1141
Notes
WOLIN, E. A., M. J. WOLIN, AND R. S. WOLFE.
iology of a Methanobacterium
Microbial.
11 l(3): 199-206.
1963. Formation
of methane by bacterial extracts. J. Biol. Chem. 238: 2882-2886.
ZEHNDER, A. J., AND K. WUHRMANN. 1977. Phys-
Limnol.
Oceanogr.,
24(6), 1979,1141-1145
Society of Limnology
@ 1979, by the American
A modified
respiration
and Oceanography,
strain AZ. Arch.
Submitted:
1 March 1979
Accepted: 22 May 1979
Inc.
procedure for determining
[‘“C]CO,
in water or sediment samples’
produced
by
strate incubated with sediment or water
samples is displaced from the sample by
acidification
and trapped in 2-phenylethylamine (PEA) absorbed on a small strip
of filter paper suspended over the sample. Respired
[l”C]CO,
is then determined by liquid scintillation
counting of
the filter strip in toluene scintillation
Organic
materials
labeled
with 14C fluid. All the evolved CO, was absorbed
have been used by many workers to de- by PEA (Hobbie and Crawford 1969), but
termine heterotrophic
activity and sub- only 82% of the [14C]C0, evolved could
strate turnover times in soils, sediments,
be accounted for by scintillation
counting
and natural waters. Initial
experiments
after correcting
for counting
efficiency
on the kinetics of substrate uptake by nat- with an internal standard. Christian and
ural populations
of heterotrophs took no Weibe (1978) observed even lower recovaccount of carbon lost as [14C]C0, by res- ery (60%) when using a scintillation
fluid
piration
and consequently
the results
containing
Triton
X-100 to estimate
consistently
underestimated
the total
[14C]C0, evolved
from soils although
turnover of substrate (Wright and Hobbie
they did not give sufficient experimental
1965; Vaccaro and Jannasch 1967; Vac- detail to conclude unequivocally
that all
care 1969; Hobbie et al. 1968). Respirathe [r4C]C0, was absorbed by the traption losses of substrate
were a large
ping agent. Harrison
et al. (1971) and
source of error in these early studies with
Griffiths
et al. (1974) have shown that
substrates such as acetate, glucose, and PEA not absorbed on filter paper quanamino acids, for which respiration can be titatively
traps C02, and Hobbie
and
the major metabolic fate. However, HobCrawford (1969) established by wet oxibie and Crawford (1969) devised a simple
dizing their filter paper strips and countand reasonably effective means of traping the absorbed
[14C]C0, in an ion
ping and quantifying
respired [ 14C]C0,
chamber that complete trapping had ocwhich has achieved widespread popularcurred. Hobbie and Crawford concluded
ity (see Christian and Hall 1977; Wright
that the lower level of radioactivity
mea1973) in kinetic studies of substrate utisured in the strips by liquid scintillation
lization in natural systems.
counting was a result of masking effects
According
to Hobbie and Crawford’s
by the paper. Because the complex of
(1969) procedure, [ l”C]CO, produced by PEA with CO, (2-phenylethylammonium
respiration
of 14C-labeled organic sub- phenylethylcarbamate)
is not very soluble in toluene (Rapkin 1969) it remains
r The work was carried out at the Central Elecassociated with the filter paper strip and
tricity Research Laboratories
and the paper is pubdoes not dissolve appreciably in the scinlished by permission of the Central Electricity
Gentillation
cocktail. Thus beta-absorption
erating Board.
Abstract-Poor
recovery
of radioactivity
when determining
[14C]C0, respired by microbes can be overcome by substituting 2-ethanolamine for 2-phenylethylamine
to absorb
[14C]C0, and by increasing the solubility
of
solution
absorbed [‘“C]CO, in the scintillation
with 2-ethoxyethanol.
1142
Notes
by the paper and by the complex (selfabsorption) may almost entirely account
for the low recovery of radioactivity.
Heterogeneous
counting on filter paper has been reviewed by Long et al.
(1976) in connection
with 3H counting,
and their remarks are partly applicable to
more energetic beta-emitters such as r4C.
Beta-absorption
occurs when the radioisotope is not homogeneously
distributed
within
the scintillation
cocktail. Some
beta particles emitted inside an aggregate of sample or within the filter matrix
are absorbed without producing measurable excitation of the solvent, resulting in
a reduction of the potential counting rate.
Because the amount of beta-absorption
is
in part related to the quantity of material
on the filter (self-absorption)
the observed counting rate also depends on the
total amount of CO, absorbed. Beta particles emitted in the bulk of the scintillation cocktail are quenched to a different
extent than those emitted from the surface of the filter; therefore internal standards cannot be used to correct for the
effects of beta-absorption.
For the same
reason, quench
correction
cannot be
made by using an external standard ratio
(ESR) method, and the sample channels
ratio (SCR) technique is of doubtful value
because SCR does not change in accordance with increasing
beta-absorption.
Another disadvantage
of heterogeneous
counting
on filter paper is the dependence of counting rate upon the orientation of the paper strip within the vial, a
consequence of which is to increase the
variability
between
replicate
samples.
