Technique of Measuring Phytoplankton Primary Production
39
sulfate solution, and they are overturned into appropriate funnels with the correspondingly marked filters for their filtration. The light bottles can be better
filtered without fixation it this can take place within half an hour of the end
of incubation. Otherwise, they can also be fixed with a weak Lugol solution at
a concentration giving a slightly yellow color in the bottle. The fixed samples
can be stored in the refrigerator for several hours without serious loss in
radioactivity by the phytoplankton. It is preferable to filter the whole volume
of water in experimental bottles. For this, the bottles are overturned into the
funnels, the taps under them are opened, and their filtration proceeds automatically. As mentioned above, the vacuum during the filtration procedure
should not exceed 100mmHg, or better, even less (50mmHg). If the density
of phytoplankton in the samples happens to be very high, so that filtration of
the whole volume of water in the experimental bottles cannot be accomplished
within a short time (5min) by a limited vacuum (<100mmHg), only a part of
it can be filtered. In this case, a measured volume (50 or 100ml) is filtered, and
the volume of the rest is estimated. Thus the radioactivity assayed on the filter
can easily recalculated for the whole volume of the sample.
When filtration is ended, the remains of '4C-carbonates should be removed
from the filters, for which two alternative procedures can be employed. One
is the rinsing of filters with 3-ml portions of the prefiltered natural water and
0.3 and 0.03% HCI solutions in the same water. This procedure may be safely
used in freshwater and in productive coastal marine waters (Mague et al. 1980;
Smith 1982; Gachter et al. 1984). In the sea, it is better to use the second, more
time-consuming, but safer, procedure. After filtration, the still-moist filters are
placed into scintillation vials and 0.2 ml 0.1 N HCI is added to them to decompose the remains of the labeled bicarbonate. The open vials are kept for 2 h at
40°C, or 1 day at room temperature. Then 0.1 ml of 10% solution of NCS or
hyamine in methanol is added, followed by the scintillation cocktail (6-7ml).
The vials thus prepared will be ready for counting radioactivity in the
liquid scintillation spectrometer 5-10h later, when their counting efficiency
stabilizes. Calculations of the photosynthesis rate of phytoplankton in samples
h- 1 (Cp) and of their primary production day-l (Cps) are accomplished using the
values of radioactivity measured in vials without quench correction (see Sect.
2.3.2.7). First the radioactivity of 14C-carbonate assimilated by photosynthesis
h- 1 (Ra) is calculated: Ra = (R/ - Ra)ltcpmh-l, if R/ is the average of radioactivity measured in two parallel light bottles, cpm per whole volume of the
sample, Ra the same measured in a dark bottle, and t exposure time, h. Then
Cp can be calculated as follows: Cp = Ra Cr 1.0610 3 K cmgCm- 3 h-l, if Ra 10 3
is the radioactivity of assimilated by phytoplankton 14C calculated 1 m- 3 ; Cr
the reverse specific radioactivity of hydrocarbonate-carbon (TCOrC) in the
sample, mgCcpm- 1 (see above, Sect. 2.3.1); 1.06 the correction coefficient for
the isotopic effect (see above, Sect. 2.3.1), and Kc the correction coefficient for
the losses of 14C assimilates during the filtration (see below, Sect. 2.3.2.7). The
primary production per day in the surface layer Cps can be calculated with the
aid of K/ coefficients (see above): Cps = Cp K/mgCm- 2 day-I.
39
sulfate solution, and they are overturned into appropriate funnels with the correspondingly marked filters for their filtration. The light bottles can be better
filtered without fixation it this can take place within half an hour of the end
of incubation. Otherwise, they can also be fixed with a weak Lugol solution at
a concentration giving a slightly yellow color in the bottle. The fixed samples
can be stored in the refrigerator for several hours without serious loss in
radioactivity by the phytoplankton. It is preferable to filter the whole volume
of water in experimental bottles. For this, the bottles are overturned into the
funnels, the taps under them are opened, and their filtration proceeds automatically. As mentioned above, the vacuum during the filtration procedure
should not exceed 100mmHg, or better, even less (50mmHg). If the density
of phytoplankton in the samples happens to be very high, so that filtration of
the whole volume of water in the experimental bottles cannot be accomplished
within a short time (5min) by a limited vacuum (<100mmHg), only a part of
it can be filtered. In this case, a measured volume (50 or 100ml) is filtered, and
the volume of the rest is estimated. Thus the radioactivity assayed on the filter
can easily recalculated for the whole volume of the sample.
When filtration is ended, the remains of '4C-carbonates should be removed
from the filters, for which two alternative procedures can be employed. One
is the rinsing of filters with 3-ml portions of the prefiltered natural water and
0.3 and 0.03% HCI solutions in the same water. This procedure may be safely
used in freshwater and in productive coastal marine waters (Mague et al. 1980;
Smith 1982; Gachter et al. 1984). In the sea, it is better to use the second, more
time-consuming, but safer, procedure. After filtration, the still-moist filters are
placed into scintillation vials and 0.2 ml 0.1 N HCI is added to them to decompose the remains of the labeled bicarbonate. The open vials are kept for 2 h at
40°C, or 1 day at room temperature. Then 0.1 ml of 10% solution of NCS or
hyamine in methanol is added, followed by the scintillation cocktail (6-7ml).
The vials thus prepared will be ready for counting radioactivity in the
liquid scintillation spectrometer 5-10h later, when their counting efficiency
stabilizes. Calculations of the photosynthesis rate of phytoplankton in samples
h- 1 (Cp) and of their primary production day-l (Cps) are accomplished using the
values of radioactivity measured in vials without quench correction (see Sect.
2.3.2.7). First the radioactivity of 14C-carbonate assimilated by photosynthesis
h- 1 (Ra) is calculated: Ra = (R/ - Ra)ltcpmh-l, if R/ is the average of radioactivity measured in two parallel light bottles, cpm per whole volume of the
sample, Ra the same measured in a dark bottle, and t exposure time, h. Then
Cp can be calculated as follows: Cp = Ra Cr 1.0610 3 K cmgCm- 3 h-l, if Ra 10 3
is the radioactivity of assimilated by phytoplankton 14C calculated 1 m- 3 ; Cr
the reverse specific radioactivity of hydrocarbonate-carbon (TCOrC) in the
sample, mgCcpm- 1 (see above, Sect. 2.3.1); 1.06 the correction coefficient for
the isotopic effect (see above, Sect. 2.3.1), and Kc the correction coefficient for
the losses of 14C assimilates during the filtration (see below, Sect. 2.3.2.7). The
primary production per day in the surface layer Cps can be calculated with the
aid of K/ coefficients (see above): Cps = Cp K/mgCm- 2 day-I.