APPLiED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1976, Copyright 0 1976 American Society for Microbiology
p. 455-457
Vol. 31, No. 3 Printed in U.SA.
Influence of Starch Source on Sporulation and Enterotoxin Production by Clostridium perfringens Type A R. LABBE,* E. SOMERS, AND C. DUNCAN Food Research Institute and Department ofBacteriology, University of Wisconsin, Madison, Wisconsin 53706
Received for publication 6 October 1975
Of 16 different starch preparations tested, Clostridium perfringens NCTC 8798 yielded maximum sporulation and enterotoxin formation when ICN-soluble starch was included in Duncan and Strong sporulation medium. In general soluble starches were better than potato, corn, or arrowroot starch with regard to these two parameters. Since it was first proposed several years ago, Duncan and Strong sporulation medium (3) has been widely used for sporulation and enterotoxin production by Clostridium perfringens type A and more recently for other toxigenic types (9). The degree of sporulation in this organism varies from strain to strain and indeed even within a given strain the percentage of sporulation may fluctuate from experiment to experiment. Starch is one component that has a decided effect on the production of heat-resistant spores (7). We have experienced variation in the ability of a strain to sporulate, depending on the source of starch used. Since sporulation and enterotoxin production have been shown to be directly related (5), we investigated the influence of various starches on these two parameters. The basal medium consisted of 0.4% yeast extract (Difco Laboratories, Detroit, Mich.), 1.5% proteose peptone (Difco), 0.1% sodium thioglycolate (Difco), and 1% Na2HPO047H2O. Starch was added at a concentration of 0.4%. This medium has been routinely used in our laboratory for several years. The inoculation sequence was as follows. One drop of a cooked meat stock culture of C. perfringens NCTC 8798 (Hobbs serotype 9 with a normally high sporulation frequency) or NCTC 10239 (Hobbs serotype 12 with a normally low sporulation frequency) was added to 10 ml of freshly steamed fluid thioglycolate medium. The latter was heat shocked for 20 min at 75 C, cooled, and incubated overnight (16 to 18 h) at 37 C. The entire contents of the fluid thioglycolate tube were then inoculated into 1 liter of basal medium with or without starch and incubated at 37 C. No special precautions were followed to obtain anaerobic conditions other than the inclusion of sodium thioglycolate and stationary incubation of the culture. Heat-resistant spore levels (1) 455
and percentage of sporulation (6) were determined as previously reported. Extracts of cells from 250 ml of culture incubated for 6 h were obtained as described (4). Enterotoxin in cell extracts was quantitated by electroimmunodiffusion (2) and protein by the method of Lowry et al. (8). All determinations were done in duplicate. Specific activity of toxin is expressed as micrograms of enterotoxin per milligram of cell extract protein. Starches were obtained from the following sources: Corn Products Co. International, Englewood Cliffs, N. J.; Matheson, Coleman and Bell, Norwood, Ohio; Fisher Scientific Co., Pittsburgh, Pa.; Sigma Chemical Co., St. Louis, Mo.; Leco Corp., St. Joseph, Mich.; Pfanstiehl Laboratories, Waukegan, Ill.; ICN Pharmaceuticals, Cleveland, Ohio; J. T. Baker Co., Phillipsburg, N. J.; Baker and Adamson, Morristown, N. J. Table 1 shows that in the case of NCTC 8798 soluble starches in general resulted in higher levels of heat-resistant spores and enterotoxin than potato, corn, or arrowroot starches. The inclusion of soluble starch (ICN Pharmaceuticals) in Duncan and Strong medium resulted in the highest level of toxin and among the highest level of heat-resistant spores. Rice starch (Matheson, Coleman and Bell) also yielded a high number of spores and the second highest level of toxin; however, another rice starch (ICN Pharmaceuticals) was much poorer in both respects. A high number of heat-resistant spores was also produced using wheat starch, but toxin yields were poor, an unexpected result. Omitting starch altogether resulted in a fairly high amount of toxin. In fact in only four experiments using starch was the level of toxin higher than in the no-starch control. In some cases starch actually decreased enterotoxin specific activity. The relatively high level of enter-
456
APPL. ENVIRON. MICROBIOL.
