The Effect of Temperature on Growth and Survival of Fusobacterium necrophorum Isolated from Bovine Liver Abscesses P.C. Simon*
ABSTRACT The ability of Fusobacterium necrophorum to survive or grow in liquid nitrogen or at temperatures between -10° and 59°C was determined. The organism remained viable but did not grow in liquid nitrogen or between -100 and 21°C. It grew between 220 and 43°C. No isolate grew at temperatures above 43°C and all three isolates survived for a minimum of 15 minutes and an average of 25 minutes at 59'C. The optimum temperature for maximum growth was 37°C. The organism survived in ampoules stored in liquid nitrogen for eight years. It survived in liver abscesses stored at -10°C for five years and as cultures in screw capped tubes for three years.
RESUME Cette experience visait a determiner l'habilite de Fusobacterium necrophorum 'a survivre ou a se multiplier lorsqu'on le garde dans l'azote liquide ou 'a des temperatures variant de -10° 'a 59°C. II survecut, sans toutefois se multiplier, dans l'azote liquide et 'a des temperatures variant de -10° 'a 21°C. Il se multiplia entre 22° et 43°C. Aucune souche ne se multiplia 'a une temperature superieure 'a 43°C; les trois souches utilisees au cours de cette experience survecurent durant au moins 15 minutes et en moyenne durant 25 minutes, 'a une temperature de 59°C. La temperature optimale
*Animal Pathology Division, Health of Animals Branch, Agriculture Canada, Pacific Area Laboratory, 3802 West 4th Avenue, Vancouver, British Columbia V6R
'P5.
Submitted August 12, 1976.
Volume 41 - April, 1977
d'une croissance maximale fut de 37°C. Le microbe survecut dans des ampoules entreposees dans l'azote liquide, durant huit ans. II survecut aussi dans des abces hepatiques entreposes 'a -10°C, durant cinq ans, et dans des tubes visses, durant trois ans.
INTRODUCTION
Cesari (2), Prevot (5) and Schmorl (6) reported that Fusobacterium necrophorum grew between 300 and 40°C. The optimum temperature for growth was 37°C. Beveridge (1) in Sydney, Australia observed growth at room temperature in summer but not in winter and noted growth was rapid at 370C. Prevot (5) found that F. necrophorum died at 55°C but he did not indicate the period of survival. Beveridge (1) and Cesari (2) found that the organism died within 15 minutes at 55°C. Fievez (3) reported that hemolytic strains survived 560 for five to ten minutes and the nonhemolytic strains for 12 to 30 minutes. During a previous experiment in this laboratory F. necrophorum survived in medium 156 (M156) for 30 weeks at 370C (7). However, growth and survival temperatures were not investigated. Determination of the minimum temperature at which F. necrophorum would grow and the maximum temperature at which it would survive may be useful in the control of the organism in the environment. Preservation of an isolate will enable one to test artificially induced immunity by the vaccine strain. Therefore, the following studies were carried out to determine these values. 169
MATERIALS AND METHODS
This study was conducted as three experiments. Experiment 1 was to determine the lowest temperature at which F. necrophorum will grow and the period of growth. Experiment 2 was to determine the highest temperature at which F. necrophorum will survive and the period of survival. Experiment 3 was to determine the period of survival of F. necrophorum in liver abscesses when stored at -10°C and in cultures when stored at -10°C or in liquid nitrogen.
ISOLATES From 22 isolates of F. necrophorum cultured from bovine liver abscesses in this laboratory and identified by the criteria of Simon and Stovell (8) three were selected after preliminary studies to represent the least, greatest, and average thermal resistance. INOCULUM
The inoculum was 0.5 ml of a 24 hr culture of F. necrophorum in M156 (7). This was mixed well with the medium before incubation. MEDIUM
Five ml of M156 in 13 x 100 mm screw capped tubes was used for cultures. Prior to inoculation, the medium was brought to the incubation temperature under study. READINGS
Growth and lag phases were assessed by optical density (OD) readings taken before incubation and at daily intervals at 520 nm using a Bausch and Lomb Spectronic 20 spectrophotometer. A significant growth consisted of an overall increase of 0.16 OD or higher (9). EXPERIMENT 1
Cultures were incubated in temperature controlled water baths in a room with an
170
ambient temperature of 12'C. The culture.s were incubated at temperatures between 170 and 30°C, 350 to 39°C and 410 to 45°C at 10 intervals. One tube of each isolate was incubated at each temperature. Growth was studied for 25 days by OD measurements.
