Hawaii Ocean Time-series (HOT)
in the School of Ocean and Earth Science and Technology at the University of Hawai'i


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SUMMARY: Picoplankton populations are subsampled from seawater collected in Niskin bottles. The cells are fixed in the field by the addition of paraformaldehyde and, in the laboratory, stained with Hoechst 33342 (a DNA specific dye) and enumerated by dual-beam flow cytometry.

1. Principle

Flow cytometric enumeration is based on the method of 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. A 225 mW UV laser is aligned colinearly with a 1 W visible (488 nm) laser to permit enumeration of both heterotrophic and autotrophic picoplankton. Methods for enumeration of autotrophs are given by Campbell and Vaulot (1993) and are not described in detail here.

2. Precautions

Because this is a procedure for enumerating preserved cells, sterilization is not a guarantee against contamination. However new, sterile plastic containers tend to be among the cleanest containers available. All reagents (preservative and stain) must be prefiltered through 0.2 µm filters and the sample should not come in contact with fingertips or other potentially contaminating surfaces. No drawing tubes are used.

Because of the small sample volume (1 ml), frozen samples thaw quickly. Therefore they should be kept in liquid nitrogen during transport from the ship to the shore-based laboratory.

3. Sampling

3.1. A small volume of sample (10-12 ml) is drawn from the Niskin bottle into a sterile 15 ml plastic centrifuge tube. Drawing tubes are not used.
3.2. 1 ml subsamples are drawn from the sample and placed in 2 ml cryovials containing 0.02 ml of 0.2 µm prefiltered 10% paraformaldehyde (final concentration 0.2%).
3.3. Cryovials are let to sit for 10 minutes and then quick frozen in liquid nitrogen. They are then stored frozen at -20 °C or colder until analyzed.

4. Analysis

4.1. Samples are stained with freshly prepared, prefiltered Hoechst 33342 (Molecular Probes Inc.) to a final concentration of 0.5 µg/ml. Samples stain for 2 h at room temperature in the dark.

215 µL sample

25 µL Hoechst 33342 (5 µg/ml)

10 µL fluorescent beads (internal standard)

4.2. Samples are analyzed by colinear dual-beam flow-cytometry using a Coulter EPICS flow instrument (Monger and Landry, 1993). Prior to analyzing samples, fluorescent beads are run to check the volume calibration and the alignment of the instrument.

100 µL subsamples are analyzed for forward angle light scatter (FALS), right angle light scatter (RALS), blue (DNA) fluorescence (BF) and red (chlorophyll) fluorescence (RF). The scatter parameters are a function of cell size and are used to separate bacteria from beads and other blue fluorescence signals. Marine bacteria form a distinct cluster on a scatter plot of BF vs. RALS (Monger and Landry, 1993). BF and RF signals are used to distinguish heterotrophic (unpigmented) from autotrophic (chlorophyll-containing) cells.

A known concentration of beads (0.98 µm, Polysciences) are used as an external standard for volume calibration. Beads (0.46 µm, Polysciences) are also added to each sample as an internal standard for per cell blue fluorescence. A record of cruise-to-cruise variation in fluorescence per cell relative to beads is maintained.

4.3. carried out on an 80486 microcomputer using the CYTOPC program developed by Dr. Daniel Vaulot (Station Biologique, Roscoff-sur-mer, France). This is not presently available commercially; see Vaulot (1989) for a description of the program. Further calculations (correction for dilution by preservative and stain) are done on a Microsoft Excel spreadsheet.

5. Reagents

  • Hoechst 33342 (5 µg/ml; prepared fresh)

  • paraformaldehyde (10%)

Add 10 g paraformaldehyde (be careful not to breathe dust) to 90 ml boiling distilled water and stir (use magnetic stir bar). Add 1 M NaOH, dropwise, with constant mixing, until the solution clears. Cool in ice to room temp, add 10 ml filtered sea water (can be cooling in ice while on stirrer). Adjust pH to 7.5 (careful, pH changes very rapidly below about 8.8; i.e., only one or two more drops needed). Use a Sterifil apparatus with 47 mm GF/F filters to filter this solution. Filter in two equal aliquots,changing the filter between aliquots to avoid clogging. The final preservative strength is about 10%. This solution should be prepared fresh for each cruise and pH checked before use.

6. Equipment and Supplies

  • 15 ml sterile polypropylene centrifuge tubes

  • 2 ml sterile cryovials

  • sterile plastic syringes and Acrodisc filters

  • 1 ml autopipet and tips

  • liquid nitrogen and container

  • Coulter EPICS flow cytometer with UV and visible lasers

  • IBM-compatible 80486 microcomputer and software

  • 0.5 µm fluorescent beads

7. References

  • Campbell, L. and D. Vaulot. 1993. Photosynthetic picoplankton community structure in the subtropical North Pacific Ocean near Hawaii (Station ALOHA). Deep-Sea Research, in press.

  • Monger, B. C. and M. R. Landry. 1993. Flow cytometric analysis of marine bacteria with Hoechst 33342. Applied and Environmental Microbiology, 59, 905-911.

  • Vaulot, D. 1989. CYTOPC: Processing software for flow cytometric data. Signal and Noise, 2, 8.