NOTES
TABLE 1. Influence of starch source on heat-resistant spore formation and enterotoxin formation by C. perfringens NCTC 8798 NCTC 10239 Type
Manufacturer
Heat-resistant spores/ml spores/mi
Sp act of toxin (jAgI mg)
Sp act of Heat-resistant toxin (ILgl soe/l soe/img)
104 105 105 104
6.6 10.8 7.2 3.9
39.8 40.0
1.5 x 105 1.2 x 105 6.3 x 104 8.2 x 104
8.5 6.1 7.1 7.9
1.7 x 106 6.1 x 106 2.4 x 10'
18.5 34.1 18.3
7.1 x 104 8.4 x 104 6.7 x 104
3.5 3.3 3.6
MC/B Leco
4.3 x 106 8.4 x 106
30.9 30.9
8.0 x 104 9.6 x 104
7.2 5.7
Rice
MC/B ICN
1.3 x 107 6.3 x 106
78.5 28.2
1.3 x 105 2.1 x 105
5.5 4.2
Wheat
Sigma
1.4 x 107
15.5
2.2 x 105
7.2
5.3 x 104
48.9
1.0 x 105
6.9
Soluble
BA
Fisher ICN Pfanstiehl
4.7 x 4.8 x 1.3 x 1.1 x
MC/B Fisher ICN Baker
1.2 3.2 3.5 2.6
Corn
106
54.1
106
107 107
31.0 106.9 55.4
106 106
32.3 46.5
106 106
MC/B Fisher Corn Products
Arrowroot
Potato
No starch
x x x x
5.0 x 1.8 x 3.1 x 9.4 x
Abbreviations: BA, Baker and Adamson; ICN, ICN Pharmaceuticals; MC/B, Matheson, Coleman and Bell.
otoxin in the no-starch control may have been due to depressed growth of the nonsporulating population (7) which would serve to increase specific activity. The low number of spores produced without starch by NCTC 8798 was expected and has been reported (7). In the case of NCTC 8798 all starches were effective in increasing the heat-resistant spore level above the no-starch control. The soluble starches were easier to use than all other types. The poor solubility of the latter resulted in the requirement for prolonged sonication to disrupt sporangium. If harvesting of mature spores had been the object of the study, such weakly solu-
ble starches, which sediment along with the spores, would have made cleaning of spores additionally tedious. Results obtained using NCTC 10239 were less definitive. The requirement for starch for heatresistant spore formation by this strain is questionable. This may be a reflection of the low sporulating ability of this strain. Unlike the case of NCTC 8798, the components of Duncan and Strong medium without starch may provide the strain with sufficient energy for sporulation by the low sporulating population. In any
event these results indicate that strain to strain variation may be as important as the starch source with regard to sporulation by C. perfringens. This research was supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wis., by research grant FD-00203-05 from the Food and Drug Administration, by Public Health Service research grant AI-11865-05 from the National Institute of Allergy and Infectious Diseases, and by contributions from the Food Research Institute by member industries. C. L. Duncan is a recipient of Public Health Service Research Career Development Award AI-70721-02 from the National Institute of Allergy and Infectious Diseases.
LITERATURE CITED 1. Duncan, C., R. Labbe, and R. Reich. 1972. Germination of heat- and alkali-altered spores of Clostridium perfringens type A by lysozyme and an intiation protein. J. Bacteriol. 109:550-559. 2. Duncan, C., and E. Somers. 1972. Quantitation of Clostridium perfringens type A enterotoxin by electroimmunodiffusion. Appl. Microbiol. 24:801-804. 3. Duncan, C., and D. Strong. 1968. Improved medium for sporulation of Clostridium perfringens. Appl. Microbiol. 16:82-89. 4. Duncan, C., and D. Strong. 1969. Ileal loop fluid accumulation and production of diarrhea in rabbits by cell-free products of Clostridium perfringens. J. Bacteriol. 100:86-94.
