168
Use of Radioisotopic Methodology in Aquatic Microbial Ecology
excess of water over the volume marks is discharged in all samples. Then equal
portions of working solution of labeled organic substrate are injected into
them. Several (two to three) parallel samples serve as controls. They are fixed
with a weak Lugol solution before the injection of isotope. All the sets of
samples thus charged are incubated at the same standard temperature of 1820°C. The incubation is halted by fixation of all samples with the same Lugol
solution when a slightly yellow color. Before filtration, several drops of 5%
thiosulfate solution are added to the samples to kill the iodine, which might
induce chemoluminescence. Then two parallel subsamples 50ml or 100mi each
are taken from each sample and filtered onto 0.45-mm pore size membrane
filters using a filtration set as shown in Fig. 2.7. The filters are subsequently
rinsed with 10ml of prefiltered natural water with 2ml of carrier solution and
then again with 5 ml of the same natural water; 1 % glutamate solution is used
as the carrier solution when employing the glutamate as the labeled substrate,
while it should be 0.1 % solution of yeast extract when using the labeled
hydrolyzate.
The filters are placed into scintillation vials and radioassayed. Their
cpm radioactivity R; expressed as cpm 100ml- 1 and corrected per control
counting is accepted as corresponding to a relative activity of heterotrophic
bacteria. In order to avoid the drawbacks which could be caused by the uptake
of organic substrates also by planktonic algae, this possibility should be evaluated by control filtration on 2-3-I-!m pore size NucIeopore filters. If the algal
uptake turns out to be large, over 15-20% of the whole, the fixed samples
should be first prefiltered at 3-l-.lm NucIeopore filters using the funnel without
external vacuum funnel of 40-mm internal diameter arranged as shown in
Fig. 4.8.
Once a standard procedure is established, it may be used for the spatial
seasonal and annual monitoring of heterotrophic activity as the parameter
characterizing the changes of trophicallevel, and the self-purification activity
in a given area. After the standardization of the procedure, the cpm indices of
heterotrophic potential might be calibrated in relation to the number of heterotrophic bacteria estimated by the microbiological plate count. This gives
the possibility to compare data obtained by this method with those obtained
in other areas by plate counts.
With the use of the above methodology, it is also possible to estimate
the specific radioactivity in organic substrates. Its value, estimated for given
counting conditions such as cpm mmol- I or cpm mg C-l, in the case of the
use of glutamate, could be calculated as the ratio of cpm radioactivity in the
batch (measured directly in the working solution and recalculated per whole
batch), and its carbon or I-.lmolar glutamate content, estimated as the sum of
its initial content in the batch plus the added amount of unlabeled glutamate
as a carrier to decrease specific activity [see above, (1)]. In the case of using
home-made protein hydrolyzate, the specific radioactivity could be easily
measured directly as described above (see Sect. 3.4.1). When using the labeled
Use of Radioisotopic Methodology in Aquatic Microbial Ecology
excess of water over the volume marks is discharged in all samples. Then equal
portions of working solution of labeled organic substrate are injected into
them. Several (two to three) parallel samples serve as controls. They are fixed
with a weak Lugol solution before the injection of isotope. All the sets of
samples thus charged are incubated at the same standard temperature of 1820°C. The incubation is halted by fixation of all samples with the same Lugol
solution when a slightly yellow color. Before filtration, several drops of 5%
thiosulfate solution are added to the samples to kill the iodine, which might
induce chemoluminescence. Then two parallel subsamples 50ml or 100mi each
are taken from each sample and filtered onto 0.45-mm pore size membrane
filters using a filtration set as shown in Fig. 2.7. The filters are subsequently
rinsed with 10ml of prefiltered natural water with 2ml of carrier solution and
then again with 5 ml of the same natural water; 1 % glutamate solution is used
as the carrier solution when employing the glutamate as the labeled substrate,
while it should be 0.1 % solution of yeast extract when using the labeled
hydrolyzate.
The filters are placed into scintillation vials and radioassayed. Their
cpm radioactivity R; expressed as cpm 100ml- 1 and corrected per control
counting is accepted as corresponding to a relative activity of heterotrophic
bacteria. In order to avoid the drawbacks which could be caused by the uptake
of organic substrates also by planktonic algae, this possibility should be evaluated by control filtration on 2-3-I-!m pore size NucIeopore filters. If the algal
uptake turns out to be large, over 15-20% of the whole, the fixed samples
should be first prefiltered at 3-l-.lm NucIeopore filters using the funnel without
external vacuum funnel of 40-mm internal diameter arranged as shown in
Fig. 4.8.
Once a standard procedure is established, it may be used for the spatial
seasonal and annual monitoring of heterotrophic activity as the parameter
characterizing the changes of trophicallevel, and the self-purification activity
in a given area. After the standardization of the procedure, the cpm indices of
heterotrophic potential might be calibrated in relation to the number of heterotrophic bacteria estimated by the microbiological plate count. This gives
the possibility to compare data obtained by this method with those obtained
in other areas by plate counts.
With the use of the above methodology, it is also possible to estimate
the specific radioactivity in organic substrates. Its value, estimated for given
counting conditions such as cpm mmol- I or cpm mg C-l, in the case of the
use of glutamate, could be calculated as the ratio of cpm radioactivity in the
batch (measured directly in the working solution and recalculated per whole
batch), and its carbon or I-.lmolar glutamate content, estimated as the sum of
its initial content in the batch plus the added amount of unlabeled glutamate
as a carrier to decrease specific activity [see above, (1)]. In the case of using
home-made protein hydrolyzate, the specific radioactivity could be easily
measured directly as described above (see Sect. 3.4.1). When using the labeled