EXPERIMENT 2 Fifteen tubes of each isolate were incubated at temperatures and periods as shown in Table I. One tube was removed at the end of predetermined incubation periods to 37°C. Optical densities were taken at the commencement of incubation at 370C and daily thereafter for six days. Since the incubation period was short for testing thermoresistance of cultures to temperatures of 43°C and above, it was necessary to keep the period of incubation as exact as possible. Therefore, care was taken that the preincubated medium was never out of the water bath for more than eight seconds during inoculation. At the end of each incubation period a tube was removed and cooled in running water to about 37°C within eight seconds. It was then incubated at 370C and OD readings were taken. EXPERIMENT 3 Eighty-one liver abscesses with about two inches of apparently healthy tissue surrounding the abscesses were stored at -100C. At half-yearly intervals for five and one-half years, three or more abscesses were allowed to thaw partially, opened aseptically and the pus
a. Liver Abscesses
from them inoculated into M156 and in-
cubated at 370C. If there was no growth of F. necrophorum another set of three abscesses was cultured until a culture was positive. At the last half-yearly interval TABLE I. Temperature of Incubation, Interval Between RemrovalTof Tubes,' and? Maximum Incubation Period for Experiment 2 Interval
Temperature of Incubation (C°)
430 440 450 500 550 570 590
Between Tests 48 hr 24 hr 24 hr 6 hr 30 min 10 min 5 min
Maximum ' Incubation
Period days days days days hr hr min
20 13 10 3 6 2 60
Can. J. comp. Med.
DAYS
OF
INCUBATrON
Fig. 1. Average optieal density of cultures of F. necrophorum in Medium 156 grown at different temperatures.
when no organism was recovered the remaining 20 abscesses were cultured. b. Cultures in Tubes - Ten tubes containing 10 ml of 24 hr cultures of each isolate in M156 in 16 x 125 mm screw capped tubes were stored at -100C. Viability of these cultures was checked monthly, bimonthly and half yearly during the first year and yearly thereafter for four years. Cultures were checked by subculturing from one tube at a time. If this culture was nonviable a second tube was cultured. A culture was considered nonviable when three separate cultures were negative.
growth was observed more ampoules were opened and cultured. At least three cultures were negative before the organism was considered nonviable.
RESULTS Since results of the tests of three isolates were comparable, for the sake of simplicity, the average is given with an explanation of significant variations.
EXPERIMENT 1 c. Cultures in Sealed Ampoules - Twentyfive 2.0 ml sealed glass ampoules, each containing 1.0 ml of a 24 hr culture of each isolate in M156 were stored in liquid nitrogen. They were tested annually for nine years to determine viability. Two ampoules per isolate were cultured at a time. If no
Volume 41
-
April, 1977
Results of growth are given in Table II. The salient features are given as a graph (Fig. 1). Though observations were made for 25 days, results are given for 19 days since the pattern of results was established prior to this time.
171
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Fig. 2. Average period of viability of F. necrophorum at temperatures between 43°C to 59°C.
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There was no growth at 170 and 18°C. Though some evidence of growth was observable at temperatures between 190 to 21°C, since such growth was not sustained and was less than 0.16 OD, growth was recorded as nil. Table II shows that at 22°C there was slight growth (0.16 OD) on the sixth day. At 230 growth commenced on the fourth day and continued till the seventh day. At 24° the length of the log phase increased to nine days. Above 240 the log phase decreased gradually to two days at 30°C. Cultures at 360, 37° and 380 reached their maximum growth in 24 hr and the maximum growth was at 37°C. At 440 and above there was no growth.
00-C C00'10 - 00.01o_noz_ 00cCO0000-----
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EXPERIMENT 2 0
H
The average periods of viability of F. necrophorum at temperatures between 430 and 59°C are shown in Figure 2. At 43°C one isolate survived for six days, another for ten days and a third for 20 days. At 440C all isolates survived for 24 hr, two for 48 hr and one for 72 hr. At 45°C all three isolates survived for 24 hr but not for 48 hr. At 500C all three isolates survived for six hours. However, these cultures showed lag phases of one, two or three days and reached their maximum total growth of 0.88 OD or less in two, three or five days. At 50°C two isolates survived for 18 hr.
172
Can. J. comp. Med.
