114
Radioisotopic Methods for the Study of Nutrition in Aquatic Animals
rotifers can also be washed out of the labeled food in the 20-f..1m plankton net
in a miniature cylinder of some lOmm diameter, arranged as shown in Fig.
3.2F. After washing, they are transferred with a fine pipette into the same
watch glass with several ml of water and fixed, then treated as described above.
The larger mesozooplanktonic food organisms like cladocerans, copepods,
the large rotifer Asplanchna, or large Anemia nauplii au:; wash~d out from the
labeled food with the use of the same device (Fig. 3.2F). When washed, they
are transferred from the cylinder of the device with the aid of a wide-mouth
pipette (Fig. 3.3B) into a beaker, where they are killed with 10% alcohol
solution or by slight heating. From the beaker a definite quantity of them
(ca. 20 to 50 sp., depending on their size) is transferred first into a watch glass,
and their exact number is counted. They are then transferred into a scintillation vial. The excess of water is removed by pipet, the end of which is closed
with a plankton net (Fig. 3.3). The sample is then ready for further treatment,
as described above.
2. Determination of organic matter content in labeled food (W)
The second value required to calculate Cr is W, the organic carbon content in
an amount of labeled food. This value can be estimated by any available
method: Van Slyke combustion, CNH analyzer, or UV combustion. For practical use in the field laboratory or on the ship, the wet combustion method could
also be applied using the following procedure. The exact volume of suspension
of the labeled food or a definite number of labeled animals is placed into small
combustion test tubes (Fig. 3.2.A). Then standard ~3-ml portions of 0.05N
solution of KjCr207 or Cr02 in 95% H 2S04 are added to them with the aid of
a special pipet (Fig. 3.2B). The test tubes are kept either in a boiling water bath
for 2 h or in the thermostat at 135°C for 30 min. Into parallel controls, 0.3 ml of
distilled water is added. After cooling, the contents are transferred into a 50ml flask, being diluted with water to 20 ml. After cooling, the mixture is poured
into another flask with a ground glass stopper, into which 0.5 ml fresh 20%
sodium iodide solution and 20ml of water have been previously added. After
5 min, the iodine formed is titrated with 0.02 N thiosulfate solution. For each
sample, three to four parallel replicates are analyzed. The chromic solution for
the combustion is prepared with addition of 100mgl- 1 AgS04 as the catalyzer.
The freshly prepared mixture is heated before use to to 135°C.
Calculation of the amount of organic carbon (W) in the combusted sample
is carried out using the formula: W = (Vo - VI) K mgC, where Vo = ml of
0.02 N thiosulfate used for blank titration, VI is the mean value for sample
titration, and K the amount of organic carbon, which corresponds to 1 ml of
0.02 N thiosulfate; for glucose totally combusted to CO2, it will be 60 f..lg C.
It is, however, better to use the value of K estimated empirically by analogous
combustion of 0.3 ml of peptone solution or solution of some amino acid
(glutamic, for example) with a known amount of organic carbon per ml.
For the calculation of Cr the values of W thus obtained are expressed per
ml of suspension, per 10 3 sp. of small animals (ciliates, rotifers, chidoruses) or
Radioisotopic Methods for the Study of Nutrition in Aquatic Animals
rotifers can also be washed out of the labeled food in the 20-f..1m plankton net
in a miniature cylinder of some lOmm diameter, arranged as shown in Fig.
3.2F. After washing, they are transferred with a fine pipette into the same
watch glass with several ml of water and fixed, then treated as described above.
The larger mesozooplanktonic food organisms like cladocerans, copepods,
the large rotifer Asplanchna, or large Anemia nauplii au:; wash~d out from the
labeled food with the use of the same device (Fig. 3.2F). When washed, they
are transferred from the cylinder of the device with the aid of a wide-mouth
pipette (Fig. 3.3B) into a beaker, where they are killed with 10% alcohol
solution or by slight heating. From the beaker a definite quantity of them
(ca. 20 to 50 sp., depending on their size) is transferred first into a watch glass,
and their exact number is counted. They are then transferred into a scintillation vial. The excess of water is removed by pipet, the end of which is closed
with a plankton net (Fig. 3.3). The sample is then ready for further treatment,
as described above.
2. Determination of organic matter content in labeled food (W)
The second value required to calculate Cr is W, the organic carbon content in
an amount of labeled food. This value can be estimated by any available
method: Van Slyke combustion, CNH analyzer, or UV combustion. For practical use in the field laboratory or on the ship, the wet combustion method could
also be applied using the following procedure. The exact volume of suspension
of the labeled food or a definite number of labeled animals is placed into small
combustion test tubes (Fig. 3.2.A). Then standard ~3-ml portions of 0.05N
solution of KjCr207 or Cr02 in 95% H 2S04 are added to them with the aid of
a special pipet (Fig. 3.2B). The test tubes are kept either in a boiling water bath
for 2 h or in the thermostat at 135°C for 30 min. Into parallel controls, 0.3 ml of
distilled water is added. After cooling, the contents are transferred into a 50ml flask, being diluted with water to 20 ml. After cooling, the mixture is poured
into another flask with a ground glass stopper, into which 0.5 ml fresh 20%
sodium iodide solution and 20ml of water have been previously added. After
5 min, the iodine formed is titrated with 0.02 N thiosulfate solution. For each
sample, three to four parallel replicates are analyzed. The chromic solution for
the combustion is prepared with addition of 100mgl- 1 AgS04 as the catalyzer.
The freshly prepared mixture is heated before use to to 135°C.
Calculation of the amount of organic carbon (W) in the combusted sample
is carried out using the formula: W = (Vo - VI) K mgC, where Vo = ml of
0.02 N thiosulfate used for blank titration, VI is the mean value for sample
titration, and K the amount of organic carbon, which corresponds to 1 ml of
0.02 N thiosulfate; for glucose totally combusted to CO2, it will be 60 f..lg C.
It is, however, better to use the value of K estimated empirically by analogous
combustion of 0.3 ml of peptone solution or solution of some amino acid
(glutamic, for example) with a known amount of organic carbon per ml.
For the calculation of Cr the values of W thus obtained are expressed per
ml of suspension, per 10 3 sp. of small animals (ciliates, rotifers, chidoruses) or