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Leucocytes and the Methylene Blue Reduction Test1
LEUCOCYTES AND THE M E T H Y L E N E BLUE REDUCTION T E ST 1 N. J. STRYNADKA AND H. R. THORNTON
Depart~nent of Dairying, University of Alberta, Edmonton, Canada
The observation is not infrequently made that some samples of milk apparently of low bacterial content have unexpectedly short reduction times. It has been customary to explain this phenomenon by assuming a reducing activity for milk leucocytes, although unequivocal acceptance of this explanation is not always possible in the absence of any more adequate measure of bacterial numbers than the methylene blue reduction test itself. In the present study an attempt has been made to use more delicate criteria of the influence of bacteria on the reduction times of such milks than the plate count or a cursory preliminary microscopic examination of the milk. HISTORICAL
Skar (6) demonstrated reduction of methylene blue in sterile milk by leucocytes from the lymph gland of a steer. He believed that a leucocyte content up to approximately 6.7 million per cc. cannot reduce methylene blue in milk in the standard test but that leucocytes effect reduction of the dye if they are kept evenly distributed by periodic agitation of the milk during incubation. Barthel (1), although recognizing the reducing power of leucocytes, is not inclined to attribute to this power great importance in milk control. Wilson (9) concurs in the opinion that leucocytes are a factor in the reduction test but was unable to effect any marked decrease in " a e r o b i c " reduction times of milks to which suspensions of rabbit leucocytes were added. Ramsdell (5) observed a general but not direct relation between the reduction of resazurin and the leucocyte content of milk but was unable to demonstrate reduction of either resazurin or methylene blue by washed leucocytes. He believed " t h e cause of reduction must be the result of the presence of substances associated with cells, or substances present in abnormal milks in amount comparable to the cell content." Devcreux and Bryan (2) and Hastings (4) regard leucocytes as being significant in the reduction test, at least in better class milks. METHODS
The technique of collection and analysis of the milk samples is described elsewhere (7) and will not be repeated here. Plate counts are not reported Received for publication March 20, 1938. 1 The data contained herein are taken from a thesis presented by N. J. Strynadka (now Inspector of Dairy Products, Dairy and Cold Storage Branch, Dominion Department of Agriculture) at the University of Alberta in partial fulfilment of the requirements for the degree of Master of Science. 561
562
• N. J .
S T R Y N A D K A A N D H . R. T H O R N T O N
as they were f o u n d to contribute practically nothing to the study. The Breed counts refer to bacteria only and the n u m b e r of examined fields f r o m which they were computed is in each case cited except when the bacterial content w a s in the millions in which case usually 5 microscopic fields per Breed smear were counted. The leucocyte counts a r e computed f r o m the examination of 60 microscopic fields per Breed smear except when the count was very high in which case fewer fields were observed. All cells other t h a n the cells of microorganisms were classed as leucocytes and differentiation of kinds of leucocytes was not attempted. All bacterial and leucocyte counts are on a per cc. basis and the methylene blue reduction times are reported in hours and minutes, 1 : 45 meaning 1 hour and 45 minutes. CORRELATION BETWEEN LEUCOCYTE COUNTS AND REDUCTION TIMES The coefficient of correlation between the leucocyte counts and the standard methylene blue reduction times of 158 samples of aseptically-drawn milk was f o u n d to be 0.698 ___ 0.027. This constant as a sole criterion is not sufficiently high, in the opinion of the authors, to justify the popular assumption that milk leucocytes reduce methylene blue in milk. T H E I N I T I A L BREED C O U N T
The initial 1000 field Breed counts are available f o r 95 of the 158 samples of milk and show that in the large m a j o r i t y sufficient bacteria were probably present to account f o r reduction through bacterial action irrespective of the leucocyte count which sometimes was v e r y high. There were a few exceptional samples having leucocyte counts over 1 million, reduction times of less t h a n 10 hours and low Breed counts. There were also 6 samples with low leucocyte and Breed counts and short reduction times. I t is probable that the initial 1000 field Breed count when applied to this type of milk is not a sufficiently precise measure (7) to justify a conclusion f r o m the foregoing data f u r t h e r t h a n t h a t in the m a j o r i t y of the samples the bacterial content was high enough to account for reduction of the dye independently of the leucocytes. T H E B A C T E R I A L C O N T E N T AT T H E T I M E OF R E D U C T I O N
I t has been shown that in samples of this class of milk about which there is no suspicion of a b n o r m a l i t y the bacterial content at the moment of reduction is m a n y millions per cc. as measured by the Breed count (8). I t would a p p e a r sound to assume that in such samples the only practically significant reducing influence is exerted by the bacterial cells. I n our present state of knowledge the n u m b e r of bacteria at the time of reduction seems to be the most precise criterion of the p r e p o n d e r a t i n g influence of the bacteria on the reduction time.