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0 0)
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At 55CC one isolate survived for one and one-half hours, one for two hours and a third for two and one-half hours. At 57°C one survived for 30 minutes, the second for 40 minutes and the third for 50 minutes. At 59°C one survived for 15 minutes, one for 25 minutes and one for 35 minutes. EXPERIMENT 3
In experiment 3 cultures in ampoules stored in liquid nitrogen survived for eight years but not for nine. At -10°C cultures in tubes survived for three years but not for four. In frozen liver abscesses stored at -10cC F. necrophorum remained viable for five years but not for five and one-half.
DISCUSSION Lahelle's (4) findings of sparse growth at 220 and 230C and no growth at 21°C correspond to the results of this study. The ability of the organism to survive at -10 ( in liver abscesses for nearly double the period it survived in artificial media at the same temperature indicates the possibility of longer survival in natural environment than in an artificial environment. Isolates survived for an average of 1.8 hr at 550C and 40 minutes at 57°C. Fievez (3) found his hemolytic strains survived for five to ten minutes at 560C. In spite of differences in the media used by Fievez his results are comparable to those reported here. The isolates used in this study were also hemolytic. Lahelle's findings of sparse growth at 440C and no growth at 450 are fairly similar to the results of this study. Lahelle did not study survivability at higher temperatures. From 240 to 38°C there was a gradual decrease in the logarithmic phase. Between 360 and 380C the growth was maximum and the logarithmic phase the shortest. The optimum temperature was 370 C. Cultures lysed gradually at all temperatures. At 240 and 250C, both growth and lysis were slow. Why there is more growth at 280 than at 30°C in four to six days is not clear. It may be due to the prolonged logarithmic phase at 28°C. A lag phase was observable at temperatures between 190° and 230C. A similar lag
Volume 41 - April, 1977
phase was observed when cultures were incubated at 37°C after exposure to temperatures between 450 and 590C. This lag phase may have been due to decreasing numbers of viable cells. Lag phases may also have been due to unfavorable environment where the organism takes time to adapt to the conditions or due to small inocula where the organism may be growing but is not detectable because of very low numbers. The attempt during this study was not to determine the number of viable cells per ml of culture but to determine if any organism survived in the culture. For this purpose it was necessary to incubate the same culture at 370C after it had been subjected to temperatures between 45° and 590C. Plate counts would have failed to detect viable cells if they were very few. Survivability of the organism is seen to be greatest in liquid nitrogen and less at -100C. The survivability decreases with higher temperatures until 220 to 24°C at which proliferation takes place. Growth occurs between 240 and 430C. Above that temperature the survival period diminishes drastically, reaching 15 minutes at 59°C.
ACKNOWLEDGMENTS The author wishes to acknowledge the invaluable assistance of Miss M. Harris in all phases of this study. REFERENCES 1. BEVERIDGE, W. I. B. A study of twelve strains of Bacillus necrophorus with observations on the oxygen intolerance of the organism. J. Bact. 38: 467491. 1934. 2. CESARI, E. Le bacille de la necrose et son r6le pathogene. Revue Path. comp. Med. exp. 24: 695-711. 1924. 3. FIEVEZ, L. etude comparee des souches de Spherophorus necrophorus isolees chez l'homme et chez l'animal. Bruxelles: Presses Academiques Europeennes. 1963. 4. LAHELLE, 0. Necrobacterium. A study of itcs bacteriology, serology and pathogenicity and its relation to Fusobacterium. Acta path. microbiol. scand. suppl. 67: 88-90. 1947. 5. PREVOT, A. R. Manuel de classification et de '16termination des bacteries anaerobies. 3e Edit. pp. 278-279. Paris: Masson. 1957. f. SCHMORL, G. Ueber ein pathogens Fadenbacterium (Streptothrix cuniculi). Deut. zeitschfur. Tiermed. 17: 375. Abst. in Zentbl. Bakt. ParasitKde I 11: 666. 1892. 7. SIMON, P. C. Cultivation and maintenance of Sphaerophorus necrophorus Part I: An anaerobic medium for aerobic incubation. Can. J. comp. Med. 38: 9496. 1974. S. SIMON, P. C. and P. L. STOVELL. Isolation of Sphaerophorus necrophorus from bovine hepatic abscesses in British Columbia. Can. J. comp. Med. 35: 103-106. 1971. 9. SIMON, P. C. Cultivation and maintenance of Spha erophorus necrophorus Part 11: Infivence of pH on maximum growth and acid production in medium 156. Can. J. comp. Med. 39: 89-93. 1975.
