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.

Literature Cited

Olson, R.J., E.R. Zettler, and M.D. DuRand (1992).: Phytoplankton 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.