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A rapid, hygienic method for the preparation of fecal samples for liquid scintillation counting
ANALYTICAL
BIOCHEMISTRY
85,
79-85 (1978)
A Rapid, Hygienic Method for the Preparation of Fecal Samples for Liquid Scintillation Counting B.J. MORRISON~AND R. A. FRANKLIN Drug Metabolism
Section, Wyeth Laboratories, Huntercombe Maidenhead, Berks, England
Lone South, Taplow,
Received October 19, 1976; accepted September 28, 1977 A method for the preparation of fecal samples for liquid scintillation couuting is described which is rapid, hygienic, and inexpensive. By the use of a novel type of homogenizer, fecal samples can be homogenized while totally enclosed within a sealed, plastic bag, so reducing the possible risk of infection. The subsequent preparation of a clear solution suitable for liquid scintillation counting is petfommd using an “in-vial” digestion technique which enabks any “CO* released during digestion to be trapped within the vial.
The preparation of fecal material for liquid scintillation counting invariably requires that the sample be homogenized prior to further treatment. Homogenization is usually carried out using a blade homogenizer (e.g., Waring blender) or a Potter-Elvehjem-type homogenizer. If a large number of samples are involved, this homogenixation step can become very time-consuming, owing to the need to frequently decontaminate the homogenizer parts. Furthermore, the operator may be exposed to the possible risk of enteric infection due to the repeated transfer of fecal homogenates and washing of the homogenizer parts. If the homogenizer is incompletely decontaminated, then there is also the possibility that experimental errors may be introduced due to radioactive cross-contamination. Several methods have been described for the subsequent treatment of the homogenate; these include combustion in a tissue oxidizer, treatment, with solubilizing agents, (such as Hyamine 10X hydroxide, NCS, and Bio-Solv BBS), or a wet oxidation technique (1,2). Of these, the wet oxidation method of Mahin and Lofberg (2) is both rapid and inexpensive compared with the other techniques. This technique is useful for tritiated samples and for ‘*C-labeled samples, where the labeled carbon atom is stable under the conditions of digestion. However, a major criticism of the technique is that where the 14Catom is likely to undergo oxidation the resultant 14C0, will be lost. ’ Resent address: Shell Research Ltd., Metabolic Chemistry Research Centre, Sittingbourne, Kent ME9 8AG, U. K. 79
Section, Sittingboume
0003-2697/78/0851-0079!$82.0@‘0 Copy&lit Au &hts
Og 1978 by APress. Inc. of reproduction in my form resewed.
80
MORRISON
AND
FRANKLIN
This communication describes a rapid, safe, and inexpensive technique for the preparation of fecal homogenates and a modification of the Mahin and Lofberg technique to overcome the problem of a r4C label readily susceptible to oxidation. MATERIALS
Chlorhexidine (Hibitane) was supplied by I.C.I. Ltd., Macclesfield, Cheshire, U.K. Plastic bags (Cryovac, 18 x 30 cm, 250-g capacity) and the Colworth Stomacher 400 paddle homogenizer were from A. J. Seward & Co. Ltd., London, U.K. The Ato-Mix laboratory blender was supplied by M.S.E. Ltd., Crawley, Sussex, U.K. Scintillation vials (Type 6001050 fitted with polyethylene skive) were obtained from Packard Instruments Ltd., Caversham, Berkshire, U.K. The septa (20 x 3 mm) used were made of a butyl rubber and supplied by West Pharmarubber Ltd., London, U.K. Perchloric acid and hydrogen peroxide, both “ Aristar” grade, were from BDH Chemicals Ltd., Poole, Dorset, U.K. The Hamilton l-ml gas-tight syringe was supplied by Phase Separations Ltd., Queensferry, Clwyd, U.K. The 14C-labeled sodium bicarbonate, 14C-labeled benzyl penicillin potassium, and tritiated water were obtained from the Radiochemical Centre Ltd., Amersham, Buckinghamshire, U.K. The scintillant used was NE 260 from Nuclear Enterprises Ltd., Edinburgh, Scotland, and all scintillation counting was performed using a Packard Tri-Carb liquid scintillation spectrometer, Model 3380, equipped with automatic external standardization. Animals. Male and female Red Patas monkeys (Erythrocebus patas) weighing 3-5 kg were supplied by Shamrock Farms Ltd., Brighton, Sussex. They were maintained on an Oxoid 41B diet supplemented by fresh fruit. METHODS
