Sampling, Biomass Estimation and Counts of Freshwater Algae - Freshwater Algae: Identification, Enumeration and Use as Bioindicators - page 65

Cleaning is normally carried out chemically by
treatment with acids and/or oxidising agents. Trial
and error will show which method is best for par-
ticular samples. Mild treatments, which leave most
frustules and some colonies intact, include treating
samples with dilute hydrochloric acid (to remove
calcareous matter) or soaking the material in 30%
hydrogen peroxide at room temperature for 24 h.
More extreme cleaning procedures involve expo-
sure to hot peroxide (see below) or treatment with
strong acids (Barber and Howarth, 1981). These
methods result in the loss of all non-siliceous algae,
the fragmentation of colonial diatoms into separate
cells and the break-up of diatom frustules. These
treatments result in the emission of toxic and cor-
rosive fumes, and must be carried out in a fume cup-
board.

5. 0.5 ml of the dilute suspension is placed on a cov-
erslip and left overnight under sterile laminar flow
to evaporate. A microscope slide is then placed on
a hot plate (90 ◦ C), a drop of Naphrax© embed-
ding medium placed on it and the coverslip bear-
ing the dry diatom sample inverted onto it. The
slide is subsequently removed from the hotplate
and allowed to cool - forming a permanent prepa-
ration.

Numerous micrographs of hot peroxide-cleaned
diatoms are shown in Chapter 4, with combined
images of living and cleaned cells illustrated for
Aulacoseira (Fig. 4.8), Fragilaria (Fig. 4.10), Tabel-
laria (Fig. 4.11), Diatoma (Fig. 4.12), Asterionella
(Fig. 4.42), Stephanodiscus (Fig. 4.58), Nitzschia
(Fig. 4.70a) and Navicula (Fig. 4.73a).

Hot hydrogen peroxide treatment 2.5.3 Species counts

A very effective procedure, this is carried out as fol-
lows:

Initial identification of major algal species within the
fixed sample is a useful preliminary to the enumera-
tion process, and may involve visual comparison to a
fresh phytoplankton sample (Section 2.5.1). Species
counts areusuallymadeinrelationtothe'livingunit',
which may be a single cell (unicellular alga), filament
of cells or globular colony. There are no hard and fast
rules as to whether colonies and filaments should be
treated as single units or constituent cells, but clearly
consistency is important within and between sam-
ples. The range of techniques available for making
algal counts has been widely reported (Wetzel and
Likens, 1991; Eaton etal ., 2005), including the use of
statistical procedures to estimate precision, required
number and randomness of algal enumeration (Lund
et al ., 1958).
Microscopical counts of algal species (cells, fila-
ments or colonies) can be carried out from

1. Approximately 2 g of environmental sample is
placed in a 100-ml beaker, 20 ml of concentrated
H 2 O 2 solution added, and the vessel left for 20 min
at room temperature. The beaker is then heated on
a hotplate at 90 ◦ C) for 2 h, keeping topped-up with
H 2 O 2 .

2. The beaker is removed from the hotplate and a few
drops of HCl (50%) added, withreleaseof chlorine
(the HCl removes surplus H 2 O 2 and carbonates.)

3. Samples are allowed to cool to room temperature,
transferred to centrifuge tubes and spun for 4 min
at 1200 rpm. The supernatant is removed and the
sample resuspended in distilled water. The process
is repeated five times, with the addition of a few
drops of weak ammonia before the final wash to
help clay removal, and final suspension in DW.

Liquid samples contained in a sedimentation
chamber

Concentrated samples contained in a counting
chamber

4. 0.5 ml of the suspension is placed in a centrifuge
tube and diluted with DW to produce a final sus-
pension that is neither totally clear nor milky, but
has many fine particles when held up to the light.

Filtered samples present on a filter membrane.