THE METHYLENE BLUE REDUCTION TEST
563
The Breed counts at the moment of reduction were f o u n d to be over 60 million in 34 of 46 samples of aseptically-drawn milk. The reduction times of 19 of these samples were less than 10 hours but the Breed counts on reduction of 7 of these samples were over 60 million. The remaining 12 milks (tables 1 and 2) are interesting exceptions. TABLE 1 Ten mil}s reacting abnormally to the reduction test
Milk ber
Reduction time
Leucocyte count
Initial 2000-field Breed count
200O-field Breed count at reduction
1 2 3 4 5 6 7 8 9 10
O: 30 o: 45 O: 55 1:45 1:45 1:45 2:10 3:30 5:O0 7:15
47,400,000 17,800,000 8,300,000 4,160~000 5,340,000
477,000 107,000~ 142,000 119,700 91,200 48,000 335,000 200,700 39,200 56,100
68,400 253,800~ 86,000 364,200 59,700 43,000 146,000 56,400 81,600 435,600~
nllm-
15,960,000
7,520,000 4,150,000 680,000 3,500,000
60-field Breed count after total of 8 hours incubation 590,000 670,800,000 6,000,000 920,000 140,000 210,000
1000-field count.
The initial plate counts of these 12 exceptional milks varied from 50 to 7600 per cc. but the initial Breed counts were in some cases rather high. The 2000 field Breed count at reduction was in only 1 sample over 0.5 million while the leucocyte counts were low (less than 1 million) in 2 samples. These data are not clear-cut evidence that the leucocytes possessed a reducing power. F r o m the point of view of numbers only it is difficult to attribute to 4 million leucocytes in one milk a reducing power of 1 : 45 and to 3.5 million in another a reducing power of 7 • 15 while it took 16 million in a third 1 : 45 to reduce the dye. Samples 9 and 12 were low in leucocytes and in bacterial content at reduction. When these two milks are considered with the six exceptional milks mentioned in the preceding section entitled T h e I n i t i a l B r e e d C o u n t (one of which had a reduction time of 6 : 4 5 , an initial 1000 field Breed count of 6000 and a leucocyte count of 180,000) the reducing power of the leucocytes is not a tempting explanation of the short reduction times. F o r research purposes in this laboratory the standard methylene blue reduction test is ro~itinely supplemented by the modified reduction test (hourly or half-hourly agitation of the tubes d u r i n g incubation). Occasionally samples of aseptically-drawn milk were encountered which had considerably longer modified than standard reduction times. I n each case where the particulars of the animal giving such milk was available a history
TABLE 2 Two milks reacting abnormally to the reduction test
~eduetion time Milk No.
