Sampling, Biomass Estimation and Counts of Freshwater Algae - Freshwater Algae: Identification, Enumeration and Use as Bioindicators - page 62

Flow cytometry provides a completely automated
approach to analysis of particulate suspensions (0.5-
40 μm diameter) such as phytoplankton popula-
tions. The phytoplankton sample is injected into the
flow cytometer and is hydrodynamically focussed
into a narrow stream that passes through a narrow
laser beam. As the cells and colonies intercept the
light source they scatter light and fluorochromes are
excited to a higher state - releasing photons of light
with spectral qualities unique to particular pigments.
Flow cytometry thus measures fluorescence per cell
or particle, in contrast to spectrophotometry - in
which emission and absorption of particular wave-
lengths are measured for a bulk volume of sample.
Phytoplankton units within the mixed sample can
be characterised in terms of forward-angle light scat-
ter (FALS) and autofluorescence, which are related
respectively to organism size and pigment concen-
tration (Cavender-Bares et al ., 1998). In addition to
obtaining information about the phytoplankton sam-
ple, algal subpopulations can also be separated out as
discretefractionsbytheincorporationofahigh-speed
sorter.
Flow cytometry has been used by research work-
ers to analyse phytoplankton in various ways
(Table 2.2).

by these procedures for initial concentration of the
sample.
Flow cytometry was used by Verspagen et al .
(2005), for example, to obtain Microcystis frac-
tions in their studies on benthic-pelagic coupling
of this alga in a eutrophic lake. Bulk Microcys-
tis populations were obtained from phytoplankton
and sediment trap samples on the cytometric basis
of size (0.5-2000 μm) and phycocyanin fluores-
cence. Sediment samples were fully mixed and ini-
tially centrifuged in a Percoll© mixture to separate
algal cells from sediment particles. Final separa-
tion was carried out on a flow cytometer, calibrated
to detect only particles containing chlorophyll- a .
The biomass of Microcystis fractions from phy-
toplankton, sediment traps and sediment sam-
ples was then determined by chlorophyll- a
analysis.

Determination of size range . One of the prob-
lems in using flow cytometry to study natural
samples is the wide range of organism sizes and
concentrations encountered. This can be partly sur-
mounted by prefiltration to restrict size range, oth-
erwise time-consuming changes in flow cytometer
configuration (lengthy changeover times) may be
required to characterise the full phytoplankton size
range. This instrumental problem can be largely
overcome by modiication of the conventional flow
cytometer (Cavendar-Bares et al ., 1998), allowing
simultaneous (real-time) analysis of the full spec-
trum of phytoplankton sizes.

Collection of algal fractions for microscopy and
bulk analysis. Although the use of flow cytom-
etry represents a different approach to classical
techniques (filtration, centrifugation) for obtaining
bulk phytoplankton samples, it is often preceded

Table 2.2 Flow Cytometry Analysis: Examples of Mixed Phytoplankton and Sediment Samples.

Target Organism Sorting Criteria Sample Analysis Reference

Prochlorococcus Fluorescence and

light scattering

Molecular analysis of
genetic heterogeneity

Urbach and Chisholm (1998)

Mixed phytoplankton Forward angle light

scatter

Real-time analysis of size
spectra: modification of
cytometer

Cavender-Bares et al . (1998)

Microcystis in water
column, sediment
traps and sediment

Unit size (0.5-2000
μm). Phycocyanin
fluorescence

Blue-green algal biomass
(chlorophyll- a )

Verspagen et al . (2005)