i. Dose administration and sample collection, All animals were fasted overnight prior to dosing, but allowed free access to water. In a volume of 5 ml/kg of isotonic saline, 10 mg/kg of [l-14C]benzyl penicillin was administered by gastric intubation. The collection of urine and feces was facilitated by housing the monkeys in specially constructed metabolism cages (National Iron and Wire Co. Ltd., Manchester, U.K.). ii. Preparation offecal homogenate. Fecal material (up to 100 g) was weighed in a Cryovac plastic bag, and a volume of 0.1% (v/v) chlorhexidine solution was added to form a homogenate of suitable viscosity. This volume varied depending on the water content of the particular fecal sample. For human and monkey samples, the proportions were usually one part chlorhexidine solution to one part feces, and for rat samples one part to two parts, respectively. These proportions produced a homogenate with the consistency of a “stiff sludge.” The plastic bag was then placed in a
SCINTILLATION
COUNTING OF FECES
81
FIG. 1, Diagram of Colworth Stomacher 400 paddle homogenizer and sample preparation bag. CH, door-closing handle;D, door; RS, rubber seal (ensures that the open end ofthe bag is tightly clamped when the door is closed): P, paddles.
Colworth Stomacher 400 paddle homogenizer (Fig. 1) and sealed by the clamping action of the door. The contents of the bag were then pulverised for 1.5-2.0 min by the reciprocating action of the paddles pounding the bag repeatedly against the door of the homogenizer. The bag was removed, and the homogenate sampled using a long-handled, spoon-spatula. Alternatively, samples were homogenized, using the same proportions as above, in an ’ ‘Ato-Mix” blade-type homogenizer for 1.5 min and then sampled. iii. Digestion of the fecal homogenate. The digestion was performed in a scintillation vial with a modified cap. A hole 3 mm in diameter was drilled through the center of the cap, and its polyethylene skive was replaced with a rubber septum. A small amount of fecal homogenate (100-200 mg wet weight) was weighed into the scintillation vial, and 0.2 ml of 70% (v/v) perchloric acid and 0.4 ml of lOO-vol hydrogen peroxide were added. The vial was then tightly capped and placed in an oven, at 7X, for 1S-2.0 hr. It was then removed and cooled at -20°C for 20 min. The vial was then kept in ice while 0.5 ml of 20 M sodium hydroxide was injected through the septum using a gas-tight syringe. The vial was left at room temperature for at least 1 hr to allow the sodium hydroxide to absorb any 14C0, evolved. Subsequently, 16 ml of NE 260 scintillant was added, and the sample was counted. iv. Measurement of urinary radioactivity. Urine was assayed by the direct addition of an aliquot of the sample to NE 260 scintillant.
82
MORRISON
AND FRANKLIN
RESULTS i. Homogenization
Technique
To evaluate the use of the Colworth Stomacher, a comparison of this novel paddle-type homogenizer was made with a more conventional blade-type homogenizer. Four monkeys were dosed orally with [l14C]benzyl penicillin, and urine and feces were collected for 4-5 days. Fecal samples from two monkeys were homogenized using a Colworth Stomacher and those from the other two using an ’ ‘Ato-Mix” homogenizer. Both types of homogenate were then assayed for radioactivity using the septum-closed vial technique described above. Ten replicates of a suitable sample from each monkey were assayed to demonstrate the thoroughness of homogenization. The results show no marked difference in the standard errors obtained for each set of values (Table 1). Furthermore the total recoveries of the administered radioactivity were good (see Table 2) indicating that the fecal analyses were not merely reproducible but were also correct in the absolute sense. ii. Digestion
Technique
To verify that any 14C0, evolved during a conventional Mahin and Lofberg digestion would normally be lost, a number of monkey fecal samples were collected, and to each was added 5 &i of 3Hz0 and 5 &i of TABLE
1
COMPARININ OFTHEREPRODUCIBILITY OFRESULTS FROMFECALSAMPLES USING Two DIFFERENTMETHODSOFHOMOGENIZATION Percentage recovery of administered radioactivity Colworth Stomacher homogenization
Ato-Mix blade homogenization
Monkey No.