Leucocyte count
Initial Breed count
Fields examined
Breed count at reduction
Fields
examined
60-field Breed count a f t e r 8 hrs. incubation
Breed count at (modified) reduction
Standard
Modifled
11
5:00
7:15
2,100,000
143,100
2,000
1,017,600
1,000
18,660,000
127,200,000
12
6:15
14:45
860,000
75,000
1,000
132,800
2,000
350,000
64,200,000
THE METHYLENE BLUE REDUCTION TEST
565
of udder abnormality was found. In two such milks, reported in table 2, it is evident that bacterial action was responsible for the modified but not the standard reduction times. These results do not confirm Skar's theory that shortened reduction times in the modified test are due to the more even distribution of the leucocytes because of agitation. A few samples of aseptically-drawn milk of short standard reduction times, including samples 11 and 12, were encountered which when shaken immediately after reduction had second reduction times varying up to 10 to 12 hours. It is, indeed, difficult to accept the reducing power of the leucocytes as the explanation of these observations. It is doubtful if the 2000 field Breed count is sufficiently accurate to justify the conclusion that phagocytosis was responsible for lower Breed counts at the time of reduction than initially in any of these milks. It is possible that bacterial reproduction was continuous in all of these milks but was not apparent because of phagocytic action of the leucocytes. Nothing was observed that caused suspicion that this phenomenon was operative and the modified reduction times of milks 11 and 12 are not in support of such a theory. THE ADDITION OF LEUCOCYTES TO MILK
In 1913 Skar (6) reported the reduction of methylene blue in sterile milk by an added suspension of leucocytes from the lymph gland of a steer. Gay TABLE 3
The reduction times of milk plus bovine blood leucocytes Tube number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
D e s c r i p t i o n of samples Milk only
9 9 9 9 9 9 9 9 9 9 9 9
cc. ce. cc. cc. cc. ce. cc. cc. ec. cc. ec. cc.
milk ÷ 1 milk÷ 1 milk+ 1 milk + 1 milk+l milk÷l milk + 1 milk+ 1 milk+ 1 milk + 1 milk+l milk+l
cc. ce. cc. cc. cc. ce. cc. cc. cc. cc. ce. cc.
leucocyte suspension* f r o m Tube 4 f r o m Tube 5 leucocyte suspension* f r o m Tube 7 f r o m Tube 8 blood serum f r o m Tube 10 f r o m Tube 11 red blood cell s u s p e n s i o n f r o m Tube 13 f r o m Tube 14
Modified reduction time 12 : 30 12 : 45 13 : 15 11 : 00 11:45 12:15 10:45 11:30 12:45 12:00 12:00 12:45 11:00 12:30 12:30
Breed count at reduction
200,000,000
190,000,000
120,000,000
126,000,000
* By c o m p u t a t i o n this milk leucocyte suspensio~ m i x t u r e contained a p p r o x i m a t e l y 50 million leucocytes p e r cc.
566
lV. J. STRYNADKAAND H. R. THORNTON
and Oram (3) demonstrated the reduction of methylene blue in sterile broth in the presence of leucocytes. The failure of the leucocytes to reduce methylene blue similarly in the presence of a streptococcus filtrate was ascribed to a leucocyte-destroying activity of "streptococcus leucocidin." I n the present study blood was drawn aseptically from the jugular vein of a Jersey cow into a flask of sterile physiological saline solution. The leucocytes were separated by repeated centrifugalization, decantation and washing in sterile saline solution until finally a clear suspension of approximately 500 million leucocytes per cc. was obtained. This suspension as well as clear serum and a suspension of the separated red blood cells was added to milk drawn asceptically from the udder of the same cow in varying proportions as outlined in table 3. The animal from which the blood and milk were drawn had not had a recognized u d d e r abnormality while the milk had a standard reduction time of approximately 25 hours and a leucocyte count of 330,000. Only modified reduction times are reported in tables 3 and 4 because the normal variation of reduction times in replicate tubes of this milk militates against an intelligent interpretation of the standard reduction time results. I t cannot be said with certainty that either the added leucocytes, red blood cells or blood serum had a measurable effect on reduction times. Vv'hatever effect there m a y have been was small and was the same for red blood cells as for leucocytes. Sufficient bacteria were present at reduction to account for reduction. Efforts to stain the leucocytes after their addition to the milk failed. I t was thought that the loss of staining properties might be accompanied by loss of reducing power. I n an endeavor to study the effect of adding to milk leucocytes retaining their staining properties 200 cc. of sterile physiological saline solution were injected intraperitoneally into a rabbit early in TABLE 4 The reduction times of mil]~ plus rabbit leucocytes
Description of samples
45 cc. milk + 10 ce. 45 ec. milk + 7 cc. 45 cc. milk + 3 ce. 45 cc. milk + 0.5 ce. Milk only 45 cc. milk + 10 cc. 45 cc. milk+ 5 cc.
leucocyte suspension leucocyte suspension leucocyte suspension leucocyte suspension saline solution saline solution
Modified reduction time Tube 1
2
11:00 11:00 11:00 10:30
12:00 12:00 11:00 I0:30
10:00
10:30
I I : O0
I i : O0
10:30
11:O0
Leucocyte count
Breed count at reduction
1,120,000" 730,000 590,000 520,000 340,000
66,400,000 68,400,000 77,200,000 70,400,000
84,000,000
* By computation this milk leucocyte suspension mixture contained 2,096,363 leucocytes per ce.