Because of this effect, and also because
of the reduction in counting rate caused
by beta-adsorption,
the precision of measurements obtained by the technique
is
likely to be lower than with a system in
which radioactivity
is homogeneously
distributed
throughout the cocktail, even
when correction
factors are applied to
compensate for an apparently
constant
reduction
in efficiency
caused by betaabsorption. These difficulties
can all be
minimized by ensuring that the absorbed
[14C]C0, is dissolved in the scintillation
cocktail. I here describe such a modification.
[‘“C]CO,
was generated in a system
slightly different from that described by
Hobbie and Crawford
(1969), but because complete absorption
of CO, was
observed,
these minor differences
do
not influence the validity of the conclusions drawn regarding counting conditions for the absorption strip. Carrier-free
[14C]NaHC03
(0.065 &i)
(Radiochem.
Centre, Amersham, U.K.) in 50 ~1 of distilled water was added to 25-ml screwcap
bottles containing
up to 500 ~1 of 0.5%
(wt/vol) NaHCO, solution and the total
volume of liquid adjusted to 550 ~1 with
distilled water. The bottles were sealed
with rubber suba-seals. [14C]C0, was absorbed by accordian-folded
strips (25 x
51 mm) of Whatman No. 1 filter paper
moistened with 200 ~1 of trapping solution and contained in small plastic cups
suspended (by means of bent paper clips)
from the suba-seals. Gas was generated
by injecting 0.5 ml of 1 M H,SO, into the
liquid.
The bottles were incubated overnight
at 4°C to allow the strips to absorb all the
evolved
CO,. After the bottles were
opened, the strips were placed in plastic
scintillation
vials, either empty or containing
5 ml of 2-ethoxyethanol
(EE).
Strips placed in these latter vials were
allowed to soak for up to 10 min with occasional gentle agitation. All the strips
were counted at 6°C in scintillation
fluid
(10 ml) containing 2-(4’-t-butylphenyl)-4(4”-biphenylyl)-1,3,4-oxadiazole
(butylPBD), 7 g-liter-l,
in toluene in an Intertechnique
SL33 automatic scintillation
spectrometer having a 22”Ra external standard source. Vials were placed in the instrument 30 min before counting to allow
thermal equilibration
and the decay of
chemiluminescence
before determining
radioactivity
with the preset discriminator settings for 3H, 14C, and 3H + 14C.
Samples were counted until at least lo5
cpm had been recorded in the 3H + 14C
channel. SCR and ESR were determined
automatically.
Chemicals used for scintillation
counting
were “scintillation”
Notes
1143
Table 1. Effect of different
trapping solutions and scintillation
systems on recovery of [14C]C0,.
[14C]C0, was released from a solution
of 0.5% NaHCO,,
500 ~1, containing
70,250 k 2,458 dpm
[‘“C]NaHCO,,
and absorbed in 0.2 ml of trapping solution on Whatman No. 1 paper as described in text.
Radioactivity
of carrier-free
[r4C]NaHC03
determined
by counting aliquots of the solution in toluene
scintillation
fluid, 10 ml, with 5.2 ml of EA/EE. A filter paper strip was placed in each vial and cpm
corrected for quenching by internal standardization
with [14C1]-n-hexadecane. As discussed in text, internal
standardization
is valid only when most of the 14C is dissolved in scintillation
fluid. Figures for dpm are
means of three replicate determinations
k SD.
Soluble
Trapping
agent
SEE1
EA/EE
(Modified
PEA
Yes
procedure)
No
Yes
No
(Standard H-C)
Complete
system dpm
Recovery
%
dp111
62,645 + 314
70,755 IL 240
101
62,181 k 952
88
9,100 k 677
60,690 k 2,911
27,472 2 3,012
86
39
22,227 k 1,873
3,793 k 58
grade (Fisons, Loughborough,
U.K.); all
other chemicals were of AR grade.
Because the insolubility
in toluene of
the complex of 2-phenylethylamine
with
CO, is the cause of inefficient
counting
of radioactivity
and other problems resulting from beta-absorption,
the amount
of soluble [14C]C02 was increased
by
changing the solubility
of the recovered CO, absorbed in PEA and by using
other absorption
agents. Hyamine
hydroxide (p-di-isobutylcresoxyethoxyethyldimethylbenzylammonium
hydroxide,
1
M in methanol) has been used to trap
[14C]C02 before liquid scintillation
counting (Snyder and Godfrey 1961; Edwards
and Kitchener 1965), but although it forms
a toluene-soluble
complex with C02, it
has the disadvantages of being a severe
quenching agent and of low trapping capacity per unit cost (Rapkin 1969). Ethanolamine (EA) is also able to completely
trap CO, (Jeffay and Alvarez 1961; Kadota
et al. 1966), but its high viscosity makes
accurate pipetting
difficult
and the salt
formed with CO, is not appreciably soluble in toluene. However, these problems
can be overcome by diluting EA with EE,
which reduces its viscosity and dissolves
the EA-CO, complex.
