Enumeration and Analysis of Picophytoplankton
Robert J. Olson and Sallie W. Chisholm
We will analyze depth profiles of the picophytoplankton by flow
cytometry for the time series cruises. Synechococcus,
Prochlorococcus and ``eukaryotic picophytoplankton'' populations will
be enumerated, and the light scattering and fluorescence properties will
be determined for each population to provide information about cell size
and pigment contents. Details of the methods used are described in Olson
et al. (1992). A brief description follows.
Sample Collection and Storage
Water samples are collected using PVC Niskin or GoFlo bottles
and transferred to acid cleaned dark plastic bottles (taking care to
shield samples from sunlight) as soon as possible after the rosette is on
deck. Samples are fixed with glutaraldehyde (0.125 % final concentration)
for 10 min. in the dark, then frozen in liquid nitrogen (a modification of
Vaulot et al., 1989). We use 1.2 ml cryovials to which 6 µl
of 25 % electron microscopy grade glutaraldehyde is added.
Flow Cytometric Analysis
Before analysis, frozen samples are thawed in a room
temperature water bath and are then analyzed within 15 min. Fluorescent
beads are added as internal standards (see below) and the sample is run on
the flow cytometer until data from about 10,000 cells is accumulated, or
for 15 min. We use a Coulter EPICS flow cytometer with excitation at 488 nm
(500 mW focussed to a 20 µm spot by a single 38-mm spherical lens) to
measure forward angle light scatter (FLS, 1.5--19 degrees), light
scattered at right angles (LS90, 73--107 degrees), phycoerythrin
fluorescence (PE, 560--630 nm), and chlorophyll fluorescence (CHL,
660--700 nm) signals. Each signal is logarithmically scaled (3 decades).
Each signal from each cell is stored in a ``list mode'' file; these files
are gated on CHL by specifying a minimum threshold CHL fluorescence which
the cell must exceed in order for its measurements to be stored.
The data are transferred to a personal computer for analysis using the
program Cyto-PC (D. Vaulot, pers. comm.). Analysis involves observing
multiple two parameter scatterplots of the data and drawing windows around
the populations of interest. First, Synechococcus and beads
(which fluoresce in both the PE and CHL spectral regions) are
discriminated from each other and from cells containing only chlorophyll
using a plot of FLS vs. PE. After these particles are ``tagged'',
Prochlorococcus and eukaryotic picoplankton are discriminated from each
other using a plot of FLS vs. CHL. The number of events within each
window and statistics (mean, mode, coefficient of variation) on each
population's optical properties are calculated. The values for optical
properties can be expressed relative to those for the internal standard beads.
Quantitation of Results
We add fluorescently labelled microbeads (``Fluoresbrite''
0.57 µm diameter carboxylate, Polysciences Lot No. 436754) to each sample
as internal standards for light scattering and fluorescence, and as a
means to calculate the volume of sample analyzed. The working stock of
beads in distilled-deionized water is sonicated for 5 minutes at the start
of the day to reduce clumping. Before each use, the tube of working stock
is mixed on a vortex mixer for 5--10 seconds to suspend the beads uniformly.
A known volume of calibrated bead stock is pipetted into the sample and
mixed in by swirling the sample tube. The bead stock is calibrated daily
by analyzing samples containing only filtered seawater and beads;
instrument settings are adjusted so that noise pulses and sample
coincidence rates are negligible, so that the number of beads in a known
volume of sample (determined gravimetrically) can be determined. The
concentration of beads in the stock is such that several thousand beads
are analyzed when we add 10 µl of beads to a 1 ml sample.
Measurement of the sample volume analyzed is accomplished using a stock of
beads of known concentration. At high acquisition rates (as when there
are many non-target particles in the samples), the electronics may miss
target cells while they are processing other signal pulses. Having an
internal standard of calibrated beads allows us to correct for these
missed cells, since the beads should be missed in the same proportion as
cells. By adding a known volume of calibrated beads to a sample, the cell
concentrations can be calculated as:
cells ml¯ = (cells counted/beads counted) * beads ml¯
Quality Control/Quality Assurance
Our group and M. R. Landry's at University of Hawaii (who will
be analyzing picoplankton samples from the transect cruises) will both be
using Coulter EPICS flow cytometers to analyze our samples. However,
there are some differences between our instruments, and for reasons of
continuity with earlier studies with differing aims, our two groups use
slightly different optical filters and preservation techniques. To
facilitate interpretation of results from the two sets of cruises, our two
groups will analyze selected sets of samples in parallel, and we have sent
a supply of the beads we use as internal standards to the University of
Hawaii, so that both sets of samples can be compared to the same standards.
- Olson, R.J., E.R. Zettler, and M.D. DuRand (1992).
analysis using flow cytometry. In: P.F. Kemp, B.F. Sherr,
- E.B. Sherr, and J.J. Cols (eds.),
- Current Methods in Aquatic
Microbial Ecology, Lewis Publ. (in press).
- Vaulot, D., C. Courties, and F. Partensky (1989).
- A simple method to
preserve oceanic phytoplankton for flow cytometric analyses.
Cytometry, 10: 629--635.