166
Use of Radioisotopic Methodology in Aquatic Microbial Ecology
for these estimations, because hydrolyzate contains a complex mixture of substances. Therefore it is impossible to decrease it, as can be done with glutamate. The needed hydrolyzate can however be easily prepared with labeled
algae, such as Chiarella, as described above (see Sect. 3.3). Its specific radioactivity can be preconditioned also at the preparation stage of the medium for
their cultivation. For this, thc contents of TCOrcarbon should be adju'Sted at
8-lOmgl- 1 by adding 0.3-0.4mCil- 1 of 14C-carbonate radioactivity.
As an indirect indicator of relative heterotrophic microbial activity, the
incorporation rate of the 3H-methyl thymidine by microbial populations also
may be used. A corresponding working solution should be prepared with
about 200 times less specific activity than in the batches supplied by Amersham, e.g., -200 mCi mmot 1 instead of 20-50 Ci mmol- 1 • It is necessary to avoid
the effects of isotopic dilution, absorption of label on filters and on glassware,
which largely complicate the use of the TDR method (see below, Sect. 4.4.3).
To prepare the needed working solution ofTDR, 0.1 ml of molar solution (e.g.,
100mmol) of unlabeled thymidine in 20% alcohol solution is added to the
batch, containing usually 5 mCiml- 1 of TDR. In this case the specific activity
of thymidine will be decreased to the necessary 100-200 mCi mmol- 1 . This
operation may be performed with 1-2-ml subsamples of the original batch
solution transferred into another small vial. Being prepared with the alcohol
solution makes it safe for such a procedure, and needs no special heat sterilization, as when preserved under cold conditions. Before its use, 0.1 ml of the
thus prepared TDR solution is diluted in 1 ml of bidistilled water. It makes the
ready-for-use working solution, 0.02-0.04-ml portions of which are injected
into the samples per 100ml of water. This solution just before its use might be
filtered through 0.2-mm pore size Sartorius membrane filter with the aid of
minifunnel attached to the syringe to obtain a lower and more stable control
background.
2. Sampling and preparation of glassware
For sampling, it is possible to use an ordinary clean plastic or Plexiglas water
bottle. To avoid microbial contamination of the samples from its walls, it can
be rinsed just before its immersion into the water with 5% HCl solution or
70% alcohol. Then the antiseptic is washed off by passing open through the
water column. The bottles are prepared by Lugol sterilization as described
above (see Sect. 2.3.2.3.) for primary production measurement. Dry sterilization is also possible, but not wet, because of possible contamination of the
bottles with the organic matter via the vapor in the autoclave. The optimal
volume of bottles for work in mesotrophic and eutrophic waters is 100-250ml,
and in oligotrophic waters 200-300ml. The bottles need to have the marks on
their walls corresponding to some exact volume: 100, 150, 200, or 300ml.
Bottles should be of the BOD type with ground glass stoppers. Lugol sterilization is suitable for these bottles. When employing dry sterilization, serum
bottles also can be used by being closed with serum rubber stoppers sterilized
by boiling.
Use of Radioisotopic Methodology in Aquatic Microbial Ecology
for these estimations, because hydrolyzate contains a complex mixture of substances. Therefore it is impossible to decrease it, as can be done with glutamate. The needed hydrolyzate can however be easily prepared with labeled
algae, such as Chiarella, as described above (see Sect. 3.3). Its specific radioactivity can be preconditioned also at the preparation stage of the medium for
their cultivation. For this, thc contents of TCOrcarbon should be adju'Sted at
8-lOmgl- 1 by adding 0.3-0.4mCil- 1 of 14C-carbonate radioactivity.
As an indirect indicator of relative heterotrophic microbial activity, the
incorporation rate of the 3H-methyl thymidine by microbial populations also
may be used. A corresponding working solution should be prepared with
about 200 times less specific activity than in the batches supplied by Amersham, e.g., -200 mCi mmot 1 instead of 20-50 Ci mmol- 1 • It is necessary to avoid
the effects of isotopic dilution, absorption of label on filters and on glassware,
which largely complicate the use of the TDR method (see below, Sect. 4.4.3).
To prepare the needed working solution ofTDR, 0.1 ml of molar solution (e.g.,
100mmol) of unlabeled thymidine in 20% alcohol solution is added to the
batch, containing usually 5 mCiml- 1 of TDR. In this case the specific activity
of thymidine will be decreased to the necessary 100-200 mCi mmol- 1 . This
operation may be performed with 1-2-ml subsamples of the original batch
solution transferred into another small vial. Being prepared with the alcohol
solution makes it safe for such a procedure, and needs no special heat sterilization, as when preserved under cold conditions. Before its use, 0.1 ml of the
thus prepared TDR solution is diluted in 1 ml of bidistilled water. It makes the
ready-for-use working solution, 0.02-0.04-ml portions of which are injected
into the samples per 100ml of water. This solution just before its use might be
filtered through 0.2-mm pore size Sartorius membrane filter with the aid of
minifunnel attached to the syringe to obtain a lower and more stable control
background.
2. Sampling and preparation of glassware
For sampling, it is possible to use an ordinary clean plastic or Plexiglas water
bottle. To avoid microbial contamination of the samples from its walls, it can
be rinsed just before its immersion into the water with 5% HCl solution or
70% alcohol. Then the antiseptic is washed off by passing open through the
water column. The bottles are prepared by Lugol sterilization as described
above (see Sect. 2.3.2.3.) for primary production measurement. Dry sterilization is also possible, but not wet, because of possible contamination of the
bottles with the organic matter via the vapor in the autoclave. The optimal
volume of bottles for work in mesotrophic and eutrophic waters is 100-250ml,
and in oligotrophic waters 200-300ml. The bottles need to have the marks on
their walls corresponding to some exact volume: 100, 150, 200, or 300ml.
Bottles should be of the BOD type with ground glass stoppers. Lugol sterilization is suitable for these bottles. When employing dry sterilization, serum
bottles also can be used by being closed with serum rubber stoppers sterilized
by boiling.