Enumeration and analysis of picophytoplankton and bacteria

Brian Binder, Sallie W. Chisholm, and R.J. Olson

 Caution:  Please note that the analytical procedures for enumerating the
 cell counts in this data set and those reported by DuRand (Diel Series)
 are significantly different so that combining the results of these two
 data set should not be done without a review of the analytical procedures.

Samples from PVC Niskins or GoFlo bottles were preserved in 0.12%
glutaraldehyde (final concentration) for 10 min. at room temperature, and then
frozen in liquid nitrogen (a modification of Vaulot et al. 1989).

In preparation for flow cytometric analysis, samples were stained using a
modification of the protocol of Monger and Landry (1993).  Immediately 
following sample thawing (at room temperature), 0.5 ml aliquots were stained 
with the DNA-specific fluorochrome Hoechst-33342 (0.5 mg/ml final con-
centration).  Stained samples were held at room temperature in the dark for 1 
hour prior to analysis; no significant changes in cell number, red 
autofluorescence, or Hoechst fluorescence were detectable between 15 minutes 
and 4 hours of staining under these conditions.  Just prior to analysis, known
volumes of two standard bead stocks were added:  0.57 mm diameter blue-
excitable beads ("Fluoresbrite YG", Polysciences, Inc.) and 0.46 mm diameter 
UV-excitable beads ("Fluoresbrite BB").  These beads were used to calculate 
cell abundances, and as internal fluorescence and light scatter standards 
(Olson et al. 1993).

Samples were analyzed using dual beam flow cytometry with a modified EPICS-753
(Coulter Corp.) equipped with a 38 mm focal length quartz plano-convex focusing
lens, a "Biosense" flow cell, and a forward angle light scatter PMT detector
with an extra-wide obscuration bar (estimated angle of collection, 7 - 15). 
Two Ar-ion lasers (Coherent, Inc.) were employed: one tuned to 488 nm (800 mW
output) and the other to 360-365 nm (300 mW output).
Blue forward angle light scatter (FALS) and 90 light scatter were measured
through 488 nm band-pass filters (10 nm band width).  Fluorescence signals all
passed a 488 nm dichroic and a 418 long-pass filter; red fluorescence (from
chlorophyll) was the 488 nm-excited signal passing a 680 nm band-pass filter 
(40nm bandwidth, Omega Optical "680 DF40"), orange fluorescence (from
phycobiliproteins) was the 488 nm-excited signal passing a 580 nm band-pass
filter (50 nm bandwidth, "580 DF50"), and blue fluorescence (from Hoechst/DNA)
was the UV-excited signal passing a 470 nm short-pass filter (with strong
blocking at 488; "470 EFSP").  Log-integrated signals were collected for all

Prochlorococcus, Synechococcus, and ultra-phytoplankton were recognized
according to their light scatter and fluorescence characteristics as described
previously (Olson et al. 1993).  We define "ultra-phytoplankton" operationally
as that population of cells that has high red fluorescence and light scatter,
but no detectable orange fluorescence.  Heterotrophic bacteria were identified
on the basis of their blue fluorescence, light scatter properties, and lack of
detectable red or orange fluorescence

Literature Cited

Monger B. C. and M. R. Landry  (1993)  Flow cytometric analysis of marine
bacteria with Hoechst 33342.  Applied and Environmental Microbiology, 59,

Olson R. J., E. R. Zettler and M. D. DuRand  (1993)  Phytoplankton analysis
using flow cytometry.  In: Handbook of methods in aquatic microbial ecology, P.
F. Kemp, B. F. Sherr, E. B. Sherr and J. J. Cole, editors, Lewis Publishers,
Boca Raton, pp. 175-186.

Vaulot D., C. Courties and F. Partensky  (1989)  A simple method to preserve
oceanic phytoplankton for flow cytometric analyses.  Cytometry, 10, 629-635.