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Factors Affecting Reliability of Catalase Test Results. I. Sanitizers1
Factors Affecting Reliability of Catalase Test Results. I. Sanitizers 1 H. E. RANDOLPH, J. L, BUCY, and T. R. FREEMAN
Department of Animal Sciences, University of Kentucky, Lexington Experimental Procedure Milk samples were obtained from individual cows from the University of Kentucky dairy herds. Concentrated solutions of sanitizers (prepared from commercial products) were added to milk to give concentrations of 5 to 50 ppm of the active materials. The concentrated solutions were prepared so that a constant volume of solution would be added to the test samples. An equal volume of distilled water water was added to the control sample (0 ppm). The eatalase activity of the sample, immediately after addition of the sanitizers and after holding at 4.5 C for 5 and 24 hr (for chlorine compounds), was determined by the inverted tube method described by Spencer and Simon (6). Graduated centrifuge tubes with one-hole rubber stoppers fitted with straight glass tubing (3) or screw caps (with 0.5 mm hole) were used. The type of tube was kept constant within any given comparative trial.
Abstract
The influeuce of commercial chemical sanitizers in concentrations of 5 to 50 ppm on the catalase test was determined. Chlorine sanitizers caused a pronounced reduction in the percentage of oxygen produced. The effect increased with increasing concentrations of the sanitizer and was more pronounced when the test was made after 5 or 24 hr of storage at 4.5 C following addition of the sanitizers, than when tested immediately. Iodophors, quaternary ammonium compounds, an acid sanitizer, and a sanitizer containing both potassium iodide and chloramine-T did not influence test results. These results indicate that chlorine sanitizers interfere with the catalase test. The reliability of the results of the test could be markedly reduced by contamination of the milk with these products, but contamination by other types of sanitizers would not alter the reliability of the test.
Results and Discussion
Chlorine compounds. The effect of sodium hypochlorite, calcium tlypochlorite, 1, 3-dichloro 5, 5-dimethylhydantoin, and dichloroisocyanuric acid (dichlor (s)-triazine-trione) in concentrations of 5, 10, 15, 20, 25, and 50 ppm available chlorine on the catalase test was determined. Tests were conducted immediately and after 5 and 24 hr storage at 4.5 C following addition of the sanitizer. Results are shown in Figure 1. All of these sanitizers caused a marked reduction in the percentages of oxygen produced in the catalase test. The effect increased with increasing concentrations of chlorine and was more pronounced when the samples were tested after holding at 4.5 C for 5 and 24 hr following addition of the sanitizer. Athough there appear to be some variations in magnitude of the effect of the different sanitizers on tile test results, the basic pattern is very similar for all of the sanitizers. I t is believed that the differences shown here are not due to variations in the inhibiting properties of the sanitizers; rather, it seems more likely that these apparent differences are due to variations in the milk samples. Observations during the course of this investigation suggest that the effect of the chlorine on the catalase test may not be as pronounced when the catalase activity
The catalase test has received much attention as a screening procedure for detection of abnormal milk (1, 5). It is one of the tests that the U. S. Public Health Service (6) has recommended and is used by many state regulatory agencies (5). Considerable work (2, 3, 6) has been done on the relationship of catalase activity to leucocyte counts. Also studied has been the effect of techniques and storage of the milk on test results. There is a lack of information, however, concerning factors which might influence the reliability of the catalase test. I n a preliminary report (4) it was shown that the addition of 10 to 50 ppm sodium hypochlorite to milk decreased the amount of oxygen produced in the test. The purpose of the present study was to obtain further information on the effect of chemical sanitizers on the reliability of the catalase test results. l~eceived for publication August 9, :1966. The investigation reported in this paper (Journal Article no. 66-5-71) is in connection with a project of the Kentucky Agricultural Experiment Station and is published with the approval of the Director. 32
I~EJJIARILITY OF CATALASE TE~T
5O
L ll~
. I I ~
Z
Tested immediately after adding sanltizer
!
!
!
