Sampling, Biomass Estimation and Counts of Freshwater Algae - Freshwater Algae: Identification, Enumeration and Use as Bioindicators - page 64

Fixation can be carried out using glutaraldehyde
(final concentration 3% v/v, neutralised to pH 7.0
with Sorensen's phosphate buffer) or buffered 4%
formaldehyde. The latter is prepared by adding con-
centrated buffered formaldehyde (40% v/v) to the
phytoplankton sample at 1:9 v/v. Fixed samples
should be kept in the refrigerator (at 4 ◦ C) in the dark,
and may either be used for species counts (volume
sampler) or for examination and photography (trawl-
net sample).

sediment. Sedimentation should be carried out in the
dark and away from any sources of heat. The top 80
ml of algal-free liquid can then be carefully siphoned
of using a pipette with a 'U' tip, to cause minimal
disturbance. The remaining 20 ml (5× concentration
from environmental sample) can be re-suspended in
the bottom of the cylinder by agitation and then stored
in an opaque glass or polyethylene container in the
dark at constant temperature (4 ◦ C) until needed for
analysis.
Although iodine increases cell density, some algal
species still may not sediment. This problem applies
especially to colonial blue-green algae that contain
gas vacuoles, where their buoyancy can be great
enough to overcome the additional weight of the
iodine. It is very important to look carefully at the
surface layer of the sample in the cylinder to make
sure no buoyant species are present before decant-
ing off the supernatant (90 ml). If buoyant blue-green
species are present, one way to overcome their buoy-
ancy is to transfer the fresh sample to a strong but
flexible polythene bottle. The bottle should be filled
right to the top with the phytoplankton sample, with
no trapped air and the top screwed down tightly. The
bottle should then be dropped onto a hard surface
from a height of about 1.5 m. The sudden increase in
pressure as the bottle hits the floor is usually enough
to collapse the gas vacuoles in the cells so that they
will then sediment without trouble.
An alternative procedure to the two-stage process
ofsedimentationfollowedbyseparateenumerationin
a counting chamber is to make counts direct from the
sedimentation chamber using an inverted microscope
(Fig. 2.12).

Lugol's iodine The preferred fixative for cell
count samples is Lugol's iodine, which preserves
cell shape/structure and also stains cells (yellowish to
dark brown; Fig. 2.16) - so they can readily be seen
in the counting chamber. Uptake of iodine (atomic
weight 126.9) is also particularly effective in increas-
ing cell density and aiding the process of sedimenta-
tion for concentration of the phytoplankton sample.
Lugol's iodine is prepared by dissolving 150 g
potassium iodide plus 50 g iodine in 980 ml dis-
tilled water (DW), then adding 20 ml glacial acetic
acid. Lugol's iodine is normally added to the lake
water sample immediately after collection, using 2-3
ml of iodine per 100 ml of sample. A typical lake
water volume collected for algal counts would be
200 ml, and this would normally be concentrated by
sedimentation soon after collection and stored in the
concentrated state.

Sample concentration

For phytoplankton counts, Lugol's iodine samples
may need to be concentrated prior to enumeration
in a counting chamber. This can be carried out by
membrane filtration, centrifugation (1000 g for 20
min) or sedimentation. 2.5.2 Chemical cleaning of diatoms

Algal sedimentation This is the preferred
technique for sample concentration since it is
non-selective in terms of size (unlike filtration) and
non-destructive (unlike filtration and centrifugation).
Sedimentation is normally carried out by pouring
the 100-ml sample into a measuring cylinder and
allowing the cylinder to stand on a vibration-free sur-
face over a 24-72 h period to allow all of the algae to

Identification of diatoms to species level in environ-
mental samples (phytoplankton, benthic communi-
ties and sediment samples) typically involves detailed
light or scanning electron microscope examination of
frustule ornamentation (Barber and Haworth, 1981).
This requires the outer layer of organic material and
associated debris to be removed, which can be carried
out in various ways.