Summary

Picophytoplankton populations and non-pigmented prokaryotes sampled within the California Current Ecosystem (CCE) are fixed in the field by the addition of paraformaldehyde and, in the laboratory, stained with Hoechst 33342 (a DNA specific dye). The cells are enumerated by an Altra flow cytometer and converted to biomass estimates (Garrison et al., 2000; Brown et al., 2008).

Methods

1. Principle

Flow cytometric analyses of picoplankton populations (Prochlorococcus, Synechococcus, P-Euk and H-Bact) are preserved with paraformaldehyde, frozen in liquid nitrogen, and stored at -80°C. Prior to analysis, batches of thawed samples are stained with Hoechst 33342 at room temperature in the dark for 1 h (Monger and Landry, 1993). This method increases substantially the precision of bacterial counts, relative to epifluorescence microscopy. Hoechst 33342 is used because binding to DNA substantially alters its fluorescence spectrum, which facilitates separation of cell fluorescence signals from the background fluorescence of the unbound dye. Aliquots are analyzed using a Beckman-Coulter EPICS Altra flow cytometer with a Harvard Apparatus syringe pump for volumetric sample delivery. Simultaneous (co-linear) excitation of the plankton is provided by two argon ion lasers, tuned to 488 nm (1 W) and the UV range (200 mW) to distinguish three major populations of photoautotrophs (Prochlorococcus, Synechococcus, and pico-eukaryotes) and the assemblage of heterotrophic prokaryotes collectively referred to as H-Bact. This latter category includes cells from the domain Archaea, as well as true Bacteria. The optical filter configuration distinguishes populations on the basis of chlorophyll a (red fluorescence, 680 nm), phycoerythrin (orange fluorescence, 575 nm), DNA (blue fluorescence, 450 nm), and forward and 90° side-scatter signatures. Calibration beads are used in each sample to standardize fluorescence and scatter parameters. Raw data (listmode files) are processed using the software FlowJo (Treestar Inc., www.flowjo.com). Prochlorococcus (PRO) and Synechococcus (SYN) abundances from FCM analyses are converted to biomass estimates using mixed-layer estimates of 32 and 101 fg C cell-1, respectively (Garrison et al., 2000; Brown et al., 2008). These cell biomass values correspond to mean equivalent spherical diameters (ESD) of 0.65 and 0.95 µm, respectively, for PRO and SYN, assuming cell carbon densities of 0.22 pg C µm-3. Carbon estimates for H-Bact are made using estimates of 11 fg C cell-1 (Garrison et al., 2000). Cells enumerated by this technique as pico-eukaryotic cannot typically all be assumed to fit the rigorous definition of being ≤ 2-µm in size (i.e., true "pico"). We therefore provide only an abundance count of this category by flow cytometry and make carbon estimates of <2-µm eukaryotes based on cells enumerated and sized by epifluorescence microscopy (See Nano- and Microplankton).

2. Sampling

2.1

Picoplankton and bacteria are sampled from 3 to 8 depths for each of the 66 CalCOFI stations located in the CCE. Seawater from the Niskin bottle is collected into a 600-ml sample bottle. When sampling, make sure the flow from the valves is low since no sample tubing is used.

2.2

A 2-ml subsample is drawn from the sample bottle and placed into a properly labeled 2-ml cryovial, and 100 µl of prefiltered 10% paraformaldehyde is added (0.5% final v/v sample concentration). Samples are left out for 10 min and then stored in liquid nitrogen. At the shore-based laboratory, the samples are stored at -80°C.

2.3

Samples are shipped overnight on dry ice to SOEST Flow Cytometry Facility, University of Hawaii at Manoa for flow cytometry analysis.

3. Analysis

3.1

Prior to analysis, batches of thawed samples are stained with Hoechst 33342 (1 µg ml-1, v/v, final concentration) at room temperature in the dark for 1 h (Monger and Landry, 1993). Calibration beads (0.5- and 1.0-µm yellow-green beads and 0.5-µm UV beads) are used in each sample to standardize fluorescence and scatter parameters. A record of inter-station and cruise-to-cruise variations in bead-normalized fluorescence per cell relative is maintained.

