Sampling, Biomass Estimation and Counts of Freshwater Algae - Freshwater Algae: Identification, Enumeration and Use as Bioindicators - page 71

Algal counts The algae present on the filter
membrane can now be observed and counted using
a normal light microscope or by epifluorescence
microscopy (see end of section). Two counting meth-
ods are commonly used.

passing small amounts (10-15 ml) of increasing
concentrations of ethanol through the filter. When
dehydration is complete the algal cells are stained
with alcoholic fast green (0.1% in 95% ethanol)
by allowing the filter to stand with the stain over-
lying it for 20 min before sucking it through.
The filter is finally washed under gentle vacuum
with a small amount of ethanol (Vollenweider,
1969).

1. Most Probable Number (MPN) estimate

This method can be used for species that are particu-
larlyabundant inthesample. UsingtheMPNestimate
runs the risk that uncommon or infrequent species
might be overlooked, and these should be estimated
using an alternative technique. The MPN technique
is also used in bacteriology and records not only the
presence but also the absence of organisms.
The procedure is as follows.

2. Take a drop of either microscope immersion oil
or good quality cedar wood oil and place it on a
microscope slide. Take an unused dry membrane
filter paper and place it on the oil droplet. After a
few minutes the oil penetrates the filter which then
goes completely clear. If this does not happen gen-
tle warmth (about 30-35 ◦ C) can be tried to hasten
the clearing process. If the filter still does not go
transparent then this method cannot be used as the
filter is unsuitable. If the filter used for the sam-
ple is the correct type then the process is repeated
but this time the filter paper plus filtered sample
is placed, face up, on the oil droplet. The slide
+ filter are now placed in a dust-free atmosphere
with or without gentle warming until the filter is
transparent. A cover slip is now placed over the
clear filter avoiding trapping any air bubbles. To
help avoid air bubbles a small drop of oil can be
placed on the underside of the cover slip which is
then slowly and carefully lowered onto the filter.
Care must be taken to avoid disturbing the cells on
the filter surface. Cedar wood oil is less expensive
than immersion oil but is darker in colour so that
subsequent microscopic observations are not quite
as clear.

First view the slide under low power to confirm that
the cells present are randomly distributed. Now,
using whatever magnification is appropriate, iden-
tify the more common species present. It is always
helpful to have previously looked at a fresh unfil-
tered sample to help identification as less robust
species can be damaged by the filtration process
making identification a little more difficult. Robust
species such as diatoms are not affected by the fil-
tration process. It can be easier to spot and identify
cells if they have been stained (e.g. with Lugol's
iodine).

Select an appropriate magnification objective so
that only the most common species are present in
the field of view for about 80% of the time. It may
be necessary to either concentrate the sample, that
is filter a larger volume through the membrane, or
diluteittoachievetherequiredconcentration.Ifthe
membrane is marked with a grid, then each square
can be numbered and a random number table used
to select particular squares for algal recording. If
gridded membranes are not available the slide is
moved about and observed in a random manner.
Fifty randomly selected fields should be observed.
Note the number of fields of view in which the
different species occur. If there are less than 80%
occurrences of the key species, then select a lower
magniicationobjective,thusgivingalargerieldof
view. If there is greater than 90% occurrence, then

3. This approach is basically the same as in option
(ii) but helps preserve the randomness of cell dis-
tribution on the filter a little better. In this case,
a drop of immersion oil is placed on a large cov-
erslip (large enough to take the whole filter pad)
and allowed to spread. The filter is then placed
face down on the coverslip, transferred to a dust-
free environment and, with or without warming,
allowed to go transparent. The coverslip with fil-
ter are then carefully inverted onto a microscope
slide.