AUTHOR:
Dr. Evelyn Sherr
Oregon State University
College of Oceanic and Atmospheric Sciences (COAS),104 Oceanography Admin Bldg
Corvallis, OR, 97331-5503
Tel: 541-Phone: 737-4369
e-mail: sherre@coas.oregonstate.edu

PI: Dr. Yvette Spitz, COAS, Oregon State University

NSF GRANT: OPP 0435956

DATE SET OVERVIEW:
This data set contains flow cytometry counts of bacteria, phytoplankton and nutrient concentrations of seawater from Niskin bottle casts taken from the RV Annika Marie during August - September, 2005 in the Chukchi and Beaufort Seas, off the coast of Barrow, Alaska. Each data set presents abundances of heterotrophic bacterial cells: total, low, high, and very high nucleic acid content; coccoid cyanobacteria (Synechococcus, SYN); small (<5 um) photosynthetic eukaryotes (PEUK); large diatoms; and chlorophyll fractions. Nutrients reported include phosphate, ammonia, nitrite, nitrate plus nitrite nitrogen, and silicate. Supporting oceanographic data include station, position, water depth, and temperature and salinity values from the corresponding CTD cast.

METHODS:
Water samples were obtained from 5-l Niskin bottles cast from the RV Annika Marie along designated offshore/onshore sample lines. For flow cytometry samples, 3 ml aliquots were pipetted into 4 ml cryovials and preserved with 0.2% (v/v) final concentration of freshly made paraformaldehyde. The samples were gently mixed and let sit in the dark at room temperature for 10 minutes before quick-freezing and storage in liquid nitrogen. On return of samples to the lab, the cryovials were stored at -80 degrees C until flow cytometric analysis was performed. Subsamples of seawater for nutrient analyses were collected in acid-washed NalgeneTM 60 ml HDPE bottles and immediately frozen at -20 degrees C for later analysis at Oregon State University (method description in Sherr et al. 2005). Samples for chlorophyll a were filtered onto Whatman GF/F glass fiber filters. Subsamples were pre-screened through 10 um mesh to separately determine amount of chlorophyll in the less than 10 um fraction. Following 24-h extraction in 90% acetone at -20 degree C, chlorophyll concentrations were determined fluorometrically with a Turner DesignsTM 10-AU fluorometer that had been calibrated with purified chlorophyll-a (Sigma Co.).

Flow cytometric analysis was done in the Sherrs' laboratory at OSU. FCM samples were thawed and kept on ice in a dark container until run on a Becton-Dickinson FACSCaliber flow cytometer with a 488 nm laser. For enumeration of small sized phytoplankton, 500 ul subsamples were processed as described in Sherr et al. (2005). Populations of coccoid cyanobacteria (Synechococcus or SYN) and of photosynthetic eukaryotes (PEUK) were distinguished by differences in fluorescence in orange (cyanobacteria) and in red (eukaryotic phytoplankton) wavelengths. It was previously determined that 75% to 85% of the red-fluorescing cells in the PEUK region of our cytograms are < 5 um in size (Sherr et al. 2005). For heterotrophic bacteria, 250 ul subsamples were diluted with 250 ul of DiW, and stained with SYBR Green I and potassium citrate for 15 min, following the protocol of Marie et al. (1997). Bacterial counts were made during a three minute sample run at low flow rate. Regions were established in cytograms of side scatter and green fluorescence to define bacterial cells with high nucleic acid content (HNA) and low nucleic content (LNA). The cytogram for each sample was individually inspected, and HNA and LNA regions manually moved to conform to the appropriate areas of the bacterial dot-plot. Mean cell-specific SYBR fluorescence was obtained for total bacteria and for HNA and LNA cells, along with abundance of cells within each group. Logical gating in Becton-Dickinson Cell Quest software was used to exclude coccoid cyanobacteria, based on orange fluorescence, from the abundance counts of heterotrophic bacteria. Each subsample was spiked immediately before processing with a known amount of either 3.0 um (for phytoplankton) or 1.0 um (for bacteria) Polysciences Fluoresbrite yellow-green beads from respective stock solutions of beads that had been precalibrated with Becton-Dickinson True-Count beads. The number of beads enumerated in each sample run was used to accurately determine the sample volume processed and thus the abundances of SYN, PEUK, and bacteria.

REFERENCES:
Marie, D., Partensky, F., Jacquet, S., Vaulot, D., 1997. Enumeration and cell cycle analysis of natural populations of marine picoplankton by flow cytometry using the nucleic acid stain SYBR Green I. Applied and Environmental Microbiology 63, 186-193.

Sherr, E.B., B.F. Sherr, & P.A. Wheeler, 2005. Distribution of coccoid cyanobacteria and small eukaryotic phytoplankton in the upwelling ecosystem off the Oregon coast during 2001 and 2002. Deep-sea Res. II 52:317-330.