THE METHYLENE BLUE REDUCTION TEST
567
the morning followed by a further injection of 100 cc. late in the afternoon. Three hours later 200 cc. of exudate were removed by aspiration. By centrifugalization, decantation and washing in sterile saline solution a suspension containing approximately 10 million leucocytes per cc. was finally obtained. Milk from the same animal as previously used was treated immediately as outlined in table 4. It is seen that the leucocytes retained their staining properties in fair degree after their introduction into the milk but no decrease in reduction time is noticeable. Since the completion of these experiments Wilson (9) reported reduction times of raw and pasteurized milks to which varying concentrations of rabbit leucocyte suspensions were added. Although he believes that reduction by leucocytes was demonstrated, he observes that " t h e results were a little i r r e g u l a r " and "extremely difficult to understand." DISCUSSION AND CONCLUSIONS
The mere presence of leucocytes in milk, even in large numbers, and the absence of bacteria in large numbers do not prove a reducing power for the leucocytes. The attempts to furnish proof of such a reducing power have to date depended on strictly quantitative measurements and in the opinion of the writers have been unsuccessful. The need for qualitative measurements seems apparent. The observations reported in the literature and in this paper are explicable in terms consistent with accepted theories of dye reduction in milI~. There are reasons for believing that the abnormal udder conditions responsible for milk of high leucocyte content are also responsible for abnormally high concentrations of reducing substances in the milk. The presence of reducing substances in abnormally high concentrations would explain the observations under discussion without soliciting aid from the leucocytes. This is not, of course, a denial of the possibility of some leucocytes possessing reducing properties but the bulk of the evidence is that leucocytes are rarely, if ever, the main or significant influence in the reduction of methylene blue in milk in practice. ACKNOWLEDGMENTS
The authors gratefully acknowledge financial support from dairy interests within the province, and the assistance of Dr. M. IV[. Cantor of the Department of Biochemistry, Dr. D. R. Climenko of the Department of Physiology and Pharmacology, and the Department of Animal Husbandry in the procurement and fractionation of the blood samples. REFEI~ENCE S
(l) •ARTHEL, C.
1917. Quoted f r o m O. Skar (1931). (2) DEVEREUX, E. D. AND ]31¢YAN, C. S. The effect of streptococcic m a s t i t i s on the methylene blue reduction test. The Milk Sanitarian. 6: 9, 1937.
568
~. J. STRYNADKA AND ]~I. R. THOI~NTON
(3) GAY, F. P. AND ORAM, FLORENCE. Streptococcus leucocidin. J. Imm. 25: 501, 1933. (4) ttASTINeS, E. G. Limitations of the methylene blue test in grading milk. The Milk Dealer, 25: 72, 1935. Wis. Agr. Station Bul. 435: 85, 1936. (5) RAMSDELL,G . A . The resazurin test for the sanitary condition of milk. Proc. 14 Ann. Cony. N. ¥ . State Ass. Dairy and Milk Insp., 57, 1936. (6) SKAR, O. Yerhalten der Leukozyten der Milch bei der Methylenblaureduktaseprobe. Zeltsch. f. Fleiseh u. Milchhyg. 23: 442, 1913. Die Reduktaseprobe. Rep. Int. Dairy Congress, Copenhagen. German ed. Sec. 2, 58, 1931. (7) STRYNADKA,BT. J. AND THORNTON, H . R . The accuracy of the direct microscopic (Breed) count of bacteria and leucoctyes in milk. JOUR. DAI~y ScI. 20: 685, 1937. (8) STRYNADKA, l~'. J-. The methylene blue reduction test. Thesis, University of Alberta Library. 1935. (9) WXLSO~, G. S. The bacteriological grading of milk. Medical Research Council, Special Rep. Series, No. 206, 245, 1935.
