146
Radioisotopic Methods for the Study of Nutrition in Aquatic Animals
3.6.3.2 Technique for Measurement of DOM Uptake
Experimental techniques are as follows
1. Preparation of 14C-Iabeled DOM
The best solution for the selection of the kind of DOM to be used in the
experiments is the home-made hydrolyzates of labeled microalgae or the
commercially supplied ready-for-use labeled protein hydrolyzates. The convenience of the former is that its inverse specific radioactivity (Cr ) can be
adjusted to the required level also at the stage of the preparation of labeled
algae (see Sect. 3.3.1). The hydrolyzates represent a mixture of low molecular
weight peptides, amino acids, sugars, and lipids equally labeled. This quality
allows them to resemble the composition of DOM which is present in natural
waters. It makes it possible to use the results of estimations on nutritional
value of these labeled hydrolyzates for the evaluation of the use of DOM in
the food webs in situ; but the latter does not apply when the individual sugars
or amino acids are employed as the labeled DOM. One advantage in the use
of the latter as the labeled DOM is that their specific radioactivity can be
preconditioned by the addition of some amounts of unlabeled kinds.
The procedure for preparation of labeled algal hydrolyzate has been
described above (see Sect. 3.3.1). The Cr values in any kind of DOM, either
home-made or commercially purchased, should be, in any case, determined by
the researcher himself under the same counting conditions which are accepted
for animal radioassay. The estimation of Cr must be performed in the concentrated hydrolyzate solution ready for the subsequent dilution for making the
working solution, but not in the latter per se because the content of Corg is too
low. The same applies to the batch preparations of commercially acquired
labeled DOM. In the latter case, the vial must be opened and diluted with
water to a volume of 2-3 ml. If it is the individual compound, the unlabeled
carrier is added into it to adjust the necessary specific radioactivity (0.3-1.0 x
10 6 cpm).lgC- 1 taking possible quenching into account). In the commercially
acquired batches, it is usually many times more. Then two to three subsamples
of 0.2-0.3 ml are taken from the volume of concentrated labeled DOM to
measure the content of organic carbon in this solution (W mgCml- 1 ). The
analysis might be performed by any accessible automated micromethod (CNH
analyzer or Technicon), but certain drawbacks with the analysis of highly
radioactive material might arise. In this case, the following simple wet combustion micromethod can be employed.
A O.l-ml aliquot of labeled DOM concentrated solution containing
between 10 and 100).lg C is injected into a small test tube, being followed with
the exact volume (between 0.3 and 0.5 ml) of 0.1 N sodium dichromate solution in 95% H 2S 04 + traces of AgS04' using the special pipet shown in Fig.
3.3C The test tube is closed with a glass stopper and heated in the boiling bath
for 2h or kept in the thermostat at 110°C for 1 h. The same portion of chromic
Radioisotopic Methods for the Study of Nutrition in Aquatic Animals
3.6.3.2 Technique for Measurement of DOM Uptake
Experimental techniques are as follows
1. Preparation of 14C-Iabeled DOM
The best solution for the selection of the kind of DOM to be used in the
experiments is the home-made hydrolyzates of labeled microalgae or the
commercially supplied ready-for-use labeled protein hydrolyzates. The convenience of the former is that its inverse specific radioactivity (Cr ) can be
adjusted to the required level also at the stage of the preparation of labeled
algae (see Sect. 3.3.1). The hydrolyzates represent a mixture of low molecular
weight peptides, amino acids, sugars, and lipids equally labeled. This quality
allows them to resemble the composition of DOM which is present in natural
waters. It makes it possible to use the results of estimations on nutritional
value of these labeled hydrolyzates for the evaluation of the use of DOM in
the food webs in situ; but the latter does not apply when the individual sugars
or amino acids are employed as the labeled DOM. One advantage in the use
of the latter as the labeled DOM is that their specific radioactivity can be
preconditioned by the addition of some amounts of unlabeled kinds.
The procedure for preparation of labeled algal hydrolyzate has been
described above (see Sect. 3.3.1). The Cr values in any kind of DOM, either
home-made or commercially purchased, should be, in any case, determined by
the researcher himself under the same counting conditions which are accepted
for animal radioassay. The estimation of Cr must be performed in the concentrated hydrolyzate solution ready for the subsequent dilution for making the
working solution, but not in the latter per se because the content of Corg is too
low. The same applies to the batch preparations of commercially acquired
labeled DOM. In the latter case, the vial must be opened and diluted with
water to a volume of 2-3 ml. If it is the individual compound, the unlabeled
carrier is added into it to adjust the necessary specific radioactivity (0.3-1.0 x
10 6 cpm).lgC- 1 taking possible quenching into account). In the commercially
acquired batches, it is usually many times more. Then two to three subsamples
of 0.2-0.3 ml are taken from the volume of concentrated labeled DOM to
measure the content of organic carbon in this solution (W mgCml- 1 ). The
analysis might be performed by any accessible automated micromethod (CNH
analyzer or Technicon), but certain drawbacks with the analysis of highly
radioactive material might arise. In this case, the following simple wet combustion micromethod can be employed.
A O.l-ml aliquot of labeled DOM concentrated solution containing
between 10 and 100).lg C is injected into a small test tube, being followed with
the exact volume (between 0.3 and 0.5 ml) of 0.1 N sodium dichromate solution in 95% H 2S 04 + traces of AgS04' using the special pipet shown in Fig.
3.3C The test tube is closed with a glass stopper and heated in the boiling bath
for 2h or kept in the thermostat at 110°C for 1 h. The same portion of chromic