190
Use of Radioisotopic Methodology in Aquatic Microbial Ecology
exact value of its cpm radioactivity is measured also as described there. This
avoids problems with quenching correction.
The total contents of TCOrcarbon in the water samples Kc should be
determined as described above (see Sect. 2.3.2.2). For dark incubations at a
temperature close to that in situ, it is first necessary to prepare black bags of
a sufficient size to place two parallel bottles. They are made of two to three
layers of thick black cloth. The incubation bath can be made either of a thermostatic aquarium placed inside the black box, or of the black box placed
inside the deck aquarium (see Fig. 2.4) or into the shore stand-incubator (see
Fig. 2.21).
It is easy to apply this method in the laboratory where the measurements
of primary production are also fixed because both primary and the microbial
production are measured with the use of similar techniques. For filtration of
the samples 0.2-0.3-llm pore size Sartorius or 0.45-llm Millipore membrane
filters can be used. Nucleopore filters are not suitable for this procedure. Their
filter diameter should be 25 mm, and the diameter of filtering surface in the
funnel is 19-20 mm. The filters must be boiled in distilled water. After this, they
may be used wet or after drying, in which case the filter should be first
immersed in a weak detergent solution to be completely moistened.
The procedure is as follows. The samples of water are collected at each
station (or at each depth) into two parallel bottles or into one bottle twice as
large, taking all the precautions described above (see Sect. 2.3.2.4). If a dense
mesozooplankton is present in the sampling site, the water from the water
bottle is discharged first into the glass jar. From this jar water is subsampled
into the experimental bottles through the syphon, which, at the end inserted
in the jar, has a funnel 25mm diameter with 150-mm mesh plankton net covering its wide end to prevent large zooplankton grazers disturbing the sample.
The aim of these precautions is to avoid the destruction of microbial aggregations in subsamples. It may artificially accelerate microbial activity and
change its composition during the relatively long incubations required for this
method.
Before the isotopic solution is injected into them, the set of samples thus
collected is placed into the black bags for about 1 h being kept at a temperature close to that in situ to let the phytoplankton use the energy accumulated
from the light and stored within the cells of algae as ATP. This decreases their
dark CO2 uptake during the subsequent incubation. While the bulk of the
algae are then avoided due to postfiltration, some of them may pass through
the Nucleopore filters of 3-4-llm pore size which are used. After the previous
dark incubation is resumed, the black bags are opened one by one in a darkened place and the bottles inside them are rapidly charged with portions of
l4C-isotopic solution. After injection, they are instantly closed with glass stoppers, the stoppers are covered with pieces of plastic film fixed on their necks
with rubber bands. The necks of the black bags are tightly closed with rubber
bands. All these operations should be done to minimize illumination of experimental bottles while being charged with the isotope.
Use of Radioisotopic Methodology in Aquatic Microbial Ecology
exact value of its cpm radioactivity is measured also as described there. This
avoids problems with quenching correction.
The total contents of TCOrcarbon in the water samples Kc should be
determined as described above (see Sect. 2.3.2.2). For dark incubations at a
temperature close to that in situ, it is first necessary to prepare black bags of
a sufficient size to place two parallel bottles. They are made of two to three
layers of thick black cloth. The incubation bath can be made either of a thermostatic aquarium placed inside the black box, or of the black box placed
inside the deck aquarium (see Fig. 2.4) or into the shore stand-incubator (see
Fig. 2.21).
It is easy to apply this method in the laboratory where the measurements
of primary production are also fixed because both primary and the microbial
production are measured with the use of similar techniques. For filtration of
the samples 0.2-0.3-llm pore size Sartorius or 0.45-llm Millipore membrane
filters can be used. Nucleopore filters are not suitable for this procedure. Their
filter diameter should be 25 mm, and the diameter of filtering surface in the
funnel is 19-20 mm. The filters must be boiled in distilled water. After this, they
may be used wet or after drying, in which case the filter should be first
immersed in a weak detergent solution to be completely moistened.
The procedure is as follows. The samples of water are collected at each
station (or at each depth) into two parallel bottles or into one bottle twice as
large, taking all the precautions described above (see Sect. 2.3.2.4). If a dense
mesozooplankton is present in the sampling site, the water from the water
bottle is discharged first into the glass jar. From this jar water is subsampled
into the experimental bottles through the syphon, which, at the end inserted
in the jar, has a funnel 25mm diameter with 150-mm mesh plankton net covering its wide end to prevent large zooplankton grazers disturbing the sample.
The aim of these precautions is to avoid the destruction of microbial aggregations in subsamples. It may artificially accelerate microbial activity and
change its composition during the relatively long incubations required for this
method.
Before the isotopic solution is injected into them, the set of samples thus
collected is placed into the black bags for about 1 h being kept at a temperature close to that in situ to let the phytoplankton use the energy accumulated
from the light and stored within the cells of algae as ATP. This decreases their
dark CO2 uptake during the subsequent incubation. While the bulk of the
algae are then avoided due to postfiltration, some of them may pass through
the Nucleopore filters of 3-4-llm pore size which are used. After the previous
dark incubation is resumed, the black bags are opened one by one in a darkened place and the bottles inside them are rapidly charged with portions of
l4C-isotopic solution. After injection, they are instantly closed with glass stoppers, the stoppers are covered with pieces of plastic film fixed on their necks
with rubber bands. The necks of the black bags are tightly closed with rubber
bands. All these operations should be done to minimize illumination of experimental bottles while being charged with the isotope.