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(B) Scheme of the Eulerian approach used in this study, with the coastal parallel current vector y and the normal to the coast vector ... Prokaryotic Cell Number and Activity Measurements Prokaryotic cells were counted using a flow cytometer (FacsCalibur, Becton Dickinson, Heidelberg, Germany) following the method of Gasol et al. (1999). Calculations were performed using the software program ��Cell Quest Pro,�� plotting the emission fluorescence of SYBR Green I (488 nm) vs. the side scatter. Picocyanobacteria were similarly counted on the basis of their signature in a plot of orange (FL2) vs. red (FL3) fluorescence. The incorporation of 3H-leucine (140 Ci mmol-1) was measured to estimate heterotrophic bacterial productivity in 10-mL water samples. Triplicate samples were incubated at a final concentration of 100 nM for at least 1 h at the in situ temperature in the dark. Incorporation was stopped by fixing the cells with formaldehyde (5% v/v). A fourth sample, serving as a blank, was fixed for at least 10 min prior to the addition of the radioactively labeled substrate. The samples were filtered onto 0.22-��m polycarbonate filters (Millipore), which were then placed in 4 mL of scintillation cocktail. The incorporated substrate was counted in a scintillation counter (Packard). Bacterial carbon production was calculated from 3H-leucine incorporation according to Simon and Azam (1987), using a leucine mol% value of 7.3 and a carbon conversion factor of 0.86. Catalyzed Reporter Deposition-Fluorescence in Situ Hybridization (CARD-FISH) and Cell Counting Catalyzed reporter deposition-fluorescence in situ hybridization was carried out using the protocol of Pernthaler et al. (2002), with modifications. Before digestion, the filters were incubated in 0.01 M HCl for 10 min to inactivate undesirable peptidases. Bacterial staining was carried out using the horseradish-peroxidase-labeled FISH probes EUBI-III (Daims et al., 1999), VER47 (Buckley and Schmidt, 2001), and SAR11-486 (Fuchs et al., 2005). For signal amplification, tyramide labeled with the fluorescent dye carboxyfluoresceine was used. Total cell numbers were estimated by 4��,6��-diamidino-2-phenylindole (DAPI)- staining of the probe-labeled samples. DAPI and EUB I-III stained cells were counted using an automated system coordinated with the epifluorescence microscope AxioImager (Zeiss, Germany) and in combination with a Colibri LED unit and a charge-coupled device camera (AxioCam MRm, Zeiss, Germany). Images were acquired using a 100�� Plan-Apochromat objective (Zeiss) and the Zeiss multi-band filter set 62HE. Automatic SB203580 chemical structure processing of the samples was achieved with the Visual Basic for Application module of AxioVision 4.6 (Zeiss, Germany) together with comprised automated sample recognition and localization, multichannel image acquisition, image processing, and cell counting routines (Zeder and Pernthaler, 2009).