probe counts correlated well,vith autoradiography results, indicating a population with at least minimal metabolic activity, The greater variability of the nucleoid-containing cell counts calls for further investigation of the processes involved, and CTC counts were well below the range of the other methods tested.