Unlike PEA, EA does not darken appreciably on storage and therefore does
not quench as severely as some batches
of PEA. EA was used as a trapping agent
%
89
13
32
5.4
for these experiments,
mixed with an
equal volume of EE. The low viscosity of
the mixture permitted accurate dispensing, and 200-4 aliquots of the mixture
were applied to filter paper strips for absorption of [14C]C0,. The capacity of the
EA/EE mixture to absorb CO, is not appreciably different from that of an equal
volume of PEA, because the higher molecular weight of the latter compound
(121) is compensated by the dilution
of
EA (mol wt 61). In all the experiments
described here, ~2% of the absorption
capacity of the trapping agent was utilized by the evolved CO,. The recovery
of radioactivity
absorbed on filter strips
moistened with PEA and then placed in
toluene scintillation
fluid for counting
(referred to as standard H-C) is compared
in Table 1 with recovery of radioactivity
from filter strips moistened with EA/EE.
This table also shows the effect of adding
EE (5 ml) to the scintillation
system on
recovery of radioactivity.
Soluble counts
were determined by replacing the original filter paper strips with nonradioactive
strips of similar dimensions and recounting. The standard H-C procedure gave
the lowest recovery of radioactivity,
although recovery was more than doubled
by adding 5 ml of EE. However, even
without additional EE, recovery was better using EA/EE as trapping agent than
the standard H-C procedure, and, when
Notes
I
100
I
I
I
200
300
400
CARRIER NaHCO, SOLUTION,pt
I
500
Fig. 1. Effect of CO2 concentration
on recovery
of fixed amount of [14C]C0,. [14C]C0, was liberated
in presence of various amounts of 0.5% NaHCO,
carrier from [14C]NaHC0,
at total activity of 63,000
cpm (see text). Absorbed radioactivity
determined
by standard H-C procedure (0) or by the modified
procedure described here (0).
[14C]C02 was trapped with EA/EE and
additional EE was included in the scintillation
cocktail, recovery of radioactivity was complete. The increase in recovery of radioactivity
obtained by using EA/
EE as trapping agent and counting in the
presence of additional EE (referred to as
modified
procedure)
over the standard
H-C procedure is paralleled
by a large
increase in dissolved counts, from 5.4%
soluble with the standard H-C procedure
to over 89% soluble in the modified system. An overall counting
efficiency
of
95.5% was determined
for the modified
procedure with an internal standard of
[ 14CJ-n -hexadecane.
Chemical quenching
in the modified
procedure by the filter paper strips resulted in small but significant (P = 0.01)
reductions of ESR and SCR, of 1.56 and
6.50%, compared with control vials in
which filter paper had been omitted and
to which [14C]NaHC03 and 200 ~1 of EA/
EE were added directly to the cocktail.
The P-energy spectrum was slightly displaced toward the lower energy (3H)
range, but no significant
difference
in
3H + 14C channel counts could be detected between vials containing
paper
strips and the controls. The orientation of
the paper strips did not affect 3H + 14C
count rate, ESR, or SCR, and these parameters were effectively
constant between vials in which the filter strips were
pressed against the bottom of the vessel
and those in which the strips were allowed to stand upright.
Figure 1 shows the effect of increasing
the amount of absorbed CO, on recovery
of radioactivity
from a constant amount of
[14C]C02. The maximum amount of CO,
released in these experiments
(57 pmol
of CO, from 500 ,ul of 0.5% NaHCO,) is
similar to that liberated after acidification
of l-2 ml of hard freshwater or seawater
samples, although
more would be released from carbonate-rich
sediments.
Because most of the recovered radioactivity in the standard H-C procedure remains associated with the filter strips (see
Table I), apparent recovery is lowered
when the amount of CO, absorbed is increased, even though a maximum of ~2%
of the trapping agent’s capacity is utilized. In contrast to these results, complete recovery independent of the amount
of CO, absorbed was recorded using the
modified procedure because most of the
radioactivity
here was in free solution
and therefore not subject to losses caused
by self-absorption.
It may be possible to use the modified
technique described here with commercially prepared scintillation
cocktails, if
it is first established
that EA-[‘4C]C02
complex dissolves in the fluid. This can
be checked by measuring radioactivity
in
trial experimental
vials with the filter paper strip in place and then again after its
removal. Counting
rate after removing
the strip should not be appreciably
less
than in the complete system. Some scintillation
cocktails
containing
solvents
other than toluene (e.g. 1,4-dioxane) may
not need the addition of EE to increase
the solubility
of the EA-CO, complex.
Previous work using the standard H-C
procedure did not necessarily underestimate respired [14C]C02 provided that an
adequate correction
factor was used to
1145
Notes
compensate
for beta-absorption
losses
and complete trapping of [‘4C]C02 was
established. Because counting rate in the
standard procedure
depends partly on
the total amount of CO, absorbed, correction factors must be determined
for
batches of samples which contain different amounts of total CO, or in which the
endogenous rate of respiration varies, for
example, in sediments or soils where biochemical
activity
may change greatly
within a few centimeters depth.
Keith A. Brown
Research
Central Electricity
Laboratories
Leatherhead
Surrey KT22 7SE U.K.
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1979