Depart~nent of Dairying, University of Alberta, Edmonton, Canada
The observation is not infrequently made that some samples of milk apparently of low bacterial content have unexpectedly short reduction times. It has been customary to explain this phenomenon by assuming a reducing activity for milk leucocytes, although unequivocal acceptance of this explanation is not always possible in the absence of any more adequate measure of bacterial numbers than the methylene blue reduction test itself. In the present study an attempt has been made to use more delicate criteria of the influence of bacteria on the reduction times of such milks than the plate count or a cursory preliminary microscopic examination of the milk. HISTORICAL
Skar (6) demonstrated reduction of methylene blue in sterile milk by leucocytes from the lymph gland of a steer. He believed that a leucocyte content up to approximately 6.7 million per cc. cannot reduce methylene blue in milk in the standard test but that leucocytes effect reduction of the dye if they are kept evenly distributed by periodic agitation of the milk during incubation. Barthel (1), although recognizing the reducing power of leucocytes, is not inclined to attribute to this power great importance in milk control. Wilson (9) concurs in the opinion that leucocytes are a factor in the reduction test but was unable to effect any marked decrease in " a e r o b i c " reduction times of milks to which suspensions of rabbit leucocytes were added. Ramsdell (5) observed a general but not direct relation between the reduction of resazurin and the leucocyte content of milk but was unable to demonstrate reduction of either resazurin or methylene blue by washed leucocytes. He believed " t h e cause of reduction must be the result of the presence of substances associated with cells, or substances present in abnormal milks in amount comparable to the cell content." Devcreux and Bryan (2) and Hastings (4) regard leucocytes as being significant in the reduction test, at least in better class milks. METHODS
The technique of collection and analysis of the milk samples is described elsewhere (7) and will not be repeated here. Plate counts are not reported Received for publication March 20, 1938. 1 The data contained herein are taken from a thesis presented by N. J. Strynadka (now Inspector of Dairy Products, Dairy and Cold Storage Branch, Dominion Department of Agriculture) at the University of Alberta in partial fulfilment of the requirements for the degree of Master of Science. 561
562
• N. J .
S T R Y N A D K A A N D H . R. T H O R N T O N
as they were f o u n d to contribute practically nothing to the study. The Breed counts refer to bacteria only and the n u m b e r of examined fields f r o m which they were computed is in each case cited except when the bacterial content w a s in the millions in which case usually 5 microscopic fields per Breed smear were counted. The leucocyte counts a r e computed f r o m the examination of 60 microscopic fields per Breed smear except when the count was very high in which case fewer fields were observed. All cells other t h a n the cells of microorganisms were classed as leucocytes and differentiation of kinds of leucocytes was not attempted. All bacterial and leucocyte counts are on a per cc. basis and the methylene blue reduction times are reported in hours and minutes, 1 : 45 meaning 1 hour and 45 minutes. CORRELATION BETWEEN LEUCOCYTE COUNTS AND REDUCTION TIMES The coefficient of correlation between the leucocyte counts and the standard methylene blue reduction times of 158 samples of aseptically-drawn milk was f o u n d to be 0.698 ___ 0.027. This constant as a sole criterion is not sufficiently high, in the opinion of the authors, to justify the popular assumption that milk leucocytes reduce methylene blue in milk. T H E I N I T I A L BREED C O U N T
The initial 1000 field Breed counts are available f o r 95 of the 158 samples of milk and show that in the large m a j o r i t y sufficient bacteria were probably present to account f o r reduction through bacterial action irrespective of the leucocyte count which sometimes was v e r y high. There were a few exceptional samples having leucocyte counts over 1 million, reduction times of less t h a n 10 hours and low Breed counts. There were also 6 samples with low leucocyte and Breed counts and short reduction times. I t is probable that the initial 1000 field Breed count when applied to this type of milk is not a sufficiently precise measure (7) to justify a conclusion f r o m the foregoing data f u r t h e r t h a n t h a t in the m a j o r i t y of the samples the bacterial content was high enough to account for reduction of the dye independently of the leucocytes. T H E B A C T E R I A L C O N T E N T AT T H E T I M E OF R E D U C T I O N
I t has been shown that in samples of this class of milk about which there is no suspicion of a b n o r m a l i t y the bacterial content at the moment of reduction is m a n y millions per cc. as measured by the Breed count (8). I t would a p p e a r sound to assume that in such samples the only practically significant reducing influence is exerted by the bacterial cells. I n our present state of knowledge the n u m b e r of bacteria at the time of reduction seems to be the most precise criterion of the p r e p o n d e r a t i n g influence of the bacteria on the reduction time.