173
ABSTRACT The ability of Fusobacterium necrophorum to survive or grow in liquid nitrogen or at temperatures between -10° and 59°C was determined. The organism remained viable but did not grow in liquid nitrogen or between -100 and 21°C. It grew between 220 and 43°C. No isolate grew at temperatures above 43°C and all three isolates survived for a minimum of 15 minutes and an average of 25 minutes at 59'C. The optimum temperature for maximum growth was 37°C. The organism survived in ampoules stored in liquid nitrogen for eight years. It survived in liver abscesses stored at -10°C for five years and as cultures in screw capped tubes for three years.
RESUME Cette experience visait a determiner l'habilite de Fusobacterium necrophorum 'a survivre ou a se multiplier lorsqu'on le garde dans l'azote liquide ou 'a des temperatures variant de -10° 'a 59°C. II survecut, sans toutefois se multiplier, dans l'azote liquide et 'a des temperatures variant de -10° 'a 21°C. Il se multiplia entre 22° et 43°C. Aucune souche ne se multiplia 'a une temperature superieure 'a 43°C; les trois souches utilisees au cours de cette experience survecurent durant au moins 15 minutes et en moyenne durant 25 minutes, 'a une temperature de 59°C. La temperature optimale
*Animal Pathology Division, Health of Animals Branch, Agriculture Canada, Pacific Area Laboratory, 3802 West 4th Avenue, Vancouver, British Columbia V6R
'P5.
Submitted August 12, 1976.
Volume 41 - April, 1977
d'une croissance maximale fut de 37°C. Le microbe survecut dans des ampoules entreposees dans l'azote liquide, durant huit ans. II survecut aussi dans des abces hepatiques entreposes 'a -10°C, durant cinq ans, et dans des tubes visses, durant trois ans.
INTRODUCTION
Cesari (2), Prevot (5) and Schmorl (6) reported that Fusobacterium necrophorum grew between 300 and 40°C. The optimum temperature for growth was 37°C. Beveridge (1) in Sydney, Australia observed growth at room temperature in summer but not in winter and noted growth was rapid at 370C. Prevot (5) found that F. necrophorum died at 55°C but he did not indicate the period of survival. Beveridge (1) and Cesari (2) found that the organism died within 15 minutes at 55°C. Fievez (3) reported that hemolytic strains survived 560 for five to ten minutes and the nonhemolytic strains for 12 to 30 minutes. During a previous experiment in this laboratory F. necrophorum survived in medium 156 (M156) for 30 weeks at 370C (7). However, growth and survival temperatures were not investigated. Determination of the minimum temperature at which F. necrophorum would grow and the maximum temperature at which it would survive may be useful in the control of the organism in the environment. Preservation of an isolate will enable one to test artificially induced immunity by the vaccine strain. Therefore, the following studies were carried out to determine these values. 169
MATERIALS AND METHODS
This study was conducted as three experiments. Experiment 1 was to determine the lowest temperature at which F. necrophorum will grow and the period of growth. Experiment 2 was to determine the highest temperature at which F. necrophorum will survive and the period of survival. Experiment 3 was to determine the period of survival of F. necrophorum in liver abscesses when stored at -10°C and in cultures when stored at -10°C or in liquid nitrogen.
ISOLATES From 22 isolates of F. necrophorum cultured from bovine liver abscesses in this laboratory and identified by the criteria of Simon and Stovell (8) three were selected after preliminary studies to represent the least, greatest, and average thermal resistance. INOCULUM
The inoculum was 0.5 ml of a 24 hr culture of F. necrophorum in M156 (7). This was mixed well with the medium before incubation. MEDIUM
Five ml of M156 in 13 x 100 mm screw capped tubes was used for cultures. Prior to inoculation, the medium was brought to the incubation temperature under study. READINGS
Growth and lag phases were assessed by optical density (OD) readings taken before incubation and at daily intervals at 520 nm using a Bausch and Lomb Spectronic 20 spectrophotometer. A significant growth consisted of an overall increase of 0.16 OD or higher (9). EXPERIMENT 1
Cultures were incubated in temperature controlled water baths in a room with an
170
ambient temperature of 12'C. The culture.s were incubated at temperatures between 170 and 30°C, 350 to 39°C and 410 to 45°C at 10 intervals. One tube of each isolate was incubated at each temperature. Growth was studied for 25 days by OD measurements.
