BACTERIA AND CYANOBACTERIA

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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. 
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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. Data are stored in list (ASCII) files.  Statistical analysis is 
     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.