50 Tested after 5 hr storageat 4.5C
k ~
I
5C
I
I
I
I
I
I
I
I
I
t
~ k
Tested after 24 hr
33
Other sanitizers. The effect of iodophors, quaternary ammonium compounds (QAC), an acid sanitizer, and a sanitizer containing both potassium iodide and chloramine-T, and chloramine-T alone also was investigated. These sanitizers were used in concentrations of 10, 25, and 50 ppm of active ingredients, and the catalase test was made immediately a n d after 5 hr storage at 4.5 C following addition of the sanitizer. There were two different iodophors and two different QAC's used. Active ingredients listed for one iodophor were polyethoxy polypropoxy ethanol-iodine complex and nonyl phenylether of polyethylene glycol-iodine complex, and for the other, phosphoric acid, nonyl phenoxy polyethoxy ethanol-iodine complex, and polyethoxy polypropoxy polyethoxy ethanoi-iodine complex. The active ingredient for one QAC was alkyl dimethyl benzyl ammonium chloride and the other contained this compound plus alkyt dimethyl ethylbenzyl ammonium chloride. None of these compounds (in the concentrations used) had a measurable effect on the ca~alase test results, either when tested immediately or after 5 hr storage at 4.5 C following addition of the sanitizer. Contamination of milk with these sanitizers, within the levels investigated, should not adversely affect the reliability of the catalase test results. Conclusions
~'20~ 10 0
I
I
I
I
I
5
10
15
20
25
I
30
I
35
!
J
L
40
45
50
PPM CHLORINE
FIG. 1. Effect of chlorine sanitizers on per cent oxygen produced in the eatalase test: calcium hypochlorite e , sodium hypochlorite V, dieh]oro (s)triazine-trioneA, diehloroisocyanuric acid i , and 1, 3-dichloro 5, 5-dimethylhydantoin ×. Results for each sanitizer represent trials with five different samples of milk. of the milk is high. This possible relationship is under further investigation. The foregoing results indicate that the reliability of the catalase test results could be greatly decreased as a result of contamination with chlorine sanitizers on the farm or in collection of the samples.
Addition of chlorine sanitizers to milk decreases the amount of oxygen produced in the catalase test; thus, the reliability of this test for detecting abnormal milk could be profoundly influenced as a result of contamination with a ehlm~ne sanitizer. Extreme caution must be used in interpreting catalase test results when the possibility of such contaminations exists. Iodophors, quaternary ammonium compounds, an acid sanitizer, and a sanitizer containing potassium iodide and chloramine-T in concentrations up to 50 ppm did not exert a measurable effect on the catalase test and, thus, should not adversely affect the reliability of the results. References
(1) Corbett, W. J. 1963. Catalase Test for Detection of Abnormal Milk. Milk In& Found. Conv. Proc., Lab. See., p. 15. (2) ]~rank, Norma A., and Pounden, W. D. 1963. Preservation and Extended Refrigeration Effects on Catalase, California, and Whiteside Mastitis Tests. J. Am. Vet. Mcd. Assoc., 14 : 878. (3) Nageswararao, G., Blobel, H., and Derbyshire, J. ]3. 1965. Catalase Test for Abnormal Milk. I. Techniques and l~a~tors Affecting the Test. J. Dairy Sci., 48: 1290. J . DAIRY SCIENCE ~OL. 50, NO. 1
34
RANDOLPH, BUCY, FREEMAN
(4) Randolph, H. E., Bucy, J. L., and Freeman, T. R. 1966. Effect of Sodium Hypochlorite on the Catalase Test. J. Dairy Sci., 49: 685. (5) Randolph, tL E., and Conner, D. J. 1966. Survey of Abnormal Milk ~)rograms Adminstered by State Regulatory Agencies. J. Milk Food Technol., 29: 188.
J. DAIRY SCIENCE VOL. 50, NO. 1
(6) Spencer, G. R., and Simon, J. 1960. The Catalase, California, and Cell Count Tests for Detecting Abnormalities in Milk. Am. J. Yet. Research, 21: 578. (7) U. S. Department of Health, Education and Welfare. 1963. Screening Tests for the Detection of Abnormal Milk. Public Health Service Publ. no. 1306. Washington, D. C.
