Techniques for the Quantification Density of Microplankton Populations
277
between 1.2 and 1.6, depending on the composition of phytoplankton. This correction accounts for most of the losses of the pico fraction comprised by
cyanobacteria and other picoalgae. This latter fraction should in any case be
counted separately for the most adequate presentation of phytoplankton composition. For its quantification, the epifluorescence microscopy technique must
be used, which is described below.
3. Direct microscopy techniques: viable counts
This technique is most useful and adequate in its application for phytoplankton quantification in areas with dense populations (biomass >3-5 gm- 3 ), which
are formed during seasonal phytoplankton blooms. In this case, there is no
need to concentrate the samples. The blooms are usually induced by a narrow
selection of algal species, which can be easily quantified at their natural density,
using simple chambers like shown in Figs. 6.3 and 6.6. The only problem is
counting the motile phytoflagellates in their dense populations; but in this case
their motility may be slowed down by the addition of empirically adjusted
quantities of ether solution, of glutaraldehyde, or of the neuroleptic substance
tisercin (mepromasine; Vedernikov and Mikaelyan 1983) to the samples. The
algal cells in this case are rapidly counted within the field of microscope or
along the stripes marked on the bottom of the chamber.
4. Epifluorescence microscopic counting
This method was developed by Sherr and Sherr (1983) and Caron (1983).
Its modern versions are described in the handbook edited by Kemp (1993).
Under some specific conditions, when the phytoplankton populations are
dense (>2-3 gm- 3 of wet biomass) and are dominated by several species, or
when they are composed mainly of nano- and picoalgae with a negligible presence of larger forms of >5-1O)lm size, this method is sufficient for adequate
quantification of an entire phytoplankton population. The composition of phytoplankton is examined in this case additionally in the sedimentation or filtration concentrates and in viable preparations. For other situations, this method
serves mostly as a complementary one to the Utremohl or the concentration
method, described above. Here, the epifluorescence method is described in its
modification by P. Sorokin.
The phytoplankton samples are fixed with glutaraldehyde, which, being
a soft fixer, induces a minor deformation of microplankters, even of the
ciliates. At the same time, it causes the denaturation of proteins in their protoplasm, thus making their cells more rigid and resistant to mechanical disintegration during the filtration procedure. For a period of several hours, it
preserves also the primary strength of the chlorophyll autofluorescence in
algae, which is extremely important for epifluorescence counting of algae. The
fixed water samples should be subjected to filtration not later than 3-4h after
sampling and fixation, but the best time is 15-30 min. Before being filtered,
the samples are stained with the fluorochrome primuline, which is combined
with the proteins of the cell's protoplasm, inducing a bluish green shining,
277
between 1.2 and 1.6, depending on the composition of phytoplankton. This correction accounts for most of the losses of the pico fraction comprised by
cyanobacteria and other picoalgae. This latter fraction should in any case be
counted separately for the most adequate presentation of phytoplankton composition. For its quantification, the epifluorescence microscopy technique must
be used, which is described below.
3. Direct microscopy techniques: viable counts
This technique is most useful and adequate in its application for phytoplankton quantification in areas with dense populations (biomass >3-5 gm- 3 ), which
are formed during seasonal phytoplankton blooms. In this case, there is no
need to concentrate the samples. The blooms are usually induced by a narrow
selection of algal species, which can be easily quantified at their natural density,
using simple chambers like shown in Figs. 6.3 and 6.6. The only problem is
counting the motile phytoflagellates in their dense populations; but in this case
their motility may be slowed down by the addition of empirically adjusted
quantities of ether solution, of glutaraldehyde, or of the neuroleptic substance
tisercin (mepromasine; Vedernikov and Mikaelyan 1983) to the samples. The
algal cells in this case are rapidly counted within the field of microscope or
along the stripes marked on the bottom of the chamber.
4. Epifluorescence microscopic counting
This method was developed by Sherr and Sherr (1983) and Caron (1983).
Its modern versions are described in the handbook edited by Kemp (1993).
Under some specific conditions, when the phytoplankton populations are
dense (>2-3 gm- 3 of wet biomass) and are dominated by several species, or
when they are composed mainly of nano- and picoalgae with a negligible presence of larger forms of >5-1O)lm size, this method is sufficient for adequate
quantification of an entire phytoplankton population. The composition of phytoplankton is examined in this case additionally in the sedimentation or filtration concentrates and in viable preparations. For other situations, this method
serves mostly as a complementary one to the Utremohl or the concentration
method, described above. Here, the epifluorescence method is described in its
modification by P. Sorokin.
The phytoplankton samples are fixed with glutaraldehyde, which, being
a soft fixer, induces a minor deformation of microplankters, even of the
ciliates. At the same time, it causes the denaturation of proteins in their protoplasm, thus making their cells more rigid and resistant to mechanical disintegration during the filtration procedure. For a period of several hours, it
preserves also the primary strength of the chlorophyll autofluorescence in
algae, which is extremely important for epifluorescence counting of algae. The
fixed water samples should be subjected to filtration not later than 3-4h after
sampling and fixation, but the best time is 15-30 min. Before being filtered,
the samples are stained with the fluorochrome primuline, which is combined
with the proteins of the cell's protoplasm, inducing a bluish green shining,