Use of the Radiocarbon Method to Determine Primary Production
95
after thorough mixing and added to the scintillation vials for radioassay. The
simplest way to do this is to count them directly by use of a cocktail miscible
with water jelly like Aquasol or some of the toluene-Triton-X cocktails with
the addition of the gellifying agent cab-o-sil (Scauen et al. 1971; see Sect.
1.2.2.2). Before selecting this simple procedure for routine use, the quenching
of radiation in su(;h (;OuntiIlg salllple~ should first be estimated. If it is less thdIl
ca. 50-60%, this method is quite possible. If quenching is too large and parallel counting displays high variability, the crude tissue homogenate obtained as
described above should be bleached and solibilized directly in the scintillation
vial. For this, the following techniques can be recommended. A subsample of
homogenate (0.5-0.8ml) is added into the glass scintillation vial by automatic
pipet and the liquid in it is evaporated at 50°C. To the dry sediments at the
bottom OAm130% hydrogen peroxide or 0.5 ml of 30% benzoyl peroxide solution is added. The vials are capped and kept at 50°C in the dark for 1 day.
Then 4ml of the solubilizer NCS is added. The vials are then kept at the same
temperature for another 16h, after which scintillation cocktail is added and
the sample radioassayed (Gagne et aI1979).
More efficient bleaching of the plant material can be achieved, if, in addition to H 20 2 004 ml of 50% perchloric acid is added to the vials. The vials are
closed and heated in the dark at 70°C for 1.5 h. If, at the end of this heating,
some samples still remain colored, an additional portion of hydrogen peroxide solution is injected into them and the heating is repeated. Then 6ml of
methoxyethanol is added into the vials. Some time later they are thoroughly
mixed and then the appropriate cocktail, either toluene-Triton-X or dioxanebased, are added (14ml). These counting samples are radioassayed. Both these
techniques are based on the use of strong oxidizers as bleaching agents
(Lobban 1974). Therefore definite losses in radioactivity as 14C02 formed due
to oxidation of a part of the radiolabeled organic matter during the bleaching
procedures may be expected, and have been evaluated within 7 to 9% (Arnold
and Littler 1985). Therefore before using these techniques, it is recommended
to estimate possible losses experimentally as follows. A portion of homogenate
with known cpm radioactivity is added into the vial. Then 0.2 ml of 1 N HCI +
OAml 30% H 20 2 are added and it is tightly closed with a cap, to which is
attached a small test tube with 0.5 ml of 10% hyamine solution added. A plug
of filter paper is inserted into it to moisten the hyamine solution. The vials are
heated for 1 day in the dark at 50°C, then the cap is taken out. The contents
of the test tube with hyamine solution, together with the filter paper, is transferred into another vial, radioassayed, and the percentage of 14C02 losses is
calculated. If this appears significant, it should be taken into account when the
radioactivity of samples is determined.
In addition to these losses, the counting samples of plant tissues, whether
or not treated with peroxides, can display significant chemoluminescence. Thus
should also be controlled (see Sect. 1.2.204).
In my experience with periphyton (Sorokin 1993), I used a rather efficient
technique of plant tissue solubilization with the aid of NaOH: 0.5-1ml por-
95
after thorough mixing and added to the scintillation vials for radioassay. The
simplest way to do this is to count them directly by use of a cocktail miscible
with water jelly like Aquasol or some of the toluene-Triton-X cocktails with
the addition of the gellifying agent cab-o-sil (Scauen et al. 1971; see Sect.
1.2.2.2). Before selecting this simple procedure for routine use, the quenching
of radiation in su(;h (;OuntiIlg salllple~ should first be estimated. If it is less thdIl
ca. 50-60%, this method is quite possible. If quenching is too large and parallel counting displays high variability, the crude tissue homogenate obtained as
described above should be bleached and solibilized directly in the scintillation
vial. For this, the following techniques can be recommended. A subsample of
homogenate (0.5-0.8ml) is added into the glass scintillation vial by automatic
pipet and the liquid in it is evaporated at 50°C. To the dry sediments at the
bottom OAm130% hydrogen peroxide or 0.5 ml of 30% benzoyl peroxide solution is added. The vials are capped and kept at 50°C in the dark for 1 day.
Then 4ml of the solubilizer NCS is added. The vials are then kept at the same
temperature for another 16h, after which scintillation cocktail is added and
the sample radioassayed (Gagne et aI1979).
More efficient bleaching of the plant material can be achieved, if, in addition to H 20 2 004 ml of 50% perchloric acid is added to the vials. The vials are
closed and heated in the dark at 70°C for 1.5 h. If, at the end of this heating,
some samples still remain colored, an additional portion of hydrogen peroxide solution is injected into them and the heating is repeated. Then 6ml of
methoxyethanol is added into the vials. Some time later they are thoroughly
mixed and then the appropriate cocktail, either toluene-Triton-X or dioxanebased, are added (14ml). These counting samples are radioassayed. Both these
techniques are based on the use of strong oxidizers as bleaching agents
(Lobban 1974). Therefore definite losses in radioactivity as 14C02 formed due
to oxidation of a part of the radiolabeled organic matter during the bleaching
procedures may be expected, and have been evaluated within 7 to 9% (Arnold
and Littler 1985). Therefore before using these techniques, it is recommended
to estimate possible losses experimentally as follows. A portion of homogenate
with known cpm radioactivity is added into the vial. Then 0.2 ml of 1 N HCI +
OAml 30% H 20 2 are added and it is tightly closed with a cap, to which is
attached a small test tube with 0.5 ml of 10% hyamine solution added. A plug
of filter paper is inserted into it to moisten the hyamine solution. The vials are
heated for 1 day in the dark at 50°C, then the cap is taken out. The contents
of the test tube with hyamine solution, together with the filter paper, is transferred into another vial, radioassayed, and the percentage of 14C02 losses is
calculated. If this appears significant, it should be taken into account when the
radioactivity of samples is determined.
In addition to these losses, the counting samples of plant tissues, whether
or not treated with peroxides, can display significant chemoluminescence. Thus
should also be controlled (see Sect. 1.2.204).
In my experience with periphyton (Sorokin 1993), I used a rather efficient
technique of plant tissue solubilization with the aid of NaOH: 0.5-1ml por-