Department of Animal Sciences, University of Kentucky, Lexington Experimental Procedure Milk samples were obtained from individual cows from the University of Kentucky dairy herds. Concentrated solutions of sanitizers (prepared from commercial products) were added to milk to give concentrations of 5 to 50 ppm of the active materials. The concentrated solutions were prepared so that a constant volume of solution would be added to the test samples. An equal volume of distilled water water was added to the control sample (0 ppm). The eatalase activity of the sample, immediately after addition of the sanitizers and after holding at 4.5 C for 5 and 24 hr (for chlorine compounds), was determined by the inverted tube method described by Spencer and Simon (6). Graduated centrifuge tubes with one-hole rubber stoppers fitted with straight glass tubing (3) or screw caps (with 0.5 mm hole) were used. The type of tube was kept constant within any given comparative trial.
Abstract
The influeuce of commercial chemical sanitizers in concentrations of 5 to 50 ppm on the catalase test was determined. Chlorine sanitizers caused a pronounced reduction in the percentage of oxygen produced. The effect increased with increasing concentrations of the sanitizer and was more pronounced when the test was made after 5 or 24 hr of storage at 4.5 C following addition of the sanitizers, than when tested immediately. Iodophors, quaternary ammonium compounds, an acid sanitizer, and a sanitizer containing both potassium iodide and chloramine-T did not influence test results. These results indicate that chlorine sanitizers interfere with the catalase test. The reliability of the results of the test could be markedly reduced by contamination of the milk with these products, but contamination by other types of sanitizers would not alter the reliability of the test.
Results and Discussion
Chlorine compounds. The effect of sodium hypochlorite, calcium tlypochlorite, 1, 3-dichloro 5, 5-dimethylhydantoin, and dichloroisocyanuric acid (dichlor (s)-triazine-trione) in concentrations of 5, 10, 15, 20, 25, and 50 ppm available chlorine on the catalase test was determined. Tests were conducted immediately and after 5 and 24 hr storage at 4.5 C following addition of the sanitizer. Results are shown in Figure 1. All of these sanitizers caused a marked reduction in the percentages of oxygen produced in the catalase test. The effect increased with increasing concentrations of chlorine and was more pronounced when the samples were tested after holding at 4.5 C for 5 and 24 hr following addition of the sanitizer. Athough there appear to be some variations in magnitude of the effect of the different sanitizers on tile test results, the basic pattern is very similar for all of the sanitizers. I t is believed that the differences shown here are not due to variations in the inhibiting properties of the sanitizers; rather, it seems more likely that these apparent differences are due to variations in the milk samples. Observations during the course of this investigation suggest that the effect of the chlorine on the catalase test may not be as pronounced when the catalase activity
The catalase test has received much attention as a screening procedure for detection of abnormal milk (1, 5). It is one of the tests that the U. S. Public Health Service (6) has recommended and is used by many state regulatory agencies (5). Considerable work (2, 3, 6) has been done on the relationship of catalase activity to leucocyte counts. Also studied has been the effect of techniques and storage of the milk on test results. There is a lack of information, however, concerning factors which might influence the reliability of the catalase test. I n a preliminary report (4) it was shown that the addition of 10 to 50 ppm sodium hypochlorite to milk decreased the amount of oxygen produced in the test. The purpose of the present study was to obtain further information on the effect of chemical sanitizers on the reliability of the catalase test results. l~eceived for publication August 9, :1966. The investigation reported in this paper (Journal Article no. 66-5-71) is in connection with a project of the Kentucky Agricultural Experiment Station and is published with the approval of the Director. 32
I~EJJIARILITY OF CATALASE TE~T
5O
L ll~
. I I ~
Z
Tested immediately after adding sanltizer
!
!
!