THE METHYLENE BLUE REDUCTION TEST
563
The Breed counts at the moment of reduction were f o u n d to be over 60 million in 34 of 46 samples of aseptically-drawn milk. The reduction times of 19 of these samples were less than 10 hours but the Breed counts on reduction of 7 of these samples were over 60 million. The remaining 12 milks (tables 1 and 2) are interesting exceptions. TABLE 1 Ten mil}s reacting abnormally to the reduction test
Milk ber
Reduction time
Leucocyte count
Initial 2000-field Breed count
200O-field Breed count at reduction
1 2 3 4 5 6 7 8 9 10
O: 30 o: 45 O: 55 1:45 1:45 1:45 2:10 3:30 5:O0 7:15
47,400,000 17,800,000 8,300,000 4,160~000 5,340,000
477,000 107,000~ 142,000 119,700 91,200 48,000 335,000 200,700 39,200 56,100
68,400 253,800~ 86,000 364,200 59,700 43,000 146,000 56,400 81,600 435,600~
nllm-
15,960,000
7,520,000 4,150,000 680,000 3,500,000
60-field Breed count after total of 8 hours incubation 590,000 670,800,000 6,000,000 920,000 140,000 210,000
1000-field count.
The initial plate counts of these 12 exceptional milks varied from 50 to 7600 per cc. but the initial Breed counts were in some cases rather high. The 2000 field Breed count at reduction was in only 1 sample over 0.5 million while the leucocyte counts were low (less than 1 million) in 2 samples. These data are not clear-cut evidence that the leucocytes possessed a reducing power. F r o m the point of view of numbers only it is difficult to attribute to 4 million leucocytes in one milk a reducing power of 1 : 45 and to 3.5 million in another a reducing power of 7 • 15 while it took 16 million in a third 1 : 45 to reduce the dye. Samples 9 and 12 were low in leucocytes and in bacterial content at reduction. When these two milks are considered with the six exceptional milks mentioned in the preceding section entitled T h e I n i t i a l B r e e d C o u n t (one of which had a reduction time of 6 : 4 5 , an initial 1000 field Breed count of 6000 and a leucocyte count of 180,000) the reducing power of the leucocytes is not a tempting explanation of the short reduction times. F o r research purposes in this laboratory the standard methylene blue reduction test is ro~itinely supplemented by the modified reduction test (hourly or half-hourly agitation of the tubes d u r i n g incubation). Occasionally samples of aseptically-drawn milk were encountered which had considerably longer modified than standard reduction times. I n each case where the particulars of the animal giving such milk was available a history
TABLE 2 Two milks reacting abnormally to the reduction test
~eduetion time Milk No.
