Enumeration of Phytoplankton, Bacteria, and Viruses in Marine Samples

Dominique Marie1, Frédéric Partensky1, Daniel Vaulot1, Corina Brussaard2

1 Station Biologique, Roscoff, France, 2 University of Bergen, Bergen, Norway
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 11.11
DOI:  10.1002/0471142956.cy1111s10
Online Posting Date:  May, 2001
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Abstract

For many years, a small but dedicated group of scientists have been using flow cytometry for the evaluation of marine microorganisms. One of these scientists now provides us with a detailed series of protocols in this area, spelling out the variations in method and instrument operation that are crucial to the successful extraction of quality flow data from marine organisms. In addition, the use of a number of less frequently employed fluorescent probes gives some insight into alternative staining procedures. As our collection of microbiologically oriented techniques increases, this knowledge database becomes invaluable.

     
 
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Table of Contents

  • Basic Protocol 1: Flow Cytometric Enumeration of Picophytoplankton Based on Scatter and Autofluorescence
  • Basic Protocol 2: Flow Cytometric Enumeration of Bacterioplankton by DNA Staining and Fluorescent Detection
  • Basic Protocol 3: Flow Cytometric Enumeration of Viroplankton by DNA Staining and Fluorescent Detection
  • Support Protocol 1: Preservation and Storage of Picophytoplankton
  • Support Protocol 2: Calibration of the Cytometer Flow Rate
  • Support Protocol 3: Preparation of Buffered 10% Paraformaldehyde Stock Solution
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Flow Cytometric Enumeration of Picophytoplankton Based on Scatter and Autofluorescence

  Materials
  • Natural marine samples or cultures, either fresh or frozen (see protocol 4 for freezing procedure)
  • 0.95‐µm fluorescent microspheres (Polysciences) diluted to ∼105 beads/ml (as assessed by epifluorescence microscopy) in distilled water
  • Seawater
  • 0.2‐µm‐pore‐size cartridge filter units
  • Flow cytometer equipped with a 488‐nm argon laser (e.g., FACSort, Becton Dickinson)
  • Additional reagents and solutions for flow cytometer calibration (see protocol 5)

Basic Protocol 2: Flow Cytometric Enumeration of Bacterioplankton by DNA Staining and Fluorescent Detection

  Materials
  • Natural marine samples or cultures, either fresh or frozen (see protocol 4 for freezing procedure)
  • 10% paraformaldehyde (see protocol 6) and/or 25% glutaraldehyde (Sigma)
  • DNA‐specific stain such as SYBR Green I, YOYO‐1, TOTO‐1, or TO‐PRO‐1 (Molecular Probes)
  • 0.95‐µm fluorescent microspheres (Polysciences) diluted to ∼105 beads/ml (as assessed by epifluorescence microscopy) in distilled water
  • Seawater
  • 0.2‐µm‐pore‐size cartridge filter units
  • Flow cytometer equipped with a 488‐nm argon laser (e.g., FACSort, Becton Dickinson)
  • Additional reagents and solutions for flow cytometer calibration (see protocol 5)

Basic Protocol 3: Flow Cytometric Enumeration of Viroplankton by DNA Staining and Fluorescent Detection

  Materials
  • Natural marine samples or cultures, either fresh or frozen (see protocol 4 for freezing procedure)
  • 10% paraformaldehyde (see protocol 6) or 25% glutaraldehyde (Sigma)
  • TE buffer, pH 7.2 ( appendix 2A)
  • DNA‐specific stain(s) such as SYBR Green I or II, OliGreen, or RiboGreen (Molecular Probes)
  • 0.95‐µm fluorescent microspheres (Polysciences) diluted to ∼105 beads/ml (as assessed by epifluorescence microscopy) in distilled water
  • Distilled water
  • 0.2‐µm‐pore‐size filtration units for plastic syringe
  • Flow cytometer equipped with a 488‐nm argon laser (e.g., FACSort, Becton Dickinson)
  • Additional reagents and solutions for flow cytometer calibration (see protocol 5)

Support Protocol 1: Preservation and Storage of Picophytoplankton

  Materials
  • 10% paraformaldehyde (see protocol 6) and/or 25% glutaraldehyde (Sigma)

Support Protocol 2: Calibration of the Cytometer Flow Rate

  Materials
  • Paraformaldehyde (e.g., Sigma)
  • Distilled water
  • 1 M NaOH
  • Phosphate‐buffered saline (PBS, appendix 2A), pH 7.5
  • 1 M HCl
  • 0.2‐µm‐pore‐size filtration unit for plastic syringe
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Figures