EXPERIMENT 2 Fifteen tubes of each isolate were incubated at temperatures and periods as shown in Table I. One tube was removed at the end of predetermined incubation periods to 37°C. Optical densities were taken at the commencement of incubation at 370C and daily thereafter for six days. Since the incubation period was short for testing thermoresistance of cultures to temperatures of 43°C and above, it was necessary to keep the period of incubation as exact as possible. Therefore, care was taken that the preincubated medium was never out of the water bath for more than eight seconds during inoculation. At the end of each incubation period a tube was removed and cooled in running water to about 37°C within eight seconds. It was then incubated at 370C and OD readings were taken. EXPERIMENT 3 Eighty-one liver abscesses with about two inches of apparently healthy tissue surrounding the abscesses were stored at -100C. At half-yearly intervals for five and one-half years, three or more abscesses were allowed to thaw partially, opened aseptically and the pus
a. Liver Abscesses
from them inoculated into M156 and in-
cubated at 370C. If there was no growth of F. necrophorum another set of three abscesses was cultured until a culture was positive. At the last half-yearly interval TABLE I. Temperature of Incubation, Interval Between RemrovalTof Tubes,' and? Maximum Incubation Period for Experiment 2 Interval
Temperature of Incubation (C°)
430 440 450 500 550 570 590
Between Tests 48 hr 24 hr 24 hr 6 hr 30 min 10 min 5 min
Maximum ' Incubation
Period days days days days hr hr min
20 13 10 3 6 2 60
Can. J. comp. Med.
DAYS
OF
INCUBATrON
Fig. 1. Average optieal density of cultures of F. necrophorum in Medium 156 grown at different temperatures.
when no organism was recovered the remaining 20 abscesses were cultured. b. Cultures in Tubes - Ten tubes containing 10 ml of 24 hr cultures of each isolate in M156 in 16 x 125 mm screw capped tubes were stored at -100C. Viability of these cultures was checked monthly, bimonthly and half yearly during the first year and yearly thereafter for four years. Cultures were checked by subculturing from one tube at a time. If this culture was nonviable a second tube was cultured. A culture was considered nonviable when three separate cultures were negative.
growth was observed more ampoules were opened and cultured. At least three cultures were negative before the organism was considered nonviable.
RESULTS Since results of the tests of three isolates were comparable, for the sake of simplicity, the average is given with an explanation of significant variations.
EXPERIMENT 1 c. Cultures in Sealed Ampoules - Twentyfive 2.0 ml sealed glass ampoules, each containing 1.0 ml of a 24 hr culture of each isolate in M156 were stored in liquid nitrogen. They were tested annually for nine years to determine viability. Two ampoules per isolate were cultured at a time. If no
Volume 41
-
April, 1977
Results of growth are given in Table II. The salient features are given as a graph (Fig. 1). Though observations were made for 25 days, results are given for 19 days since the pattern of results was established prior to this time.
171
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Fig. 2. Average period of viability of F. necrophorum at temperatures between 43°C to 59°C.
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There was no growth at 170 and 18°C. Though some evidence of growth was observable at temperatures between 190 to 21°C, since such growth was not sustained and was less than 0.16 OD, growth was recorded as nil. Table II shows that at 22°C there was slight growth (0.16 OD) on the sixth day. At 230 growth commenced on the fourth day and continued till the seventh day. At 24° the length of the log phase increased to nine days. Above 240 the log phase decreased gradually to two days at 30°C. Cultures at 360, 37° and 380 reached their maximum growth in 24 hr and the maximum growth was at 37°C. At 440 and above there was no growth.
00-C C00'10 - 00.01o_noz_ 00cCO0000-----
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EXPERIMENT 2 0
H
The average periods of viability of F. necrophorum at temperatures between 430 and 59°C are shown in Figure 2. At 43°C one isolate survived for six days, another for ten days and a third for 20 days. At 440C all isolates survived for 24 hr, two for 48 hr and one for 72 hr. At 45°C all three isolates survived for 24 hr but not for 48 hr. At 500C all three isolates survived for six hours. However, these cultures showed lag phases of one, two or three days and reached their maximum total growth of 0.88 OD or less in two, three or five days. At 50°C two isolates survived for 18 hr.
172
Can. J. comp. Med.
0,
0 0)
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0.