Leucocyte count
Initial Breed count
Fields examined
Breed count at reduction
Fields
examined
60-field Breed count a f t e r 8 hrs. incubation
Breed count at (modified) reduction
Standard
Modifled
11
5:00
7:15
2,100,000
143,100
2,000
1,017,600
1,000
18,660,000
127,200,000
12
6:15
14:45
860,000
75,000
1,000
132,800
2,000
350,000
64,200,000
THE METHYLENE BLUE REDUCTION TEST
565
of udder abnormality was found. In two such milks, reported in table 2, it is evident that bacterial action was responsible for the modified but not the standard reduction times. These results do not confirm Skar's theory that shortened reduction times in the modified test are due to the more even distribution of the leucocytes because of agitation. A few samples of aseptically-drawn milk of short standard reduction times, including samples 11 and 12, were encountered which when shaken immediately after reduction had second reduction times varying up to 10 to 12 hours. It is, indeed, difficult to accept the reducing power of the leucocytes as the explanation of these observations. It is doubtful if the 2000 field Breed count is sufficiently accurate to justify the conclusion that phagocytosis was responsible for lower Breed counts at the time of reduction than initially in any of these milks. It is possible that bacterial reproduction was continuous in all of these milks but was not apparent because of phagocytic action of the leucocytes. Nothing was observed that caused suspicion that this phenomenon was operative and the modified reduction times of milks 11 and 12 are not in support of such a theory. THE ADDITION OF LEUCOCYTES TO MILK
In 1913 Skar (6) reported the reduction of methylene blue in sterile milk by an added suspension of leucocytes from the lymph gland of a steer. Gay TABLE 3
The reduction times of milk plus bovine blood leucocytes Tube number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
D e s c r i p t i o n of samples Milk only
9 9 9 9 9 9 9 9 9 9 9 9
cc. ce. cc. cc. cc. ce. cc. cc. ec. cc. ec. cc.
milk ÷ 1 milk÷ 1 milk+ 1 milk + 1 milk+l milk÷l milk + 1 milk+ 1 milk+ 1 milk + 1 milk+l milk+l
cc. ce. cc. cc. cc. ce. cc. cc. cc. cc. ce. cc.
leucocyte suspension* f r o m Tube 4 f r o m Tube 5 leucocyte suspension* f r o m Tube 7 f r o m Tube 8 blood serum f r o m Tube 10 f r o m Tube 11 red blood cell s u s p e n s i o n f r o m Tube 13 f r o m Tube 14
Modified reduction time 12 : 30 12 : 45 13 : 15 11 : 00 11:45 12:15 10:45 11:30 12:45 12:00 12:00 12:45 11:00 12:30 12:30
Breed count at reduction
200,000,000
190,000,000
120,000,000
126,000,000
* By c o m p u t a t i o n this milk leucocyte suspensio~ m i x t u r e contained a p p r o x i m a t e l y 50 million leucocytes p e r cc.
566
lV. J. STRYNADKAAND H. R. THORNTON
and Oram (3) demonstrated the reduction of methylene blue in sterile broth in the presence of leucocytes. The failure of the leucocytes to reduce methylene blue similarly in the presence of a streptococcus filtrate was ascribed to a leucocyte-destroying activity of "streptococcus leucocidin." I n the present study blood was drawn aseptically from the jugular vein of a Jersey cow into a flask of sterile physiological saline solution. The leucocytes were separated by repeated centrifugalization, decantation and washing in sterile saline solution until finally a clear suspension of approximately 500 million leucocytes per cc. was obtained. This suspension as well as clear serum and a suspension of the separated red blood cells was added to milk drawn asceptically from the udder of the same cow in varying proportions as outlined in table 3. The animal from which the blood and milk were drawn had not had a recognized u d d e r abnormality while the milk had a standard reduction time of approximately 25 hours and a leucocyte count of 330,000. Only modified reduction times are reported in tables 3 and 4 because the normal variation of reduction times in replicate tubes of this milk militates against an intelligent interpretation of the standard reduction time results. I t cannot be said with certainty that either the added leucocytes, red blood cells or blood serum had a measurable effect on reduction times. Vv'hatever effect there m a y have been was small and was the same for red blood cells as for leucocytes. Sufficient bacteria were present at reduction to account for reduction. Efforts to stain the leucocytes after their addition to the milk failed. I t was thought that the loss of staining properties might be accompanied by loss of reducing power. I n an endeavor to study the effect of adding to milk leucocytes retaining their staining properties 200 cc. of sterile physiological saline solution were injected intraperitoneally into a rabbit early in TABLE 4 The reduction times of mil]~ plus rabbit leucocytes
Description of samples
45 cc. milk + 10 ce. 45 ec. milk + 7 cc. 45 cc. milk + 3 ce. 45 cc. milk + 0.5 ce. Milk only 45 cc. milk + 10 cc. 45 cc. milk+ 5 cc.
leucocyte suspension leucocyte suspension leucocyte suspension leucocyte suspension saline solution saline solution
Modified reduction time Tube 1
2
11:00 11:00 11:00 10:30
12:00 12:00 11:00 I0:30
10:00
10:30
I I : O0
I i : O0
10:30
11:O0
Leucocyte count
Breed count at reduction
1,120,000" 730,000 590,000 520,000 340,000
66,400,000 68,400,000 77,200,000 70,400,000
84,000,000
* By computation this milk leucocyte suspension mixture contained 2,096,363 leucocytes per ce.