Monkey No.
288
Mean 2 SEM
312
360
301
34.42 38.06 43.31 63.14 49.06 50.45 45.78 46.67 48.58 48.11
38.56 43.29 47.83 40.59 43.78 45.41 44.39 47.42 43.55 39.40
21.96 32.72 29.11 32.47 34.22 35.19 28.01 35.26 25.78 34.75
38.27 39.15 36.54 37.11 33.07 34.14 37.95 38.24 37.01 37.04
46.76 “- 2.43
43.42 r 0.99
30.95 f 1.44
36.85 k 0.60
SCINTILLATION
COUNTING TABLE
83
OF FECES
2
FECAL AND URINARY RECOVERY OF ‘Y: FROM MONKEYS DOSED WITA WCJBENZYL PENICILLIN: A COMPARISON OF Two METHOD OF FECAL HOMOGENIZATION Percentage recovery of administered radioactivity Feces Monkey No.
Colworth Stomacher homogenization
288 312 360 301
48.04 57.34 -
Ato-h4ix homogenization 50.79 64.11
Urine
Total
30.73 27.15 22.38 19.13
78.77 84.49 73.17 83.24
NaH1*COs. The samples were then homogenized and assayed in triplicate using the Mahin and L&berg “in-vial” wet oxidation technique. The method used was as described above except that the scintillation vials had umnodi&d caps and no sodium hydroxide was added. A good recovery of tritium was obtained, 95.312 1.01% (SEM), but the recovery of 14Cwas low, 8.58 -+ 0.66% (SEM). A method of trapping the 14COzwithin the vial was sought, and the septum-closed vial technique, described in the Methods section, was developed. Monkey fecal homogenates were prepared, and small amounts were added to scintillation vials. To each vial 0.5 &i of aHeOand 0.5 &Zi of NaH”CO, were added, and the samples were assayed using the septum-closed vial technique. The recovery of tritium was 94.63 ? 0.50% (SEM) and the recovery of 14Cwas 98.73 -c 0.86% @EM). Finally the capacity of the septum-closed vial technique for digesting fecal material containing a compound with a labile 14C atom was determined by using a previously assayed fecal sample from a monkey dosed with [PC]benzyl penicillin [a compound that was expected to release “Cq during digestion (3)]. No significant variations in the expected results were obtained for aliquots up to 600 mg (see Table 3). Counting efficiencies, under dual-label-counting conditions, for tritium and “C were 7.9-13% and 58.3-61.5%, respectively, for the conventional Mahin and L&berg technique. Following the septum-closed vial method these counting efWencies were slightly reduced to 4.6-7.9% for tritium, and to 48.3-58.3% for 14C.For single-labeled “C samples, prepared using the septum-closed vial technique, the counting efficiency was in the range 69.1-77.2%. DISCUSSION
Monkey feces can be a ready source of several types of enteric infections,
the most important
of which are Shigellosis
and Salmonellosis
84
MORRISON
AND FRANKLIN TABLE
INFLUENCE
3
OF WEIGHT OF FECAL MATERIAL DIGESTED UPON APPARENT RECOVERY OF RADIOCARBON FROM [I-YIBENZYL PENICILLIN USING THE SEPTUM-CLOSED VIAL TECHNIQUE’
Weight 63)
Percentage recovery
0.152
0.493
46.20 45.99 43.89 45.49 45.44
0.601
46.15
0.204 0.327 0.401
Expected recoveryb m 49.07 49.07 49.07 49.07 49.07 49.07
k 0.59 (SEM) ‘- 0.59 + 0.59 zk 0.59 f 0.59 k 0.59
a Results expressed as a mean of three samples. * Of administered dose.
