Sampling, Biomass Estimation and Counts of Freshwater Algae - Freshwater Algae: Identification, Enumeration and Use as Bioindicators - page 63

Speciesresolutionofunicellularpopulations .Flow
cytometry has been particularly useful for col-
lecting and analysing pico-phytoplankton frac-
tions, where algae can be differentiated from
heterotrophic bacteria in relation to size (Zubkov
et al . 2003). It is also useful where mixed pop-
ulations of different algal species are of a sim-
ilar size but differ physiologically. Urbach and
Chisholm (1998), for example, used flow cytom-
etry to isolate populations of Prochlorococcus
based on their red fluorescence (660-700 nm),
absence of orange fluorescence (540-630 nm) and
their characteristic forward light scatter. Cells of
Synechococcus , which exhibit phycoerythrin aut-
ofluorescence (540-630 nm), were rejected during
sorting. Molecular analysis of the purified samples
of Prochlorococcus led these authors to demon-
strate a high degree of genetic heterogeneity within
local populations of this organism.

2.5.1 Sample preservation and processing

Living phytoplankton

Visual examination of the fresh (live) phytoplankton
sample is always a useful preliminary to analysis of
fixed samples, particularly in relation to delicate flag-
ellated algae - which may be distorted by the fixation
process. Although smaller species are liable to be
missed, the net sample is particularly useful for this -
since the sample is concentrated during collection,
and (if transported in the dark at 10 ◦ C) will remain
fresh for a few hours.
Sample bottles should not be filled right to the top
as there is no possibility for gas exchange once the
bottle has been closed. If the bottle is only about 75%
full then some exchange with the overlying air is pos-
sible for at least a short period. This is especially true
for net hauls which, by their nature, are quite con-
centrated and have a high respiratory activity. These
samples should be diluted to reduce oxygen demand
and prevent the sample going anaerobic. If these pre-
cautionsarenotcarriedouttheremaybeseveredegra-
dation in the sample by the time it is observed under
a microscope. With live samples, zooplankton can be
filtered off to avoid excessive grazing during transit.

Although flow cytometry has considerable poten-
tial for freshwater studies, it has been used mainly in
relation to marine phytoplankton, where automated
collection of multiple samples over large areas of
ocean has been particularly useful. The domination of
many oceanic planktonic communities by unicellular
blue-greens makes the technique specially relevant to
marine biology.

Chemical fixation

2.5 Biodiversity of mixed-species
populations: microscope counts
and biovolumes

Samples to be kept for longer storage require preser-
vation, which normally involves treatment with either
aldehyde or iodine fixative. Chemical fixation kills
cells and prevents algal disintegration (caused by
autolysis and bacterial degradation). It also prevents
algal removal by any zooplankton present in the sam-
ple and increases algal cell density (which aids sam-
ple concentration by sedimentation). The major dis-
advantages of fixation are that algal shape and colour
may be altered.

Microscopical analysis of species populations within
the collected lake water sample involves algal
identification (see Chapter 4) followed by counts of
individual cells and colonies. In practice, it is often
necessary to chemically preserve the algae and to
concentrate the sample to make counting statistically
valid.
Counts of algal species are normally carried out
from volume (Section 2.2.2) or collecting tube (Sec-
tion 2.2.3) samples, but net samples provide useful
material to obtain a quick overview of major algal
species present and to see algae in the living state.

Aldehyde fixation Although aldehyde fixatives
tend to cause rupture, deformation and shrinkage of
delicate algae, most species are preserved with min-
imal change to biovolume, shape and original col-
oration.