THE METHYLENE BLUE REDUCTION TEST
567
the morning followed by a further injection of 100 cc. late in the afternoon. Three hours later 200 cc. of exudate were removed by aspiration. By centrifugalization, decantation and washing in sterile saline solution a suspension containing approximately 10 million leucocytes per cc. was finally obtained. Milk from the same animal as previously used was treated immediately as outlined in table 4. It is seen that the leucocytes retained their staining properties in fair degree after their introduction into the milk but no decrease in reduction time is noticeable. Since the completion of these experiments Wilson (9) reported reduction times of raw and pasteurized milks to which varying concentrations of rabbit leucocyte suspensions were added. Although he believes that reduction by leucocytes was demonstrated, he observes that " t h e results were a little i r r e g u l a r " and "extremely difficult to understand." DISCUSSION AND CONCLUSIONS
The mere presence of leucocytes in milk, even in large numbers, and the absence of bacteria in large numbers do not prove a reducing power for the leucocytes. The attempts to furnish proof of such a reducing power have to date depended on strictly quantitative measurements and in the opinion of the writers have been unsuccessful. The need for qualitative measurements seems apparent. The observations reported in the literature and in this paper are explicable in terms consistent with accepted theories of dye reduction in milI~. There are reasons for believing that the abnormal udder conditions responsible for milk of high leucocyte content are also responsible for abnormally high concentrations of reducing substances in the milk. The presence of reducing substances in abnormally high concentrations would explain the observations under discussion without soliciting aid from the leucocytes. This is not, of course, a denial of the possibility of some leucocytes possessing reducing properties but the bulk of the evidence is that leucocytes are rarely, if ever, the main or significant influence in the reduction of methylene blue in milk in practice. ACKNOWLEDGMENTS
The authors gratefully acknowledge financial support from dairy interests within the province, and the assistance of Dr. M. IV[. Cantor of the Department of Biochemistry, Dr. D. R. Climenko of the Department of Physiology and Pharmacology, and the Department of Animal Husbandry in the procurement and fractionation of the blood samples. REFEI~ENCE S
(l) •ARTHEL, C.
1917. Quoted f r o m O. Skar (1931). (2) DEVEREUX, E. D. AND ]31¢YAN, C. S. The effect of streptococcic m a s t i t i s on the methylene blue reduction test. The Milk Sanitarian. 6: 9, 1937.
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(3) GAY, F. P. AND ORAM, FLORENCE. Streptococcus leucocidin. J. Imm. 25: 501, 1933. (4) ttASTINeS, E. G. Limitations of the methylene blue test in grading milk. The Milk Dealer, 25: 72, 1935. Wis. Agr. Station Bul. 435: 85, 1936. (5) RAMSDELL,G . A . The resazurin test for the sanitary condition of milk. Proc. 14 Ann. Cony. N. ¥ . State Ass. Dairy and Milk Insp., 57, 1936. (6) SKAR, O. Yerhalten der Leukozyten der Milch bei der Methylenblaureduktaseprobe. Zeltsch. f. Fleiseh u. Milchhyg. 23: 442, 1913. Die Reduktaseprobe. Rep. Int. Dairy Congress, Copenhagen. German ed. Sec. 2, 58, 1931. (7) STRYNADKA,BT. J. AND THORNTON, H . R . The accuracy of the direct microscopic (Breed) count of bacteria and leucoctyes in milk. JOUR. DAI~y ScI. 20: 685, 1937. (8) STRYNADKA, l~'. J-. The methylene blue reduction test. Thesis, University of Alberta Library. 1935. (9) WXLSO~, G. S. The bacteriological grading of milk. Medical Research Council, Special Rep. Series, No. 206, 245, 1935.