108
Radioisotopic Methods for the Study of Nutrition in Aquatic Animals
an hour. After this, it is neutralized to pH 7.5-8 with 5% NaOH solution
prepared without CO2• The TC02 content in seawater thus treated is estimated
by titration with 0.05N HCl (see Sect. 2.3.2.2). Then the calculated amount of
nontreated seawater is added to adjust the TC02 content, if necessary.
The media thus prepared are sterilized in a boiling water bath for half an
hour. After cooling, the pH is adjusted to 7.8-8.2 and the calculated amount
of the working solution of 14C-carbonate is added (for preparation of the
latter and estimation of its cpm radioactivity see Sect. 2.3.2.1). To obtain
the necessary level of Cr in the algae, between 3x and 5 x 10 6 cpm of
radioactivity (2-3 f.lCi) per 1 mg C of TCOz must be added to the culture
medium. The algae grow in 1.5-2-1 flasks capped with paper fixed with rubber
bands. During growth, the pH of the media should be controlled and kept at
less than 8.5 (8-8.4) by addition of 0.1 N HCl solution. The mature algae are
separated from the medium either by filtration with Sartorius membrane
filters 0.8-1-f.lm pore size in a funnel with 40-45 mm diameter working
surface, or by centrifugation. Concentrated suspensions of microalgae
ready for use in feeding experiments should thus contain some 0.3 to 0.5 mg
Cml- 1 biomass. The suspension can be preserved at low temperature for 1-2
weeks.
If the above concentration procedures are not available or not efficient for a given kind of microalgae (very small or mucoid, for example),
the feeding experiments can also be accomplished using nonconcentrated
cultures still containing labeled 14C carbonate as a food; but, in this case, an
appropriate correction for the possible 14C uptake by animals incubated in the
presence of 14COrcarbonate should be introduced during the calculations of
Re·
If necessary, it is possible to label assemblages of natural phytoplankton,
when dealing with an ambient density over 3-5 mg 1-1 of wet biomass. With the
aid of the inverse filtration procedure it can be concentrated three to five
times. Inverse filtration is accomplished with the use of 2-3 f.lm pore size Nucleopore filters with a large (80mm diameter) working area. Filtration proceeds
gently under a pressure difference of only 20-30mm of water column with the
aid of special funnel (see Fig. 6.1). The concentrated sample is injected with
the calculated amount of 14C carbonate solution (5-1Of.lCil- 1 ) and incubated
in the shade for 2-3 days. This suspension of natural phytoplankton is then
used directly in the feeding experiments without separation from the labeled
medium containing 14C carbonate as described above.
Thalli of macrophytes and the filamentous macro algae are labeled by
growing whole small plants in jars with pre filtered natural water containing
14C carbonate (approximately 4-6 f.lCi per 1 mg of TCOz). The plant is
uniformly labeled if it increases its weight by 1.5 times.
Fresh plant detritus free of bacteria might be prepared from such a labeled
thallus simply by crushing in a mortar with water and glass powder or sand
added to obtain a fine suspension, which is washed by resuspending and
Radioisotopic Methods for the Study of Nutrition in Aquatic Animals
an hour. After this, it is neutralized to pH 7.5-8 with 5% NaOH solution
prepared without CO2• The TC02 content in seawater thus treated is estimated
by titration with 0.05N HCl (see Sect. 2.3.2.2). Then the calculated amount of
nontreated seawater is added to adjust the TC02 content, if necessary.
The media thus prepared are sterilized in a boiling water bath for half an
hour. After cooling, the pH is adjusted to 7.8-8.2 and the calculated amount
of the working solution of 14C-carbonate is added (for preparation of the
latter and estimation of its cpm radioactivity see Sect. 2.3.2.1). To obtain
the necessary level of Cr in the algae, between 3x and 5 x 10 6 cpm of
radioactivity (2-3 f.lCi) per 1 mg C of TCOz must be added to the culture
medium. The algae grow in 1.5-2-1 flasks capped with paper fixed with rubber
bands. During growth, the pH of the media should be controlled and kept at
less than 8.5 (8-8.4) by addition of 0.1 N HCl solution. The mature algae are
separated from the medium either by filtration with Sartorius membrane
filters 0.8-1-f.lm pore size in a funnel with 40-45 mm diameter working
surface, or by centrifugation. Concentrated suspensions of microalgae
ready for use in feeding experiments should thus contain some 0.3 to 0.5 mg
Cml- 1 biomass. The suspension can be preserved at low temperature for 1-2
weeks.
If the above concentration procedures are not available or not efficient for a given kind of microalgae (very small or mucoid, for example),
the feeding experiments can also be accomplished using nonconcentrated
cultures still containing labeled 14C carbonate as a food; but, in this case, an
appropriate correction for the possible 14C uptake by animals incubated in the
presence of 14COrcarbonate should be introduced during the calculations of
Re·
If necessary, it is possible to label assemblages of natural phytoplankton,
when dealing with an ambient density over 3-5 mg 1-1 of wet biomass. With the
aid of the inverse filtration procedure it can be concentrated three to five
times. Inverse filtration is accomplished with the use of 2-3 f.lm pore size Nucleopore filters with a large (80mm diameter) working area. Filtration proceeds
gently under a pressure difference of only 20-30mm of water column with the
aid of special funnel (see Fig. 6.1). The concentrated sample is injected with
the calculated amount of 14C carbonate solution (5-1Of.lCil- 1 ) and incubated
in the shade for 2-3 days. This suspension of natural phytoplankton is then
used directly in the feeding experiments without separation from the labeled
medium containing 14C carbonate as described above.
Thalli of macrophytes and the filamentous macro algae are labeled by
growing whole small plants in jars with pre filtered natural water containing
14C carbonate (approximately 4-6 f.lCi per 1 mg of TCOz). The plant is
uniformly labeled if it increases its weight by 1.5 times.
Fresh plant detritus free of bacteria might be prepared from such a labeled
thallus simply by crushing in a mortar with water and glass powder or sand
added to obtain a fine suspension, which is washed by resuspending and