3.2

Aliquots (100 µl) are analyzed using a Beckman-Coulter EPICS Altra flow cytometer with a Harvard Apparatus syringe pump for volumetric sample delivery. Simultaneous (co-linear) excitation of the plankton is provided by two argon ion lasers, tuned to 488 nm (1 W) and the UV range (200 mW). The optical filter configuration distinguishes populations on the basis of chlorophyll a (red fluorescence, 680 nm), phycoerythrin (orange fluorescence, 575 nm), DNA (blue fluorescence, 450 nm), and forward and 90° side-scatter signatures. BF and RF signals are used to distinguish DNA-containing heterotrophic (non-pigmented) from autotrophic (chlorophyll-containing) cells.

Raw data (listmode files) are processed using the software FlowJo (Treestar Inc., www.flowjo.com). Cell abundance estimates are corrected for volume dilutions from added preservative and stain using Microsoft Office Excel software.

4. Calculations

FCM abundance estimates (cells mL-1) for heterotrophic prokaryotes (H-Bact), Prochlorococcus (PRO) and Synechococcus (SYN), are converted to carbon biomass equivalents (µg C L-1) using mixed-layer estimates of 11, 32 and 101 fg C cell-1, respectively (Garrison et al., 2000).

µg C L-1 = A * F * 103 / 109 = A * F / 106


A = abundance (cells mL-1)


F = per cell carbon estimate (fg C cell-1)


 103 is the unit conversion for mL to liter


109 is the unit conversion for fempto (fg C) to micro (µg C)

5. Equipment/Supplies

  • 10L PVC bottle (productivity-clean)
  • Sample collection bottles
  • 1 ml autopipet and sterile tips
  • 2 ml Wheaton Cryle Freestanding sterile externally threaded cryogenic vials (Fisher Cat No. 03-341-18J)
  • Liquid Nitrogen Dewar and -80°C Freezer for storing samples
  • 0.5- and 1.0-µm yellow-green calibration beads and 0.5-µm UV calibration beads (Invitrogen Corp. or Polysciences Inc.)
  • EPICS Altra flow cytometer with a Harvard Apparatus syringe pump (Beckman-Coulter)
  • Software Beckman-Coulters Expo32 MultiComp and FlowJo (Treestar Inc., www.flowjo.com)

6. Reagents

  • Hoechst 33342 (5 µg/ml; prepared fresh) (Molecular Probes Inc.)
  • Paraformaldehyde (10%)

7. References

  • Brown, S.L., M.R. Landry, K.E. Selph, E.J. Yang, Y.M. Rii, R.R. Bidigare. 2008. Diatoms in the desert: Plankton community response to a subtropical mesoscale eddy in the subtropical North Pacific. Deep-Sea Research Part II 55: 1321-1333 [doi: 10.1016/j.dsr2.2008.02.012].
  • Garrison, D.L., M.M. Gowing, M.P. Hughes, L. Campbell, D.A. Caron, M.R. Dennet, A. Shalapyonok, R.J. Olson, M.R. Landry, S.L. Brown, H. Liu, F. Azam, G.F. Steward, H.W. Ducklow, and D.C. Smith. 2000. Microbial food web structure in the Arabian Sea: a US JGOFS study. Deep-Sea Research Part II 47: 1387-1422.
  • Menden-Deuer, S. and E.J. Lessard. 2000. Carbon to volume relationships for dinoflagellates, diatoms, and other protist plankton. Limnology and Oceanography 45: 569-679.
  • Monger, B.C. and M.R. Landry. 1993. Flow cytometric analysis of marine bacteria with Hoechst 33342. Applied Environmental Microbiology 59: 905-911.
  • Sherr, E.B. and B.F. Sherr. 1993. Preservation and storage of samples for enumeration of heterotrophic protists. In Handbook of Methods in Aquatic Microbial Ecology. P.F. Kemp, J.J. Cole, B.F. Sherr, and E.B. Sherr (Eds.) pp. 207-212, CRC Press Boca Raton, Florida.