Dataset: flow cytometer
Deployment: W0212A

Flow Cytometry Methods:
For flow cytometry samples, 3 ml aliquots were pipetted into 4 ml cryovials and preserved with 0.2% (w/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. Duplicate subsamples of seawater for nutrient analyses were collected in acid-washed NalgeneTM 60 ml HDPE bottles and immediately frozen at -20 degrees C for nutrient analysis. For determination of chlorophyll-a concentration, subsamples of whole seawater were vacuum filtered onto 25 mm Whatman GF/F filters, and the filters stored frozen in glass VacutainerTM tubes (-20 degrees C) until processed on shore.

Sample analysis
Nutrient and chlorophyll-a: Analysis of nutrient and chlorophyll-a concentrations during the GLOBEC cruises were carried out by the NEP-LTOP service group; results are available as a data report (Wetz et al. 2004). Analyses for concentration of nitrate plus nitrite, phosphate and silicic acid (silicate) were performed using a hybrid Technicon AutoAnalyzerII(tm) and Alpkem RFA300(tm) system following protocols modified from Gordon et al. (1995). Standard curves with four different concentrations were run daily at the beginning and end of each run. Fresh standards were made prior to each run by diluting a primary standard with low-nutrient surface seawater. Deionized water (DiW) was used as a blank, and triplicate DiW blanks were run at the beginning and end of each run, to correct for any baseline shifts. In this protocol, the coefficients of variation for duplicates at low nutrient concentrations are typically < 1% (Fleischbein et al., 1999); while at high nutrient concentrations, coefficients of variation are 2-3 % for nitrate and silicate (Corwith and Wheeler 2002). Chlorophyll was extracted for > 12 h in the dark at -20 °C (Parsons et al., 1984) using either 90% acetone or 95% methanol as the solvent. Fluorescence was measured with a Turner Designs(TM) 10-AU fluorometer that had been calibrated with purified chlorophyll-a (Sigma Co.).

Flow cytometric analysis of cell abundances: In the laboratory, 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 µl 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. We have previously determined that 75% to 85% of the red-fluorescing cells in the PEUK region of our cytograms are < 5 µm in size (Sherr et al. 2005). Prochlorococcus appears on the red-fluorescence axis of the flow cytograms to the left of the PEUK region, which was confirmed by a few observations of Prochlorococcus in cytograms of samples collected at a basin station in September 2001.

For heterotrophic bacteria, 250 µl subsamples were diluted with 250 µl 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 µm (for phytoplankton) or 1.0 µm (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.

Notes:
The Thesaurus does not contain the noted fields (N1-N6). The data for N1, N2, N3, N5 and N6 are provided as cells per ml (rather than the thesaurus recommended units of cells per cubic meter). Note N4, the "FL_ratio_HNAtoLNA" is the ratio of fluorescence of High Nucleic Acid particles to Low Nucleic Acid particles in that sample.

References cited:
Corwith, H.L., Wheeler, P.A., 2002. El Nino related variations in nutrient and chlorophyll distributions off Oregon. Progress in Oceanography 54, 361-380. Fleischbein, J., Hill, J., Huyer, A., Smith, R.L., Wheeler P.A., 1999. Hydrographic data from the GLOBEC long-term observation program off Oregon, 1997 and 1998. Data Report 172, Ref. 99-1. OSU.

Gordon, L. I., Jennings, J. C., Ross, A. A., Krest, J. M., 1995. A suggested protocol for continuous flow automated analysis of seawater nutrients (phosphate, nitrate, nitrite and silicic acid) in the WOCE Hydrographic Program and the Joint Global Ocean Fluxes Study, WOCE Operations Manual, WOCE Report No. 68/91. Revision 1995.

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.

Parsons, T. R., Maita, Y., Lalli, C.M. 1984. A Manual of Chemical and Biological Methods for Seawater Analysis, 173 pp., Pergamon Press, Oxford, UK.

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

Wetz, M.S., J. Hill, H. Corwith, and P. A. Wheeler. 2004. Nutrient and Extracted Chlorophyll Data from the GLOBEC Long-Term Observation Program, 1997-2004 Data Report 193

Figure 1. Station locations for the five GLOBEC NEP-LTOP sampling lines off Oregon and northern California: NH - Newport Hydroline off Newport, Oregon; HH - Heceta Head line off Heceta Head, Oregon; FM - Five Mile Point line 20 km south of Coos Bay, Oregon; RR - Rogue River line off the mouth of the Rogue River at Gold Beach, Oregon; and CC - Crescent City line off of Crescent City California just south of the Oregon/California border indicated on the map. Depth contours of 50 m, 200 m, and 2000 m are shown.

<p>For flow cytometry samples, 3 ml aliquots were pipetted into 4 ml cryovials and preserved with 0.2% (w/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 &deg;C until flow cytometric analysis was performed. Duplicate subsamples of seawater for nutrient analyses were collected in acid-washed NalgeneTM 60 ml HDPE bottles and immediately frozen at -20 &deg;C for nutrient analysis. For determination of chlorophyll-a concentration, subsamples of whole seawater were vacuum filtered onto 25 mm Whatman GF/F filters, and the filters stored frozen in glass VacutainerTM tubes (-20 &deg;C) until processed on shore.</p>
<p>Nutrient and chlorophyll-a: Analysis of nutrient and chlorophyll-a concentrations during the GLOBEC cruises were carried out by the NEP-LTOP service group, results are available as a data report (Wetz et al. 2004). Analyses for concentration of nitrate plus nitrite, phosphate and silicic acid (silicate) were performed using a hybrid Technicon AutoAnalyzerII(tm) and Alpkem RFA300(tm) system following protocols modified from Gordon et al. (1995). Standard curves with four different concentrations were run daily at the beginning and end of each run. Fresh standards were made prior to each run by diluting a primary standard with low-nutrient surface seawater. Deionized water (DiW) was used as a blank, and triplicate DiW blanks were run at the beginning and end of each run, to correct for any baseline shifts. In this protocol, the coefficients of variation for duplicates at low nutrient concentrations are typically &lt; 1% (Fleischbein et al., 1999), while at high nutrient concentrations, coefficients of variation are 2-3 % for nitrate and silicate (Corwith and Wheeler 2002). Chlorophyll was extracted for &gt; 12 h in the dark at -20 &deg;C (Parsons et al., 1984) using either 90% acetone or 95% methanol as the solvent. Fluorescence was measured with a Turner Designs(TM) 10-AU fluorometer that had been calibrated with purified chlorophyll-a (Sigma Co.).</p>
<p>Flow cytometric analysis of cell abundances: In the laboratory, 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 &micro;l 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. We have previously determined that 75% to 85% of the red-fluorescing cells in the PEUK region of our cytograms are &lt; 5 &micro;m in size (Sherr et al. 2005). Prochlorococcus appears on the red-fluorescence axis of the flow cytograms to the left of the PEUK region, which was confirmed by a few observations of Prochlorococcus in cytograms of samples collected at a basin station in September 2001.</p>
<p>For heterotrophic bacteria, 250 &micro;l subsamples were diluted with 250 &micro;l 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.</p>
<p>Each subsample was spiked immediately before processing with a known amount of either 3.0 &micro;m (for phytoplankton) or 1.0 &micro;m (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.</p>