50 Tested after 5 hr storageat 4.5C
k ~
I
5C
I
I
I
I
I
I
I
I
I
t
~ k
Tested after 24 hr
33
Other sanitizers. The effect of iodophors, quaternary ammonium compounds (QAC), an acid sanitizer, and a sanitizer containing both potassium iodide and chloramine-T, and chloramine-T alone also was investigated. These sanitizers were used in concentrations of 10, 25, and 50 ppm of active ingredients, and the catalase test was made immediately a n d after 5 hr storage at 4.5 C following addition of the sanitizer. There were two different iodophors and two different QAC's used. Active ingredients listed for one iodophor were polyethoxy polypropoxy ethanol-iodine complex and nonyl phenylether of polyethylene glycol-iodine complex, and for the other, phosphoric acid, nonyl phenoxy polyethoxy ethanol-iodine complex, and polyethoxy polypropoxy polyethoxy ethanoi-iodine complex. The active ingredient for one QAC was alkyl dimethyl benzyl ammonium chloride and the other contained this compound plus alkyt dimethyl ethylbenzyl ammonium chloride. None of these compounds (in the concentrations used) had a measurable effect on the ca~alase test results, either when tested immediately or after 5 hr storage at 4.5 C following addition of the sanitizer. Contamination of milk with these sanitizers, within the levels investigated, should not adversely affect the reliability of the catalase test results. Conclusions
~'20~ 10 0
I
I
I
I
I
5
10
15
20
25
I
30
I
35
!
J
L
40
45
50
PPM CHLORINE
FIG. 1. Effect of chlorine sanitizers on per cent oxygen produced in the eatalase test: calcium hypochlorite e , sodium hypochlorite V, dieh]oro (s)triazine-trioneA, diehloroisocyanuric acid i , and 1, 3-dichloro 5, 5-dimethylhydantoin ×. Results for each sanitizer represent trials with five different samples of milk. of the milk is high. This possible relationship is under further investigation. The foregoing results indicate that the reliability of the catalase test results could be greatly decreased as a result of contamination with chlorine sanitizers on the farm or in collection of the samples.
Addition of chlorine sanitizers to milk decreases the amount of oxygen produced in the catalase test; thus, the reliability of this test for detecting abnormal milk could be profoundly influenced as a result of contamination with a ehlm~ne sanitizer. Extreme caution must be used in interpreting catalase test results when the possibility of such contaminations exists. Iodophors, quaternary ammonium compounds, an acid sanitizer, and a sanitizer containing potassium iodide and chloramine-T in concentrations up to 50 ppm did not exert a measurable effect on the catalase test and, thus, should not adversely affect the reliability of the results. References
(1) Corbett, W. J. 1963. Catalase Test for Detection of Abnormal Milk. Milk In& Found. Conv. Proc., Lab. See., p. 15. (2) ]~rank, Norma A., and Pounden, W. D. 1963. Preservation and Extended Refrigeration Effects on Catalase, California, and Whiteside Mastitis Tests. J. Am. Vet. Mcd. Assoc., 14 : 878. (3) Nageswararao, G., Blobel, H., and Derbyshire, J. ]3. 1965. Catalase Test for Abnormal Milk. I. Techniques and l~a~tors Affecting the Test. J. Dairy Sci., 48: 1290. J . DAIRY SCIENCE ~OL. 50, NO. 1
34
RANDOLPH, BUCY, FREEMAN
(4) Randolph, H. E., Bucy, J. L., and Freeman, T. R. 1966. Effect of Sodium Hypochlorite on the Catalase Test. J. Dairy Sci., 49: 685. (5) Randolph, tL E., and Conner, D. J. 1966. Survey of Abnormal Milk ~)rograms Adminstered by State Regulatory Agencies. J. Milk Food Technol., 29: 188.
J. DAIRY SCIENCE VOL. 50, NO. 1
(6) Spencer, G. R., and Simon, J. 1960. The Catalase, California, and Cell Count Tests for Detecting Abnormalities in Milk. Am. J. Yet. Research, 21: 578. (7) U. S. Department of Health, Education and Welfare. 1963. Screening Tests for the Detection of Abnormal Milk. Public Health Service Publ. no. 1306. Washington, D. C.