278
Assay of Some Common Hydrobiological Techniques
which delineates the margins and shape of the cells while not masking the
reddish or brownish shining of chlorophyll. Acridine orange can also be used
for the same porpose.
The filtration proceeds through black Nucleopore filters of 0.4-0.6-/.lm
pore size. Larger pores become visible during microscopic examination at high
magnification; but if the latter cannot to used, filtration can be performed
even through filters with 1- to 3-/.lm pore size in order to count larger, more
random forms better. The use of filters with pores of less than 0.4-0.5/.lm
increases detritus fluorescence. Filtration must be performed under a controlled external vacuum as low as possible, which prevents the destruction of
gentle naked forms or forms with a complex shell configuration, like Chaetoceros or planktonic Nitzschia species. When filtration ends, the vacuum is
instantly voided, and the filters are immediately embedded in the oil preparation to prevent the drying and shrinkage of the algal cells. The oil preparation should be immediately examined under the epifluorescence microscope.
In this case, the red chlorophyll autofluorescence and the blue-green primuline shining will be at their brightest, thus making the counting procedure
easier; if this is impossible, the frozen oil preparations can be preserved for up
to several weeks.
With the use of this technique, practically all size forms of planktonic algae
and other planktonic phototrophs, e.g., the symbiotic ciliate Mesodinium
rub rum, which is among the most common and abundant components of phototrophic microplankton either in brackish coastal waters or in the open seas
and in oceanic upwelling areas. Often, this form completely replaces the
phytoplankton. This small ciliate of ~ 30 /.lm size possesses the chrisomonadic
chloroplast. Sometimes it attains a huge biomass up to 50 g m- 3 • It can be
quantified only with the epifluorescence technique or by viable counting.
Larger forms like diatoms and dinoflagellates are quantified under the objective 20, the nanoalgae under the objective 40, and the picoalgae under oil
immersion 90.
The procedure of epifluorescence counting is performed as follows:
it is best (and cheapest) to cut out Nucleopore filters 0.4-0.6-/.lm
pore size and 20mm in diameter from the filtering films with a sharp corer,
placing many layers of films between the paper layers. Then the filters
are stained with Sudan black. The saturated solution of this stain is prepared
in ethanol. The filters soaked in this stain solution are placed in a beaker
covered with a glass plate and kept for several hours at 90 DC until the liquid
almost completely evaporates or they are kept for several days at room temperature. Then the filters are washed free of the stain in ethanol and dried.
Before use, they are soaked in a weak Trilon B solution (one drop per 100ml).
The same diameter membrane filters of 0.6-0.8-/.lm pore size should also be
prepared by being soaked in the same Trilon B solution. These filters are used
as for laying under the black Nucleopore filters to provide uniform filtration
of the sample.
Assay of Some Common Hydrobiological Techniques
which delineates the margins and shape of the cells while not masking the
reddish or brownish shining of chlorophyll. Acridine orange can also be used
for the same porpose.
The filtration proceeds through black Nucleopore filters of 0.4-0.6-/.lm
pore size. Larger pores become visible during microscopic examination at high
magnification; but if the latter cannot to used, filtration can be performed
even through filters with 1- to 3-/.lm pore size in order to count larger, more
random forms better. The use of filters with pores of less than 0.4-0.5/.lm
increases detritus fluorescence. Filtration must be performed under a controlled external vacuum as low as possible, which prevents the destruction of
gentle naked forms or forms with a complex shell configuration, like Chaetoceros or planktonic Nitzschia species. When filtration ends, the vacuum is
instantly voided, and the filters are immediately embedded in the oil preparation to prevent the drying and shrinkage of the algal cells. The oil preparation should be immediately examined under the epifluorescence microscope.
In this case, the red chlorophyll autofluorescence and the blue-green primuline shining will be at their brightest, thus making the counting procedure
easier; if this is impossible, the frozen oil preparations can be preserved for up
to several weeks.
With the use of this technique, practically all size forms of planktonic algae
and other planktonic phototrophs, e.g., the symbiotic ciliate Mesodinium
rub rum, which is among the most common and abundant components of phototrophic microplankton either in brackish coastal waters or in the open seas
and in oceanic upwelling areas. Often, this form completely replaces the
phytoplankton. This small ciliate of ~ 30 /.lm size possesses the chrisomonadic
chloroplast. Sometimes it attains a huge biomass up to 50 g m- 3 • It can be
quantified only with the epifluorescence technique or by viable counting.
Larger forms like diatoms and dinoflagellates are quantified under the objective 20, the nanoalgae under the objective 40, and the picoalgae under oil
immersion 90.
The procedure of epifluorescence counting is performed as follows:
it is best (and cheapest) to cut out Nucleopore filters 0.4-0.6-/.lm
pore size and 20mm in diameter from the filtering films with a sharp corer,
placing many layers of films between the paper layers. Then the filters
are stained with Sudan black. The saturated solution of this stain is prepared
in ethanol. The filters soaked in this stain solution are placed in a beaker
covered with a glass plate and kept for several hours at 90 DC until the liquid
almost completely evaporates or they are kept for several days at room temperature. Then the filters are washed free of the stain in ethanol and dried.
Before use, they are soaked in a weak Trilon B solution (one drop per 100ml).
The same diameter membrane filters of 0.6-0.8-/.lm pore size should also be
prepared by being soaked in the same Trilon B solution. These filters are used
as for laying under the black Nucleopore filters to provide uniform filtration
of the sample.