116
Radioisotopic Methods for the Study of Nutrition in Aquatic Animals
Now the vials are ready to be filled with the scintillation cocktail. To facilitate
hydrolysis, it is always helpful to treat the samples in the vials in the ultrasonic
disintegrator just before adding the chemical solubilizers. I cannot recommend
using oxidizers like perchloric acid or hydrogen peroxide as basic solubilizers,
which is often done in nutritional studies using radioisotopes, as they could
induce various artifacts like chemoluminescence or significant 14C losses due
to oxidation of the labeled organic matter. Only if it is urgently necessary to
bleach the samples to diminish their color quenching can benzoyl peroxide
can be used.
The homogenous counting samples of the body material of larger consumers can be prepared in two different ways. First they must be homogenized
mechanically (with the aid of a mortar or mixer) or ultrasonically. Then the
aliquots of the homogenate thus obtained, containing quasi equal amounts of
dry material, are distributed into the scintillation vials and treated with solubilizers or KOH as described above for small crustaceans. The exact content
of organic material per sample, as well as the optimal volumes and kind of
solubilizers, should be selected empirically in dependence on the kinds and
sizes of the objects, the animal grazers.
Another way is the preparation of alkaline hydrolyzate from whole
animals' bodies. Where it is possible, the soft tissues are separated from their
mineral skeletons (clams); if not, the whole animals could be subjected to alkaline hydrolysis together with the skeletons (corals, small fish). As the most
practical solubilizing agent from the point of view of efficiency (and price
also), sodium hydroxide can be used. Very large colonial animals like corals
must first be crushed to 1-2 g pieces to decrease the volume of hydrolyzing
KOH solution. Hydrolysis is carried out in a weighed beaker of corresponding capacity. The fresh animals (or animal), after the experiment ends, are
placed into the beaker and weighed. Then the calculated volume of 5-8%
KOH solution is added. An optimal ratio between the weight of animal material and the volume of KOH solution should be established empirically for
each kind of animal. The beaker with KOH solution added is heated in the
thermostat or on an hot, plate at a temperature close to boiling until the liquid
is completely evaporated. Then approximately the same volume of distilled
water is added into the beaker and it is thoroughly shaken by hand or in the
shaker to dissolve the hydrolyzate thus formed. The latter is transferred into
the measuring cylinder and its volume adjusted corresponding to the wet
weight of the initial animal material to equalize the amount of dry material
per volume unit of the hydrolyzate. After thorough mixing, the empirically
preselected volumes of this alkaline hydrolyzate are distributed into the scintillation vials, which are inserted into the thermostat and dried at 40-50°C.
Then 1 N HCI solution is added drop by drop until the formation of CO2 can
no longer be seen. The liquid in the vials is then evaporated in the same
way again, and 0.5-1 ml methanol or methoxyethanol is added to dissolve the
sediments. Now the vials are ready to be filled with the scintillation cocktail
and radioassayed. The mean value of radioactivity thus estimated after
Radioisotopic Methods for the Study of Nutrition in Aquatic Animals
Now the vials are ready to be filled with the scintillation cocktail. To facilitate
hydrolysis, it is always helpful to treat the samples in the vials in the ultrasonic
disintegrator just before adding the chemical solubilizers. I cannot recommend
using oxidizers like perchloric acid or hydrogen peroxide as basic solubilizers,
which is often done in nutritional studies using radioisotopes, as they could
induce various artifacts like chemoluminescence or significant 14C losses due
to oxidation of the labeled organic matter. Only if it is urgently necessary to
bleach the samples to diminish their color quenching can benzoyl peroxide
can be used.
The homogenous counting samples of the body material of larger consumers can be prepared in two different ways. First they must be homogenized
mechanically (with the aid of a mortar or mixer) or ultrasonically. Then the
aliquots of the homogenate thus obtained, containing quasi equal amounts of
dry material, are distributed into the scintillation vials and treated with solubilizers or KOH as described above for small crustaceans. The exact content
of organic material per sample, as well as the optimal volumes and kind of
solubilizers, should be selected empirically in dependence on the kinds and
sizes of the objects, the animal grazers.
Another way is the preparation of alkaline hydrolyzate from whole
animals' bodies. Where it is possible, the soft tissues are separated from their
mineral skeletons (clams); if not, the whole animals could be subjected to alkaline hydrolysis together with the skeletons (corals, small fish). As the most
practical solubilizing agent from the point of view of efficiency (and price
also), sodium hydroxide can be used. Very large colonial animals like corals
must first be crushed to 1-2 g pieces to decrease the volume of hydrolyzing
KOH solution. Hydrolysis is carried out in a weighed beaker of corresponding capacity. The fresh animals (or animal), after the experiment ends, are
placed into the beaker and weighed. Then the calculated volume of 5-8%
KOH solution is added. An optimal ratio between the weight of animal material and the volume of KOH solution should be established empirically for
each kind of animal. The beaker with KOH solution added is heated in the
thermostat or on an hot, plate at a temperature close to boiling until the liquid
is completely evaporated. Then approximately the same volume of distilled
water is added into the beaker and it is thoroughly shaken by hand or in the
shaker to dissolve the hydrolyzate thus formed. The latter is transferred into
the measuring cylinder and its volume adjusted corresponding to the wet
weight of the initial animal material to equalize the amount of dry material
per volume unit of the hydrolyzate. After thorough mixing, the empirically
preselected volumes of this alkaline hydrolyzate are distributed into the scintillation vials, which are inserted into the thermostat and dried at 40-50°C.
Then 1 N HCI solution is added drop by drop until the formation of CO2 can
no longer be seen. The liquid in the vials is then evaporated in the same
way again, and 0.5-1 ml methanol or methoxyethanol is added to dissolve the
sediments. Now the vials are ready to be filled with the scintillation cocktail
and radioassayed. The mean value of radioactivity thus estimated after