Use of the Radiocarbon Method to Determine Primary Production
83
batch under the same counting conditions as are routinely used during liquid
scintillation radioassay of the plant tissue preparations, taking into account the
probable range of counting efficiencies. To meet these conditions, the R; values
for the batch are measured in a scale of counting samples containing a corresponding range of amounts of plant tissues added in the same form as will be
employed to measure their radioactivity after 14C photosynthetic uptake
experiments (hydrolyzate, homogenate, see below). For this, into a series of
scintillation vials (ca. six to eight) various portions of nonlabeled tissue
homogenate (hydrolyzate) are added, within the range to be used during the
radioassay radioactivity in the plant material. Then 0.3ml 5% solution of
hyamine in methanol or in toluene is added. In a series representing the nonquenched counting sample, only the hyamine-methanol solution is added into
the vial. Then 10ml of scintillation cocktail is added into all the vials, of the
same grade to be used for routine work radioactivity in plant tissues. The
working solution of 14C-bicarbonate is diluted 250-500 times in the flask with
0.2% solution of the carrier, Na2C03 in O.01N NaOH, and 0.5ml portions of
this dilution are injected with automatic pipet into the range of scintillation
vials prepared as described above. After mixing, they are counted in the liquid
scintillation spectrometer. The R; values in cpm per 1 ml of initial batch 14C_
bicarbonate solution are calculated according to the values of cpm counts
measured in each of vials, then the curve of R; is derived on the ordinate opposite the amounts of plant tissue material, mg of dry weight on the abscissa.
With the aid of this curve, the R; values may be estimated for each experiment, avoiding the problems of quench correction and the need to calculate
their measurement for each experimental vessel, as was practiced by many
authors. If the amounts of radioactive tissue material in the scintillation vials
can be equalized (which is comparatively easy), it is possible to have constant
R; values for all experiments on condition that the amount of working solution injected into each experimental vessel is proportional to its volume. Moreover, in this case, it is possible to attain a constant Cr value for all incubations
done within 1-2 weeks because the C; values (T CO2 carbon contents) in water
within a given research site are usually rather constant (see below). This
simplified method for R; measurements, which avoids the time-consuming procedure of quench corrections, can be used when working with individual thalli
of vascular hydrophytes or with their associations, including epiphytes; but
if photosynthesis estimations by the 14C method are made with periphytonic
overgrowths, or microphytobenthos as object, estimation, or even more, standardization of the amounts of plant tissues in the counting samples may
be difficult or impossible to achieve. In this case, the R; values for Cr calculations are taken as measured in the nonquenched vial without plant material
added; but then all the individual counting samples with radioactivity material prepared to estimate 14C uptake by plant associations should be corrected
for quenching with the aid of an internal standard (see Sect. 1.2.2.3). They
may also be corrected for quenching with the aid of an external standard,
but in this case the same should be done when counting the 14C-carbonate
83
batch under the same counting conditions as are routinely used during liquid
scintillation radioassay of the plant tissue preparations, taking into account the
probable range of counting efficiencies. To meet these conditions, the R; values
for the batch are measured in a scale of counting samples containing a corresponding range of amounts of plant tissues added in the same form as will be
employed to measure their radioactivity after 14C photosynthetic uptake
experiments (hydrolyzate, homogenate, see below). For this, into a series of
scintillation vials (ca. six to eight) various portions of nonlabeled tissue
homogenate (hydrolyzate) are added, within the range to be used during the
radioassay radioactivity in the plant material. Then 0.3ml 5% solution of
hyamine in methanol or in toluene is added. In a series representing the nonquenched counting sample, only the hyamine-methanol solution is added into
the vial. Then 10ml of scintillation cocktail is added into all the vials, of the
same grade to be used for routine work radioactivity in plant tissues. The
working solution of 14C-bicarbonate is diluted 250-500 times in the flask with
0.2% solution of the carrier, Na2C03 in O.01N NaOH, and 0.5ml portions of
this dilution are injected with automatic pipet into the range of scintillation
vials prepared as described above. After mixing, they are counted in the liquid
scintillation spectrometer. The R; values in cpm per 1 ml of initial batch 14C_
bicarbonate solution are calculated according to the values of cpm counts
measured in each of vials, then the curve of R; is derived on the ordinate opposite the amounts of plant tissue material, mg of dry weight on the abscissa.
With the aid of this curve, the R; values may be estimated for each experiment, avoiding the problems of quench correction and the need to calculate
their measurement for each experimental vessel, as was practiced by many
authors. If the amounts of radioactive tissue material in the scintillation vials
can be equalized (which is comparatively easy), it is possible to have constant
R; values for all experiments on condition that the amount of working solution injected into each experimental vessel is proportional to its volume. Moreover, in this case, it is possible to attain a constant Cr value for all incubations
done within 1-2 weeks because the C; values (T CO2 carbon contents) in water
within a given research site are usually rather constant (see below). This
simplified method for R; measurements, which avoids the time-consuming procedure of quench corrections, can be used when working with individual thalli
of vascular hydrophytes or with their associations, including epiphytes; but
if photosynthesis estimations by the 14C method are made with periphytonic
overgrowths, or microphytobenthos as object, estimation, or even more, standardization of the amounts of plant tissues in the counting samples may
be difficult or impossible to achieve. In this case, the R; values for Cr calculations are taken as measured in the nonquenched vial without plant material
added; but then all the individual counting samples with radioactivity material prepared to estimate 14C uptake by plant associations should be corrected
for quenching with the aid of an internal standard (see Sect. 1.2.2.3). They
may also be corrected for quenching with the aid of an external standard,
but in this case the same should be done when counting the 14C-carbonate