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Seawater samples (500 mL) for microscopical analysis were gently collected from the CTD and immediately preserved for slide preparation according to a modified protocol of Sherr and Sherr (1993). The samples were first preserved with 260 uL of alkaline Lugol\u2019s solution, immediately followed by 10 mL of buffered formalin and 500 uL of sodium thiosulfate, with gentle mixing between each addition. Preserved samples were shielded from light and left to rest at room temperature for 1 h. After the rest period, 1 mL of proflavin (0.33% w/v) was added and the samples were stored in the dark for an additional hour. Just prior to filtration, the preserved samples were stained with 1 mL of DAPI (0.01 mg mL-1) and immediately transferred to the filtration manifold. A 50-mL aliquot (small volume, SV) of the sample was filtered through a 25-mm black polycarbonate filter with 0.8-um pore size, and the remaining 450 mL aliquot (large volume, LV) was filtered through a 25-mm black polycarbonate filter with 8.0-um pores. A 10-mm nylon backing filter was placed under all polycarbonate filters to promote even cell distribution, and filtered the samples under gentle vacuum pressure (<100 mm Hg). Each filter was then mounted onto glass slides with one drop of Type DF immersion oil and a No. 2 cover slip, and the prepared slides were frozen at -80\u00b0C for later analysis in the lab.
Cell abundance estimates of eukaryotic phytoplankton by taxa and size-class, based on epifluorescence microscopy. Samples were collected on the MV1008 cruise in the Costa Rica Dome (CRD) region of the Eastern Tropical Pacific Ocean.
Slides were digitally imaged using a Zeiss Axiovert 200 M inverted compound microscope equipped for high-throughput epifluorescence microscopy with a motorized focus drive, stage, objective and filters. Digital images were acquired with a Zeiss AxioCam MRc black and white 8-bit CCD camera. SV samples (50 mL aliquots) were viewed at 630X magnification, and LV samples (450 mL aliquots) were viewed at 200X magnification. A minimum of 20 random positions were imaged for each slide, with each position consisting of three to four fluorescent channels: Chl a, DAPI, FITC (SV and LV samples) and phycoerythrin (SV samples only).
\nImages were analyzed using ImagePro software to semi-automate the enumeration of eukaryotic cells larger than 1.5 um in length (Taylor et al., 2012). Whenever possible, 20 positions and >300 cells were counted for each slide. Each cell was manually identified and grouped into identifiable taxonomic groups. Autotrophic cells were identified by the presence of chlorophyll a (red autofluorescence under blue light excitation), generally clearly packaged in defined chloroplasts, and obvious heterotrophic cells with recently consumed prey were manually excluded from the autotroph classification.
\nBCO-DMO Processing Notes:
\n- size_class and taxon columns were transposed from columns into rows.