DNA/RNA Analysis of Phytoplankton by Flow Cytometry

Dominique Marie1, Nathalie Simon1, Laure Guillou1, Frédéric Partensky1, Daniel Vaulot1

1 INSU et Université Pierre et Marie Curie, Roscoff, France
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 11.12
DOI:  10.1002/0471142956.cy1112s11
Online Posting Date:  May, 2001
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The past 10 years or so have seen the combination of molecular and biochemical techniques within the confines of cytometry. The use of flow cytometry in microbiology is finally coming of age. This unit carefully defines the criteria for evaluation of DNA and RNA in phytoplankton. Of course not everyone works with phytoplankton, but the methods outlined are very appropriately representative for other organisms. In addition, the unit discusses the methods for evaluating cell cycle and discriminating specific taxa using fluorescent oligonucleotide probes targeted to 18S rRNA.

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Table of Contents

  • Basic Protocol 1: Determining Genome Size and Ploidy Level of Phytoplankton
  • Basic Protocol 2: Cell Cycle Analysis of Phytoplankton
  • Basic Protocol 3: Identifying Phytoplankton Using Whole‐Cell Hybridization with Taxon‐Specific Fluorescent Oligonucleotide Probes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Determining Genome Size and Ploidy Level of Phytoplankton

  • Cell suspension (1–5 × 105 cells/ml)
  • Nucleus isolation buffer (NIB; see recipe; add 5 µl of 1 M sodium bisulfite per ml buffer immediately before use and filter through 0.2‐µm pore‐size filter)
  • Internal reference (i.e., suspension of nuclei or cells from species for which genome size is known, at ∼1 × 105 cells/ml)
  • 1% (w/v) RNase A (type IA, Sigma) in distilled water (heat 10 min at 90°C to degrade contaminating DNase)
  • Nucleic acid–specific stain (see Table 11.12.1 for concentrations)
  • 0.2‐µm pore‐size filters for plastic syringes
  • 10 µm nylon mesh
  • Flow cytometer with laser emitting UV, violet, and blue lines (also see )
    Table 1.2.1   MaterialsDyes Used for Determination of Genome Size and Cell Cycle Analysis of Planktonic Cells

    Dye MW Absorption maximum (λ A; nm) Fluorescence maximum (λ F; nm) Working conc. Final conc.
    Ethidium bromide 394  518  605 0.1 mg/ml  5 µg/ml
    Propidium iodide 668  535  617 0.1 mg/ml  5 µg/ml
    Mithramycin 1085  433  570  1 mg/ml 30 µg/ml
    Chromomycin A 1183  420  560  1 mg/ml 30 µg/ml
    DAPI 350  357  451 0.1 mg/ml  1 µg/ml
    Hoechst 33342 652  365  502 0.1 mg/ml  1 µg/ml
    TOTO‐1 1303  514  533  1 µM 30 nM
    YOYO‐1 1271 491/450 a 509/550a a  1 µM 30 nM
    TO‐PRO‐1 645  515  531  1 µM 30 nM
    YO‐PRO‐1 629 491/450 a 509/550 a  1 µM 30 nM
    PicoGreen  NA b  480  520  1/10 1/1000
    SYBR Green I  NA b  494  521  1/100 1/10,000

     aNumber before slash is according to manufacturer; number after slash is according to Hirons et al. ( ).
     bNA, molecular weight not available from manufacturer.

Basic Protocol 2: Cell Cycle Analysis of Phytoplankton

  • 0.05% (w/v) glutaraldehyde/1% (w/v) paraformaldehyde
  • Cell suspension (1–5 × 105 cells/ml)
  • 1% (w/v) RNase A (type IA, Sigma) in distilled water (heat 10 min at 90°C to degrade contaminating DNase)
  • 1 M potassium citrate
  • SYBR Green I working solution (dilute commercial stock solution from Molecular Probes 1:100 in distilled water)
  • 1× 105 beads/ml suspension of 0.95‐µm fluorescent microspheres (Polysciences)
  • Flow cytometer equipped with a laser emitting at 488 nm (also see )

Basic Protocol 3: Identifying Phytoplankton Using Whole‐Cell Hybridization with Taxon‐Specific Fluorescent Oligonucleotide Probes

  • Phytoplanktonic cells of interest
  • Culture medium for marine phytoplankton (sea water with appropriate nutrients; see http://ccmp.bigelow.org/CI/CI_01.html)
  • 10% (w/v) paraformaldehyde stock (store at −20°C)
  • 70:30 (v/v) ethanol/PBS, −20°C (see appendix 2A for PBS)
  • Hybridization buffer (see recipe) of optimal formamide concentration
  • 50 ng/µl fluorescein isothiocyanate (FITC)–labeled probe to be tested (see and unit 8.3)
  • Washing buffer (see recipe) with the same stringency as the hybridization buffer
  • Phosphate‐buffered saline (PBS; appendix 2A), pH 9.0
  • 1 × 105 beads/ml suspension of 0.95‐µm fluorescent microspheres (Polysciences)
  • Hybridization oven, or any oven or incubator whose temperature can be precisely tuned
  • Flow cytometer equipped with 488‐nm argon laser (also see )
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Literature Cited

Literature Cited
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  The authors wish to acknowledge support by grants from the European Community MAST III programs (MAS3‐CT96‐015‐MEDEA, MAS3‐CT97‐0128‐PROMOLEC), and from the JGOFS‐France PROSOPE program. The FACSort flow cytometer was funded in part by CNRS‐INSU and the Région Bretagne.
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