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At 55CC one isolate survived for one and one-half hours, one for two hours and a third for two and one-half hours. At 57°C one survived for 30 minutes, the second for 40 minutes and the third for 50 minutes. At 59°C one survived for 15 minutes, one for 25 minutes and one for 35 minutes. EXPERIMENT 3
In experiment 3 cultures in ampoules stored in liquid nitrogen survived for eight years but not for nine. At -10°C cultures in tubes survived for three years but not for four. In frozen liver abscesses stored at -10cC F. necrophorum remained viable for five years but not for five and one-half.
DISCUSSION Lahelle's (4) findings of sparse growth at 220 and 230C and no growth at 21°C correspond to the results of this study. The ability of the organism to survive at -10 ( in liver abscesses for nearly double the period it survived in artificial media at the same temperature indicates the possibility of longer survival in natural environment than in an artificial environment. Isolates survived for an average of 1.8 hr at 550C and 40 minutes at 57°C. Fievez (3) found his hemolytic strains survived for five to ten minutes at 560C. In spite of differences in the media used by Fievez his results are comparable to those reported here. The isolates used in this study were also hemolytic. Lahelle's findings of sparse growth at 440C and no growth at 450 are fairly similar to the results of this study. Lahelle did not study survivability at higher temperatures. From 240 to 38°C there was a gradual decrease in the logarithmic phase. Between 360 and 380C the growth was maximum and the logarithmic phase the shortest. The optimum temperature was 370 C. Cultures lysed gradually at all temperatures. At 240 and 250C, both growth and lysis were slow. Why there is more growth at 280 than at 30°C in four to six days is not clear. It may be due to the prolonged logarithmic phase at 28°C. A lag phase was observable at temperatures between 190° and 230C. A similar lag
Volume 41 - April, 1977
phase was observed when cultures were incubated at 37°C after exposure to temperatures between 450 and 590C. This lag phase may have been due to decreasing numbers of viable cells. Lag phases may also have been due to unfavorable environment where the organism takes time to adapt to the conditions or due to small inocula where the organism may be growing but is not detectable because of very low numbers. The attempt during this study was not to determine the number of viable cells per ml of culture but to determine if any organism survived in the culture. For this purpose it was necessary to incubate the same culture at 370C after it had been subjected to temperatures between 45° and 590C. Plate counts would have failed to detect viable cells if they were very few. Survivability of the organism is seen to be greatest in liquid nitrogen and less at -100C. The survivability decreases with higher temperatures until 220 to 24°C at which proliferation takes place. Growth occurs between 240 and 430C. Above that temperature the survival period diminishes drastically, reaching 15 minutes at 59°C.
ACKNOWLEDGMENTS The author wishes to acknowledge the invaluable assistance of Miss M. Harris in all phases of this study. REFERENCES 1. BEVERIDGE, W. I. B. A study of twelve strains of Bacillus necrophorus with observations on the oxygen intolerance of the organism. J. Bact. 38: 467491. 1934. 2. CESARI, E. Le bacille de la necrose et son r6le pathogene. Revue Path. comp. Med. exp. 24: 695-711. 1924. 3. FIEVEZ, L. etude comparee des souches de Spherophorus necrophorus isolees chez l'homme et chez l'animal. Bruxelles: Presses Academiques Europeennes. 1963. 4. LAHELLE, 0. Necrobacterium. A study of itcs bacteriology, serology and pathogenicity and its relation to Fusobacterium. Acta path. microbiol. scand. suppl. 67: 88-90. 1947. 5. PREVOT, A. R. Manuel de classification et de '16termination des bacteries anaerobies. 3e Edit. pp. 278-279. Paris: Masson. 1957. f. SCHMORL, G. Ueber ein pathogens Fadenbacterium (Streptothrix cuniculi). Deut. zeitschfur. Tiermed. 17: 375. Abst. in Zentbl. Bakt. ParasitKde I 11: 666. 1892. 7. SIMON, P. C. Cultivation and maintenance of Sphaerophorus necrophorus Part I: An anaerobic medium for aerobic incubation. Can. J. comp. Med. 38: 9496. 1974. S. SIMON, P. C. and P. L. STOVELL. Isolation of Sphaerophorus necrophorus from bovine hepatic abscesses in British Columbia. Can. J. comp. Med. 35: 103-106. 1971. 9. SIMON, P. C. Cultivation and maintenance of Spha erophorus necrophorus Part 11: Infivence of pH on maximum growth and acid production in medium 156. Can. J. comp. Med. 39: 89-93. 1975.
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