(43). Since the animal itself may show no outward clinical symptoms of the infection, the risk may not be immediately apparent. However, by using a “Colworth Stomacher” paddle homogenizer this risk of possible infection may be greatly reduced. The fecal sample is at no time in direct contact with the homogenizer, each sample being homogenized within its own individual plastic bag. The only transfer required is of a small aliquot to a scintillation vial. To reduce further the risk of infection, chlorhexidine, a potent bactericidal agent has been added to the suspending vehicle. Thus this technique of homogenization enables large numbers of samples to be prepared both rapidly and hygienically, and furthermore, the homogenates may be stored compactly in individual plastic bags. The basic technique for the wet oxidation of biological material as described by Mahin and Lofberg provides a convenient method for assaying radioactivity. However, while any tritiated water vapor evolved during digestion would be condensed by cooling to -2O”C, a 14Clabel could be lost as gaseous 14C02. Even when the parent compound is chemically stable to the digestion conditions, it is possible that metabolites may be produced which are completely oxidized in the technique. Benzyl penicillin potassium (potassium-6-phenyl[ I-‘*C]acetamido penicillinate) was chosen as a representative type of compound which would undergo chemical decomposition leading to loss of 14C0, during digestion. (Perchloric acid and hydrogen peroxide would hydrolyze the benzyl penicillin to yield [lJ4C]phenylacetic acid which would be readily decarboxylated to produce 14C0,). Using the septum-closed vial technique described in this paper it has been possible to absorb this 14C0, within the vial using sodium hydroxide as a trapping agent, and by using NE 260 scintillant, no problems of base-induced chemiluminescence have been encountered. Thus this
SCINTILLATION
COUNTING
OF FECES
85
technique extends the Mahin and Lofberg method by overcoming its major criticism. Moreover its use in combination with the novel method of sample homogenization leads to a rapid, hygienic, and inexpensive method for the preparation of fecal samples for liquid scintillation counting. REFERENCES 1. Pollay, M., and Stevens, F. A. (1970) in The Current Status of Liquid Scintillation Counting (Bransome, E.D., ed.), pp. 207-211, Grune & Stratton, New York. 2. Mahin, D. T., and Lotberg, R. T. (1966) Anal. Biochem. 16, 500-509. 3. Peck, R. L., and Folkers, K. (1949) in The Chemistry of Penicillin (Clarke, H. T., Johnson, J. R., and Robinson, R., ed.), p. 53. Princeton University Press, Princeton. 4. Fiennes, R. (1%7) in Zoonoses of Primates, pp. 103-114. Wiedenfield and Nicholson, London. 5. Boulter, E. A. (1972) in Safety in the Animal House (Seamer, J., ed.), pp. 17-29. Laboratory Animals Ltd., London.
BIOCHEMISTRY
85,
79-85 (1978)
A Rapid, Hygienic Method for the Preparation of Fecal Samples for Liquid Scintillation Counting B.J. MORRISON~AND R. A. FRANKLIN Drug Metabolism
Section, Wyeth Laboratories, Huntercombe Maidenhead, Berks, England
Lone South, Taplow,
Received October 19, 1976; accepted September 28, 1977 A method for the preparation of fecal samples for liquid scintillation couuting is described which is rapid, hygienic, and inexpensive. By the use of a novel type of homogenizer, fecal samples can be homogenized while totally enclosed within a sealed, plastic bag, so reducing the possible risk of infection. The subsequent preparation of a clear solution suitable for liquid scintillation counting is petfommd using an “in-vial” digestion technique which enabks any “CO* released during digestion to be trapped within the vial.
The preparation of fecal material for liquid scintillation counting invariably requires that the sample be homogenized prior to further treatment. Homogenization is usually carried out using a blade homogenizer (e.g., Waring blender) or a Potter-Elvehjem-type homogenizer. If a large number of samples are involved, this homogenixation step can become very time-consuming, owing to the need to frequently decontaminate the homogenizer parts. Furthermore, the operator may be exposed to the possible risk of enteric infection due to the repeated transfer of fecal homogenates and washing of the homogenizer parts. If the homogenizer is incompletely decontaminated, then there is also the possibility that experimental errors may be introduced due to radioactive cross-contamination. Several methods have been described for the subsequent treatment of the homogenate; these include combustion in a tissue oxidizer, treatment, with solubilizing agents, (such as Hyamine 10X hydroxide, NCS, and Bio-Solv BBS), or a wet oxidation technique (1,2). Of these, the wet oxidation method of Mahin and Lofberg (2) is both rapid and inexpensive compared with the other techniques. This technique is useful for tritiated samples and for ‘*C-labeled samples, where the labeled carbon atom is stable under the conditions of digestion. However, a major criticism of the technique is that where the 14Catom is likely to undergo oxidation the resultant 14C0, will be lost. ’ Resent address: Shell Research Ltd., Metabolic Chemistry Research Centre, Sittingbourne, Kent ME9 8AG, U. K. 79
Section, Sittingboume
0003-2697/78/0851-0079!$82.0@‘0 Copy&lit Au &hts
Og 1978 by APress. Inc. of reproduction in my form resewed.