Videos

Literature Cited

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   Bratbak, G., Heldal, M., Norland, S., and Thingstad, T.F. 1990. Viruses as partners in spring bloom microbial trophodynamics. Appl. Environ. Microbiol. 56:1400‐1405.
   Button, D.K. and Robertson, B.R. 1989. Kinetics of bacterial processes in natural aquatic systems based on biomass as determined by high‐resolution flow cytometry. Cytometry 10:558‐563.
   Campbell, L. and Vaulot, D. 1993. Photosynthetic picoplankton community structure in the subtropical North Pacific Ocean near Hawaii (station ALOHA). Deep‐Sea Res. 40:2043‐2060.
   Campbell, L., Nolla, H.A., and Vaulot, D. 1994. The importance of Prochlorococcus to community structure in the central North Pacific Ocean. Limnol. Oceanogr. 39:954‐961.
   Cavender‐Bares, K.K., Frankel, S.L., and Chisholm, S.W. 1998. A dual sheath flow cytometer for shipboard analyses of phytoplankton communities from the oligotrophic oceans. Limnol. Oceanogr. 43:1383‐1388.
   Chisholm, S.W., Olson, R.J., Zettler, E.R., Waterbury, J., Goericke, R., and Welschmeyer, N. 1988. A novel free‐living prochlorophyte occurs at high cell concentrations in the oceanic euphotic zone. Nature 334:340‐343.
   Chisholm, S.W., Frankel, S.L., Goericke, R., Olson, R.J., Palenik, B., Waterbury, J.B., West‐Johnsrud, L., and Zettler, E.R. 1992. Prochlorococcus marinus nov. gen. nov. sp.: An oxyphototrophic marine prokaryote containing divinyl chlorophyll a and b. Arch. Microbiol. 157:297‐300.
   Courties, C., Vaquer, A., Trousselier, M., Lautier, J., Chrétiennot‐Dinet, M‐J., Neveux, J., Machado, C., and Claustre, H. 1994. Smallest eukaryotic organism. Nature 370:255.
   Dubelaar, G.B.J., Groenewegen, A.C., Stokdijk, W., Van Den Engh, G.J., and Visser, J.W.M. 1989. Optical plankton analyser: A flow cytometer for plankton analysis, II: Specifications. Cytometry 10:529‐539.
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   Hara, S., Terauchi, K., and Koike, I. 1991. Abundance of viruses in marine waters: Assessment by epifluorescence and transmission electron microscopy. Appl. Environ. Microbiol. 57:2731‐2734.
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   Kriss, A.E. and Rukina, E.A. 1947. Bacteriophages in the sea. Dokl. Akad. Nauk SSSR. 57:833‐836.
   Lebaron, P., Catala, P., and Parthuisot, N. 1998. Effectiveness of SYTOX Green stain for bacterial viability assessment. Appl. Environ. Microbiol. 98:2697‐2700.
   Li, W.K.W. and Wood, A.M. 1988. Vertical distribution of North Atlantic ultraphytoplankton: Analysis by flow cytometry and epifluorescence microscopy. Deep Sea Res. 35:1615‐1638.
   Li, W.K.W., Jellett, J.F., and Dickie, P.M. 1995. DNA distribution in planktonic bacteria stained with TOTO or TO‐PRO. Limnol. Oceanogr. 40:1485‐1495.
   Marie, D., Vaulot, D., and Partensky, F. 1996. Application of the novel nucleic acid dyes YOYO‐1, YO‐PRO‐1 and PicoGreen analysis of marine prokaryotes. Appl. Environ,. Microbiol. 62:1649‐1655.
   Marie, D., Partensky, F., Jacquet, S., and 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. Appl. Environ. Microbiol. 93:186‐193.
   Marie, D., Brussaard, C.P.D., Thyrhaug, R., Bratbak, G., and Vaulot, D. 1999. Enumeration of marine viruses in culture and natural samples by flow cytometry. Appl. Environ. Microbiol. 65:45‐52.
   Monger, B.C. and Landry, M.R. 1993. Flow cytometric analysis of marine bacteria with Hoechst 33342. Appl. Environ. Microbiol. 59:905‐911.
   Olson, R.J., Chisholm, S.W., Zettler, E.R., and Armbrust, E.V. 1988. Analysis of Synechococcus pigment types in the sea using single and dual beam flow cytometry. Deep Sea Res. 35:425‐440.
   Partensky, F., Blanchot, J., Lantoine, F., Neveux, J., and Marie, D. 1996. Vertical structure of picophytoplankton at different trophic sites of the tropical northeastern Atlantic Ocean. Deep Sea Res. 43:1191‐1213.
   Partensky, F., Hess, W.R., and Vaulot, D. 1999. Prochlorococcus, a marine photosynthetic prokaryote of global significance. Microbiol. Mol. Biol. Rev. 63:106‐127.
   Proctor, L.M. and Fuhrman, J.A. 1990. Viral mortality of marine bacteria and cyanobacteria. Nature 343:60‐62.
   Robertson, B.R., Button, D.K., and Kloch, A.L. 1998. Determination of the biomasses of small bacteria at low concentrations in a mixture of species with forward light scatter measurements by flow cytometry. Appl. Environ. Microbiol. 64:3900‐3909.
   Shapiro, L.P. and Haugen, E.M. 1988. Seasonal distribution and temperature tolerance of Synechococcus in Boothbay Harbor, Maine. Estuarine Coastal Shelf Sci. 26:517‐525.
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   Vaulot, D. 1989. CYTOPC: Processing software for flow cytometric data. Signal Noise. 2:8.
   Vaulot, D., Courties, C., and Partensky, F. 1989. A simple method to preserve oceanic phytoplankton for flow cytometric analyses. Cytometry 10:629‐635.
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Internet Resources
  http://www.sb‐roscoff.fr/Phyto/cyto.html
   Lists marine applications of flow cytometry and provides a downloadable copy of the Cyto Win software.
  http://carl.im.uib.no
   Provides information on marine viruses.
  http://www.flowcytometry.org
   Contains a wide range of resources for marine applications of flow cytometry.
  http://CCMP.bigelow.org
  Catalogs and maintains algal strains to be used for protocol development.
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