SAMPLE PREPARATION 
1. Barium Carbonate (C4). The inactive material was reagent grade. C14 
labeled material was prepared from a suitable mixture of sucrose and standard- 
ized benzene-carboxylic-C!4 acid (1) by Van Slyke-Folch wet oxidation (6) 
followed by routine conversion of the evolved CO, to BaCOs. 
Particles fine enough for suspension counting were made from coarse BaCO; 
crystals by grinding for 3-5 min between the ground-glass surfaces in a small 
tissue homogenizer. Absolute alcohol was used to wet the particles during 
grinding. Toluene was used to rinse them into the counting bottle. Quantities 
were 0.5 ml of alcohol and 7.5 ml of toluene. This procedure for grinding and 
rinsing was used for the other materials except in a few cases that will be 
discussed. 
2. Phenylalanine (C'*). C14-labeled material of suitable specific activity was 
made by dilution of Tracerlab’s 2-amino-3-phenylpropionic-1-C'™ acid with 
inactive compound and precipitating it from methanol by neutralization of the 
hydrochloride with ammonium hydroxide. Assay of the product was obtained 
by Van Slyke-Folch wet oxidation, followed by direct plate counting of the 
derived BaCO; in an internal-sample flow counter. This counter was carefully 
standardized with some of the BaCO; mentioned previously. 
3. Sodium acetate (H'). A 1-ml portion of a 400 mg/ml aqueous solution of 
tritium-labeled sodium acetate was evaporated to dryness. Grinding and rins- 
ing were done with 8 ml toluene. Alcohol was avoided because of its solvent 
power for sodium acetate. 
4, Barium sulfate (S*). Inactive material was prepared from commercial 
sodium sulfate, which was dissolved in water, acidified with hydrochloric acid, 
and treated with barium chloride to precipitate the desired product (7). Active 
material was prepared by adding a suitable dilution of S%°O,- in HCl as sup- 
plied by Oak Ridge to the aqueous sodium sulfate before treatment with barium 
chloride. Quantitative yields were obtained. 
5. Benzidinium sulfate (S*), Inactive material was prepared from commercial 
sodium sulfate dissolved in an alcohol-water solution by adding benzidine 
hydrochloride to precipitate benzidinium sulfate (8). As above, for the prepa- 
ration of the active material, a dilution of the Oak Ridge S** in HCl was added 
to the solution of sodium sulfate before the precipitation. 
6. Calcium oxalate (Ca‘*), Inactive material was prepared by a standard 
procedure (9) from commercial calcium chloride. To make the labeled com- 
pound, a suitable dilution of Ca** in HCl supplied by Oak Ridge was added to 
the above preparation before the final precipitation. 
7. Calcium carbonate (Ca‘*5). Inert material was prepared by precipitation from 
an aqueous solution of calcium chloride to which was added a saturated solution 
of ammonium carbonate. The labeled compound was made by adding the 
dilution of Ca‘® mentioned above before precipitation. 
8. Tissues (C!4). A number of tissues, selected to give a wide range in color 
and composition, were taken from rats injected with C!4-nitrogen mustard. 
These were liver, spleen, testes, muscle and bone. Homogenates were pre- 
pared by freezing the tissue in liquid nitrogen and pulverizing while still frozen. 
Tissues from normal animals were used to provide inactive material. 
Initial studies were made with dried material. This was obtained by 
lyophilizing aliquots of the tissue homogenates for 20 to 24 hr. The water col- 
lected from this drying was assayed for any activity that may have been lost 
from the tissue during the process. An insignificant amount of activity was 
found. 
The usual grinding and rinsing procedures were used at first, but later work 
utilized the tissue homogenates directly. The sample was ground in 2 ml abso- 
lute alcohol and then rinsed into the counting bottle with 6 ml toluene. 
9. Bacteria (C4). Lactobacillus arabinosus cells were harvested from a 48-hr- 
old synthetic culture medium (10) and washed with saline and water. They 
were then lyophilized for 24 hr and yielded 260 mg inactive material. 
Labeled bacteria were obtained as above, by growing the Lactobacillus in a 
synthetic culture medium containing C!4-nicotinic acid. 
lyophilized and then suspended or sus- 
pended directly as wet tissue homo- 
genate. Preparation of the C!4-homo- 
genates and the suspensions is described 
under Sample Preparation. 
An efficiency for each suspension was 
determined by the addition of internal 
standard and a recount. Since this 
efficiency is the same as the self-effi- 
ciency of the sample, the f-value being 
1, dpm/mg were calculated from each 
suspension count. A comparison of 
the averaged results for each tissue 
when the suspension was prepared from 
wet and lyophilized material is shown 
in Table 2. 
As mentioned previously, bottles of 
two sizes were used for this study. No 
difference in f-value was found between 
the two; this might be expected from 
consideration of the Beer-Lambert Law. 
With the same amount of suspended 
material, changing from the large bot- 
tle to the small would have the effect of 
increasing concentration while decreas- 
ing path length with absorption and 
scatter remaining the same. The small 
bottle numbers among its advantages: 
availability, cheapness, duplication of 
whiteness of glass, and ease of cleaning. 
H’. When 42.3 mg sodium acetate 
was suspended in an alcohol-free scin- 
tillator, the self-calibrated efficiency 
for the suspension was 3.1% and for 
the tritium internal standard 11.1%. 
This f-value of 0.28 is evidence for an 
expected large degree of self-absorption 
of the weak beta spectrum by the par- 
ticles of the suspension because of their 
relatively large size compared with the 
range of the tritium beta particles. 
S*° and Ca*®, The maximum beta 
energies of S*° (167 kev) and Ca‘*® (255 
kev) are both greater than that of C14 
(155 kev). One must then conclude 
from the previously mentioned C!4 data 
that, with suitable grinding, the f-fac- 
tors for S** and Ca‘ willbe 1.0. Unfor- 
tunately, homogeneous internal stand- 
ards for these isotopes were not avail- 
able to check this. 
Barium sulfate, benzidinium sulfate, 
calcium oxalate, and calcium carbonate 
gave very normal-appearing suspen- 
sions and seemed, from the Oak Ridge 
assays, to count with high efficiency, 
even when as much as 120 mg of mate- 
rial was suspended in the counting 
volume. 
Settling Studies 
An interesting facet of the behavior 
of these suspensions is their settling 
and the accompanying drop in counting 
rate. This drop is presumed to be due 
to the drift away from 100% geometry 
toward 50% geometry while settling to 
the bottom of the counting bottle. 
Figure 2 is a plot of the drop in count- 
ing rate vs. elapsed time for 20 mg of 
calcium oxalate in a typical experiment. 
At early times the drop is exponential 
and then levels off. Other concentra- 
tions and materials used behave this 
way too. 
The slope of the exponential portion 
of the settling curve was determined 
for various concentrations of some of 
the suspensions. It was found to be 
reasonably independent of concentra- 
tion, suggesting that the grinding proc- 
ess was reproducible. Average slope 
values were 3,640 sec for BaCOs, 1,060 
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