80
MORRISON
AND
FRANKLIN
This communication describes a rapid, safe, and inexpensive technique for the preparation of fecal homogenates and a modification of the Mahin and Lofberg technique to overcome the problem of a r4C label readily susceptible to oxidation. MATERIALS
Chlorhexidine (Hibitane) was supplied by I.C.I. Ltd., Macclesfield, Cheshire, U.K. Plastic bags (Cryovac, 18 x 30 cm, 250-g capacity) and the Colworth Stomacher 400 paddle homogenizer were from A. J. Seward & Co. Ltd., London, U.K. The Ato-Mix laboratory blender was supplied by M.S.E. Ltd., Crawley, Sussex, U.K. Scintillation vials (Type 6001050 fitted with polyethylene skive) were obtained from Packard Instruments Ltd., Caversham, Berkshire, U.K. The septa (20 x 3 mm) used were made of a butyl rubber and supplied by West Pharmarubber Ltd., London, U.K. Perchloric acid and hydrogen peroxide, both “ Aristar” grade, were from BDH Chemicals Ltd., Poole, Dorset, U.K. The Hamilton l-ml gas-tight syringe was supplied by Phase Separations Ltd., Queensferry, Clwyd, U.K. The 14C-labeled sodium bicarbonate, 14C-labeled benzyl penicillin potassium, and tritiated water were obtained from the Radiochemical Centre Ltd., Amersham, Buckinghamshire, U.K. The scintillant used was NE 260 from Nuclear Enterprises Ltd., Edinburgh, Scotland, and all scintillation counting was performed using a Packard Tri-Carb liquid scintillation spectrometer, Model 3380, equipped with automatic external standardization. Animals. Male and female Red Patas monkeys (Erythrocebus patas) weighing 3-5 kg were supplied by Shamrock Farms Ltd., Brighton, Sussex. They were maintained on an Oxoid 41B diet supplemented by fresh fruit. METHODS
i. Dose administration and sample collection, All animals were fasted overnight prior to dosing, but allowed free access to water. In a volume of 5 ml/kg of isotonic saline, 10 mg/kg of [l-14C]benzyl penicillin was administered by gastric intubation. The collection of urine and feces was facilitated by housing the monkeys in specially constructed metabolism cages (National Iron and Wire Co. Ltd., Manchester, U.K.). ii. Preparation offecal homogenate. Fecal material (up to 100 g) was weighed in a Cryovac plastic bag, and a volume of 0.1% (v/v) chlorhexidine solution was added to form a homogenate of suitable viscosity. This volume varied depending on the water content of the particular fecal sample. For human and monkey samples, the proportions were usually one part chlorhexidine solution to one part feces, and for rat samples one part to two parts, respectively. These proportions produced a homogenate with the consistency of a “stiff sludge.” The plastic bag was then placed in a
SCINTILLATION
COUNTING OF FECES
81
FIG. 1, Diagram of Colworth Stomacher 400 paddle homogenizer and sample preparation bag. CH, door-closing handle;D, door; RS, rubber seal (ensures that the open end ofthe bag is tightly clamped when the door is closed): P, paddles.
Colworth Stomacher 400 paddle homogenizer (Fig. 1) and sealed by the clamping action of the door. The contents of the bag were then pulverised for 1.5-2.0 min by the reciprocating action of the paddles pounding the bag repeatedly against the door of the homogenizer. The bag was removed, and the homogenate sampled using a long-handled, spoon-spatula. Alternatively, samples were homogenized, using the same proportions as above, in an ’ ‘Ato-Mix” blade-type homogenizer for 1.5 min and then sampled. iii. Digestion of the fecal homogenate. The digestion was performed in a scintillation vial with a modified cap. A hole 3 mm in diameter was drilled through the center of the cap, and its polyethylene skive was replaced with a rubber septum. A small amount of fecal homogenate (100-200 mg wet weight) was weighed into the scintillation vial, and 0.2 ml of 70% (v/v) perchloric acid and 0.4 ml of lOO-vol hydrogen peroxide were added. The vial was then tightly capped and placed in an oven, at 7X, for 1S-2.0 hr. It was then removed and cooled at -20°C for 20 min. The vial was then kept in ice while 0.5 ml of 20 M sodium hydroxide was injected through the septum using a gas-tight syringe. The vial was left at room temperature for at least 1 hr to allow the sodium hydroxide to absorb any 14C0, evolved. Subsequently, 16 ml of NE 260 scintillant was added, and the sample was counted. iv. Measurement of urinary radioactivity. Urine was assayed by the direct addition of an aliquot of the sample to NE 260 scintillant.
