Isolation of viruses (Betteral et al., 2005).
A clonal pathogen isolate was obtained from the viral concentrate generated at station 744 in April 2003. The isolation was performed by dilution of the virus in microtiter plates containing 200 µL of exponentially growing stock culture of Chaetoceros c.f. gracilis. Plates were incubated for 10 days and the contents of the most diluted wells showing lysis were transferred to new microtiter plates. This procedure was repeated three times (Sandaa et al. 2001). The resultant lysate was filtrated through 0.2 µm pore-size polycarbonate membrane filters and designated as the clonal pathogen suspension CspNIV (Chaetoceros nuclear inclusion virus). Serial transfers of lysate were performed 5 times to amplify viruses that specifically attack C. cf gracilis.
Phytoplankton Chl a biomass
Chl a concentrations were determined for unfractionated, <20 mm, and <3 mm surface water samples, with size-fractionation accomplished by gravity filtration through 47 mm Nucleopore filters. Duplicate 100 ml samples for each fraction were vacuum filtered (<150 mm Hg) onto 25 mm Whatman GF/C filters and chl a extracted in 90% acetone for 24 h at 4oC in the dark. Chl a concentration was determined fluorometrically using a Turner Designs 10-AU fluorometer.
Simulated in-situ incubations
Primary production (PP) was measured as assimilation of 14C-sodium bicarbonate using deck incubators cooled with flowing surface water. Surface water was collected using a 8-L bucket and dispensed to 250-ml glass incubation bottles, with 3 - 5 mCuries of 14C-sodium bicarbonate (ICN Pharmaceuticals, Inc.) added to each bottle. Total activity added was determined from a time-zero aliquot withdrawn from one of the incubation bottles. Neutral density screens providing 55, 33, 20, 9, 4, and 1% transmission were used to attenuate sunlight during incubations. Clear bottles gave 100% transmission and all data were adjusted for dark uptake in an opaque bottle. Incubations were run for 24 h to determine net 14C-PP, with endpoints at 4-6 h used to estimate gross 14C-PP. At the end of the incubation periods, 50-ml aliquots of whole sample were vacuum filtered (<150 mm Hg) onto 25 mm Whatman GF/C filters. At the 24-h endpoint, additional 50-ml aliquots were sequentially passed through 20 mm Nucleopore, 3 mm Nucleopore, and Whatman GF/C filters to partition PP as >20 mm, <20 >3 mm, and <3 mm fractions. Filters were rinsed several times with filtered water of equivalent salinity as the samples and gently acidified with 0.01 N HCl to remove residual inorganic label. Activities were determined on a Packard Instruments Tri-Carb 1600TR liquid scintillation counter. Total CO2 was determined using a Li-Cor LI-7000 CO2/H2O Analyzer. The depth of the euphotic layer was estimated as 1.5/Secchi depth as recommended by Harding et al. (2002).
Phytoplankton counts.
The abundance of microphytoplankton was determined for stations located along the main axis of the Bay. At each station, 125 mL of surface water was preserved using acid Lugol’s fixative, 1% final concentration. Preserved samples were enumerated using 5 ml Zeiss settling chambers and an inverted microscope with phase contrast optics (Leitz Diavert or Olympus IX51; 250X and 400X, respectively). For each sample, randomly selected fields (20 when using the Leitz Diavert; 35 for the Olympus IX51) were examined, and all cells enumerated to genus or species. As a result, ~ 0.1 mL of sample was counted for each sample, giving a detection level of ~10 cells mL-1.
