Use of 14C for Assessment of Nutritional Problems
137
the kind of animal filterer) part of the suspended food particles can settle.
Simultaneously, the specific radioactivity of food organisms, bacteria, or
microalgae inevitably decreases during incubation due to the incorporation of
unlabeled carbon compounds from the water (Gutelmacher 1974). Therefore,
r" must be estimated in subsamples taken from the parallel blank vessel
without animals; at an intermediate time in the incubation, it will equal to its
mean value during the incubation time. For such an experiment, suspended
particles must be selected which form a homogenous suspension and precipitate very slowly. Another possible source of error concerns small pelagic
filterers like cladocerans which excrete fragile feces that remain in suspension.
In an experiment lasting longer than their digestion time, they also excrete
radio labeled feces, which are then counted with the remaining suspended
labeled food during radioassay, when incubation is resumed. To avoid these
errors during experiments with animal filterers which produce no rigid rapidly
settling feces, their exposure time to the suspended labeled food must be a
little shorter than the digestion time (see Sect. 3.5.1) to prevent the appearance of labeled feces. Correspondingly, the volume of water in the experimental vessels per animal should be calculated so that during a short exposure
the food concentration will drop by 15-30%.
Taking the above stipulations into account, the experiment is carried out
as follows. Three vessels are filled with water prefiltered through a O.2-~m pore
size membrane filter, if the labeled food is bacteria, or 0.45 ~m for algae. The
volume of water, the duration of the experiment, and the number of animals
placed into the vessel are selected in a plot experiment so that the food concentration decreases during the experiment by 20-30%. Stable homogenous
suspensions of particulated food are prepared from suspensions of labeled cultures of microalgae or bacteria. Algae for these experiments must be selected
from those forms that maintain stable suspension, such as small Chlamydomonas, lsochrysis, or young Chlorella. Natural water containing labeled bacterioplankton can also be used in such an experiment, but prefiltered through
1.5-2-~m pore size Nucleopore filter without external vacuum, using the
funnel for reverse filtration (Fig. 6.2) to avoid bacterial aggregates. The concentration of food should be about 1.5-2 times more than optimal (see Sect.
3.5.5), e.g., 0.3-0.6mgCI- 1 or ca. 3 to lOmgl- 1 of wet biomass. The amounts of
concentrated suspension of labeled algae or bacteria to be added to the experimental vessels for the initial concentration can easily be calculated from
previous estimation of their Cr values (see Sect. 3.4.1). This latter value is not
estimated, the wet biomass of (biovolume) can be rapidly determined by direct
epifluorescence microscopic count on the black Nucleopore filters stained with
fluorochromes (see below, Sect. 6.2). The algae might be also counted and measured in the suspension using a cytometric counting chamber, under the ordinary microscope at magnification 200 x 400 (see Fig. 6.3).
The animals selected for the experiment must be exposed for 1-2 h to the
same concentration of the same kind of nonlabeled food as is selected to be
used in the experimental vessels. After the vessels with water containing
137
the kind of animal filterer) part of the suspended food particles can settle.
Simultaneously, the specific radioactivity of food organisms, bacteria, or
microalgae inevitably decreases during incubation due to the incorporation of
unlabeled carbon compounds from the water (Gutelmacher 1974). Therefore,
r" must be estimated in subsamples taken from the parallel blank vessel
without animals; at an intermediate time in the incubation, it will equal to its
mean value during the incubation time. For such an experiment, suspended
particles must be selected which form a homogenous suspension and precipitate very slowly. Another possible source of error concerns small pelagic
filterers like cladocerans which excrete fragile feces that remain in suspension.
In an experiment lasting longer than their digestion time, they also excrete
radio labeled feces, which are then counted with the remaining suspended
labeled food during radioassay, when incubation is resumed. To avoid these
errors during experiments with animal filterers which produce no rigid rapidly
settling feces, their exposure time to the suspended labeled food must be a
little shorter than the digestion time (see Sect. 3.5.1) to prevent the appearance of labeled feces. Correspondingly, the volume of water in the experimental vessels per animal should be calculated so that during a short exposure
the food concentration will drop by 15-30%.
Taking the above stipulations into account, the experiment is carried out
as follows. Three vessels are filled with water prefiltered through a O.2-~m pore
size membrane filter, if the labeled food is bacteria, or 0.45 ~m for algae. The
volume of water, the duration of the experiment, and the number of animals
placed into the vessel are selected in a plot experiment so that the food concentration decreases during the experiment by 20-30%. Stable homogenous
suspensions of particulated food are prepared from suspensions of labeled cultures of microalgae or bacteria. Algae for these experiments must be selected
from those forms that maintain stable suspension, such as small Chlamydomonas, lsochrysis, or young Chlorella. Natural water containing labeled bacterioplankton can also be used in such an experiment, but prefiltered through
1.5-2-~m pore size Nucleopore filter without external vacuum, using the
funnel for reverse filtration (Fig. 6.2) to avoid bacterial aggregates. The concentration of food should be about 1.5-2 times more than optimal (see Sect.
3.5.5), e.g., 0.3-0.6mgCI- 1 or ca. 3 to lOmgl- 1 of wet biomass. The amounts of
concentrated suspension of labeled algae or bacteria to be added to the experimental vessels for the initial concentration can easily be calculated from
previous estimation of their Cr values (see Sect. 3.4.1). This latter value is not
estimated, the wet biomass of (biovolume) can be rapidly determined by direct
epifluorescence microscopic count on the black Nucleopore filters stained with
fluorochromes (see below, Sect. 6.2). The algae might be also counted and measured in the suspension using a cytometric counting chamber, under the ordinary microscope at magnification 200 x 400 (see Fig. 6.3).
The animals selected for the experiment must be exposed for 1-2 h to the
same concentration of the same kind of nonlabeled food as is selected to be
used in the experimental vessels. After the vessels with water containing