82
MORRISON
AND FRANKLIN
RESULTS i. Homogenization
Technique
To evaluate the use of the Colworth Stomacher, a comparison of this novel paddle-type homogenizer was made with a more conventional blade-type homogenizer. Four monkeys were dosed orally with [l14C]benzyl penicillin, and urine and feces were collected for 4-5 days. Fecal samples from two monkeys were homogenized using a Colworth Stomacher and those from the other two using an ’ ‘Ato-Mix” homogenizer. Both types of homogenate were then assayed for radioactivity using the septum-closed vial technique described above. Ten replicates of a suitable sample from each monkey were assayed to demonstrate the thoroughness of homogenization. The results show no marked difference in the standard errors obtained for each set of values (Table 1). Furthermore the total recoveries of the administered radioactivity were good (see Table 2) indicating that the fecal analyses were not merely reproducible but were also correct in the absolute sense. ii. Digestion
Technique
To verify that any 14C0, evolved during a conventional Mahin and Lofberg digestion would normally be lost, a number of monkey fecal samples were collected, and to each was added 5 &i of 3Hz0 and 5 &i of TABLE
1
COMPARININ OFTHEREPRODUCIBILITY OFRESULTS FROMFECALSAMPLES USING Two DIFFERENTMETHODSOFHOMOGENIZATION Percentage recovery of administered radioactivity Colworth Stomacher homogenization
Ato-Mix blade homogenization
Monkey No.
Monkey No.
288
Mean 2 SEM
312
360
301
34.42 38.06 43.31 63.14 49.06 50.45 45.78 46.67 48.58 48.11
38.56 43.29 47.83 40.59 43.78 45.41 44.39 47.42 43.55 39.40
21.96 32.72 29.11 32.47 34.22 35.19 28.01 35.26 25.78 34.75
38.27 39.15 36.54 37.11 33.07 34.14 37.95 38.24 37.01 37.04
46.76 “- 2.43
43.42 r 0.99
30.95 f 1.44
36.85 k 0.60
SCINTILLATION
COUNTING TABLE
83
OF FECES
2
FECAL AND URINARY RECOVERY OF ‘Y: FROM MONKEYS DOSED WITA WCJBENZYL PENICILLIN: A COMPARISON OF Two METHOD OF FECAL HOMOGENIZATION Percentage recovery of administered radioactivity Feces Monkey No.
