212
Use of Radioisotopic Methodology in Aquatic Microbial Ecology
If the batch is expected to be used during a prolonged period (up to
2-3 months), ethanol should be added up to a 5 0 1o-final concentration in
the batch to slow down self-radiolysis. Some authors recommend using a 2%
concentration on condition that 1O-20-ml portions of it are used for the injections into the experimental samples together with alcohol they contain. This
practice is not to be recommended, because in this case a quite significant
amount of external organic substrate is added to the sample which stimulates
microbial growth. Therefore, the batches preserved with alcohol have to be
evaporated and redisolved before use in any case. In the case of prolonged
storage, the concentration of ethanol in the batch might be adjusted up to 30%.
Before starting field work, an approximate portion of isotopic solution is
withdrawn from a batch bottle and transferred into a 20-ml sterile penicillin
bottle. Then it is evaporated under the stream of filtered air, and redissolved
in sterile distilled water to adjust its radioactivity to the level of about 200/..l
Ciml- 1 • After this the exact dpm radioactivity of this CH)-TDR working
solution should be measured as described above. Using the nominal Rc of the
batch, the absolute content of TDR in it should be calculated to know its
volume, containing OAnmol of this substance, to be added into a 20-ml water
sample to create a final concentration of about 20nmoll- l . The penicillin bottle
with the stock of working solution thus prepared is stored at 2-3°C and is
closed with a sterile serum stopper. Before use, the necessary amount of this
solution is withdrawn with the sterile syringe supplied with the little plastic
intermediate funnel with a 0.2-mm pore size cellulose membrane filter, or it
could be withdrawn with an ordinary sterile syringe and then passed through
the same filter fixed in a small plastic funnel of 1 cm diameter of working
surface. The working solution stored in the penicillin bottle may be used for
about a week.
2. The procedure of R; estimation
A water sample is taken two 50-ml serum glass bottles with plastic stoppers
sterilized with Lugol solution as described above (see Sect. 2.3.2.3). The bottles
are filled with the sample up to the 40-ml marks on the experimental bottle,
and 20-ml on the control. Then the portions of working CH)-TDR solution
containing respectively 0.8 and OAnmol of TDR are injected into them to
reach a final concentration in the subsamples of 20nmoll- I ; 1.2ml of 20% formalin is added into the control bottle before the injection of the working solution of isotope. The experimental bottles thus charged are incubated at the in
situ temperature for 20-30min if the latter is over 20°e. If it is between 10
and 20°C, the bottles should be incubated for 1-2 h, and if less than 5°C for
5-10 h. The reaction is halted by addition of the carrier solution which
contains 10mg of nonlabeled thymidine in 50ml: 0.2ml is added to the experimental bottle and 0.1 ml to the control. The bottles are chilled on ice. If the
latter operation is unavailable in the field, the experimental bottles are fixed
with the 0.5% (end concentration) formalin buffered at pH 8. Before processing, the bottles are in any case chilled. The chilled samples are distributed
Use of Radioisotopic Methodology in Aquatic Microbial Ecology
If the batch is expected to be used during a prolonged period (up to
2-3 months), ethanol should be added up to a 5 0 1o-final concentration in
the batch to slow down self-radiolysis. Some authors recommend using a 2%
concentration on condition that 1O-20-ml portions of it are used for the injections into the experimental samples together with alcohol they contain. This
practice is not to be recommended, because in this case a quite significant
amount of external organic substrate is added to the sample which stimulates
microbial growth. Therefore, the batches preserved with alcohol have to be
evaporated and redisolved before use in any case. In the case of prolonged
storage, the concentration of ethanol in the batch might be adjusted up to 30%.
Before starting field work, an approximate portion of isotopic solution is
withdrawn from a batch bottle and transferred into a 20-ml sterile penicillin
bottle. Then it is evaporated under the stream of filtered air, and redissolved
in sterile distilled water to adjust its radioactivity to the level of about 200/..l
Ciml- 1 • After this the exact dpm radioactivity of this CH)-TDR working
solution should be measured as described above. Using the nominal Rc of the
batch, the absolute content of TDR in it should be calculated to know its
volume, containing OAnmol of this substance, to be added into a 20-ml water
sample to create a final concentration of about 20nmoll- l . The penicillin bottle
with the stock of working solution thus prepared is stored at 2-3°C and is
closed with a sterile serum stopper. Before use, the necessary amount of this
solution is withdrawn with the sterile syringe supplied with the little plastic
intermediate funnel with a 0.2-mm pore size cellulose membrane filter, or it
could be withdrawn with an ordinary sterile syringe and then passed through
the same filter fixed in a small plastic funnel of 1 cm diameter of working
surface. The working solution stored in the penicillin bottle may be used for
about a week.
2. The procedure of R; estimation
A water sample is taken two 50-ml serum glass bottles with plastic stoppers
sterilized with Lugol solution as described above (see Sect. 2.3.2.3). The bottles
are filled with the sample up to the 40-ml marks on the experimental bottle,
and 20-ml on the control. Then the portions of working CH)-TDR solution
containing respectively 0.8 and OAnmol of TDR are injected into them to
reach a final concentration in the subsamples of 20nmoll- I ; 1.2ml of 20% formalin is added into the control bottle before the injection of the working solution of isotope. The experimental bottles thus charged are incubated at the in
situ temperature for 20-30min if the latter is over 20°e. If it is between 10
and 20°C, the bottles should be incubated for 1-2 h, and if less than 5°C for
5-10 h. The reaction is halted by addition of the carrier solution which
contains 10mg of nonlabeled thymidine in 50ml: 0.2ml is added to the experimental bottle and 0.1 ml to the control. The bottles are chilled on ice. If the
latter operation is unavailable in the field, the experimental bottles are fixed
with the 0.5% (end concentration) formalin buffered at pH 8. Before processing, the bottles are in any case chilled. The chilled samples are distributed