Colworth Stomacher homogenization
288 312 360 301
48.04 57.34 -
Ato-h4ix homogenization 50.79 64.11
Urine
Total
30.73 27.15 22.38 19.13
78.77 84.49 73.17 83.24
NaH1*COs. The samples were then homogenized and assayed in triplicate using the Mahin and L&berg “in-vial” wet oxidation technique. The method used was as described above except that the scintillation vials had umnodi&d caps and no sodium hydroxide was added. A good recovery of tritium was obtained, 95.312 1.01% (SEM), but the recovery of 14Cwas low, 8.58 -+ 0.66% (SEM). A method of trapping the 14COzwithin the vial was sought, and the septum-closed vial technique, described in the Methods section, was developed. Monkey fecal homogenates were prepared, and small amounts were added to scintillation vials. To each vial 0.5 &i of aHeOand 0.5 &Zi of NaH”CO, were added, and the samples were assayed using the septum-closed vial technique. The recovery of tritium was 94.63 ? 0.50% (SEM) and the recovery of 14Cwas 98.73 -c 0.86% @EM). Finally the capacity of the septum-closed vial technique for digesting fecal material containing a compound with a labile 14C atom was determined by using a previously assayed fecal sample from a monkey dosed with [PC]benzyl penicillin [a compound that was expected to release “Cq during digestion (3)]. No significant variations in the expected results were obtained for aliquots up to 600 mg (see Table 3). Counting efficiencies, under dual-label-counting conditions, for tritium and “C were 7.9-13% and 58.3-61.5%, respectively, for the conventional Mahin and L&berg technique. Following the septum-closed vial method these counting efWencies were slightly reduced to 4.6-7.9% for tritium, and to 48.3-58.3% for 14C.For single-labeled “C samples, prepared using the septum-closed vial technique, the counting efficiency was in the range 69.1-77.2%. DISCUSSION
Monkey feces can be a ready source of several types of enteric infections,
the most important
of which are Shigellosis
and Salmonellosis
84
MORRISON
AND FRANKLIN TABLE
INFLUENCE
3
OF WEIGHT OF FECAL MATERIAL DIGESTED UPON APPARENT RECOVERY OF RADIOCARBON FROM [I-YIBENZYL PENICILLIN USING THE SEPTUM-CLOSED VIAL TECHNIQUE’
Weight 63)
Percentage recovery
0.152
0.493
46.20 45.99 43.89 45.49 45.44
0.601
46.15
0.204 0.327 0.401
Expected recoveryb m 49.07 49.07 49.07 49.07 49.07 49.07
k 0.59 (SEM) ‘- 0.59 + 0.59 zk 0.59 f 0.59 k 0.59
a Results expressed as a mean of three samples. * Of administered dose.
(43). Since the animal itself may show no outward clinical symptoms of the infection, the risk may not be immediately apparent. However, by using a “Colworth Stomacher” paddle homogenizer this risk of possible infection may be greatly reduced. The fecal sample is at no time in direct contact with the homogenizer, each sample being homogenized within its own individual plastic bag. The only transfer required is of a small aliquot to a scintillation vial. To reduce further the risk of infection, chlorhexidine, a potent bactericidal agent has been added to the suspending vehicle. Thus this technique of homogenization enables large numbers of samples to be prepared both rapidly and hygienically, and furthermore, the homogenates may be stored compactly in individual plastic bags. The basic technique for the wet oxidation of biological material as described by Mahin and Lofberg provides a convenient method for assaying radioactivity. However, while any tritiated water vapor evolved during digestion would be condensed by cooling to -2O”C, a 14Clabel could be lost as gaseous 14C02. Even when the parent compound is chemically stable to the digestion conditions, it is possible that metabolites may be produced which are completely oxidized in the technique. Benzyl penicillin potassium (potassium-6-phenyl[ I-‘*C]acetamido penicillinate) was chosen as a representative type of compound which would undergo chemical decomposition leading to loss of 14C0, during digestion. (Perchloric acid and hydrogen peroxide would hydrolyze the benzyl penicillin to yield [lJ4C]phenylacetic acid which would be readily decarboxylated to produce 14C0,). Using the septum-closed vial technique described in this paper it has been possible to absorb this 14C0, within the vial using sodium hydroxide as a trapping agent, and by using NE 260 scintillant, no problems of base-induced chemiluminescence have been encountered. Thus this
SCINTILLATION
COUNTING
OF FECES
85
technique extends the Mahin and Lofberg method by overcoming its major criticism. Moreover its use in combination with the novel method of sample homogenization leads to a rapid, hygienic, and inexpensive method for the preparation of fecal samples for liquid scintillation counting. REFERENCES 1. Pollay, M., and Stevens, F. A. (1970) in The Current Status of Liquid Scintillation Counting (Bransome, E.D., ed.), pp. 207-211, Grune & Stratton, New York. 2. Mahin, D. T., and Lotberg, R. T. (1966) Anal. Biochem. 16, 500-509. 3. Peck, R. L., and Folkers, K. (1949) in The Chemistry of Penicillin (Clarke, H. T., Johnson, J. R., and Robinson, R., ed.), p. 53. Princeton University Press, Princeton. 4. Fiennes, R. (1%7) in Zoonoses of Primates, pp. 103-114. Wiedenfield and Nicholson, London. 5. Boulter, E. A. (1972) in Safety in the Animal House (Seamer, J., ed.), pp. 17-29. Laboratory Animals Ltd., London.