VOL. 31, 1976 5. Duncan, C., D. Strong, and M. Sebald. 1972. Sporulation and enterotoxin production by mutants of Clostridium perfringens. J. Bacteriol. 110:378-391. 6. Labbe, R., and C. Duncan. 1974. Sporulation and enterotoxin production by Clostridium perfringens under conditions of controlled pH and temperature. Can. J. Microbiol. 20:1493-1501. 7. Labbe, R., and C. Duncan. 1975. Influence of carbohy-
NOTES
457
drates on growth and sporulation of Clostridium perfringens type A. Appl. Microbiol. 29:345-351. 8. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 9. Skjelkvale, R., and C. Duncan. 1975. Enterotoxin formation by different toxigenic types of Clostridium perfringens. Infect. Immun. 11:563-575.
p. 455-457
Vol. 31, No. 3 Printed in U.SA.
Influence of Starch Source on Sporulation and Enterotoxin Production by Clostridium perfringens Type A R. LABBE,* E. SOMERS, AND C. DUNCAN Food Research Institute and Department ofBacteriology, University of Wisconsin, Madison, Wisconsin 53706
Received for publication 6 October 1975
Of 16 different starch preparations tested, Clostridium perfringens NCTC 8798 yielded maximum sporulation and enterotoxin formation when ICN-soluble starch was included in Duncan and Strong sporulation medium. In general soluble starches were better than potato, corn, or arrowroot starch with regard to these two parameters. Since it was first proposed several years ago, Duncan and Strong sporulation medium (3) has been widely used for sporulation and enterotoxin production by Clostridium perfringens type A and more recently for other toxigenic types (9). The degree of sporulation in this organism varies from strain to strain and indeed even within a given strain the percentage of sporulation may fluctuate from experiment to experiment. Starch is one component that has a decided effect on the production of heat-resistant spores (7). We have experienced variation in the ability of a strain to sporulate, depending on the source of starch used. Since sporulation and enterotoxin production have been shown to be directly related (5), we investigated the influence of various starches on these two parameters. The basal medium consisted of 0.4% yeast extract (Difco Laboratories, Detroit, Mich.), 1.5% proteose peptone (Difco), 0.1% sodium thioglycolate (Difco), and 1% Na2HPO047H2O. Starch was added at a concentration of 0.4%. This medium has been routinely used in our laboratory for several years. The inoculation sequence was as follows. One drop of a cooked meat stock culture of C. perfringens NCTC 8798 (Hobbs serotype 9 with a normally high sporulation frequency) or NCTC 10239 (Hobbs serotype 12 with a normally low sporulation frequency) was added to 10 ml of freshly steamed fluid thioglycolate medium. The latter was heat shocked for 20 min at 75 C, cooled, and incubated overnight (16 to 18 h) at 37 C. The entire contents of the fluid thioglycolate tube were then inoculated into 1 liter of basal medium with or without starch and incubated at 37 C. No special precautions were followed to obtain anaerobic conditions other than the inclusion of sodium thioglycolate and stationary incubation of the culture. Heat-resistant spore levels (1) 455
and percentage of sporulation (6) were determined as previously reported. Extracts of cells from 250 ml of culture incubated for 6 h were obtained as described (4). Enterotoxin in cell extracts was quantitated by electroimmunodiffusion (2) and protein by the method of Lowry et al. (8). All determinations were done in duplicate. Specific activity of toxin is expressed as micrograms of enterotoxin per milligram of cell extract protein. Starches were obtained from the following sources: Corn Products Co. International, Englewood Cliffs, N. J.; Matheson, Coleman and Bell, Norwood, Ohio; Fisher Scientific Co., Pittsburgh, Pa.; Sigma Chemical Co., St. Louis, Mo.; Leco Corp., St. Joseph, Mich.; Pfanstiehl Laboratories, Waukegan, Ill.; ICN Pharmaceuticals, Cleveland, Ohio; J. T. Baker Co., Phillipsburg, N. J.; Baker and Adamson, Morristown, N. J. Table 1 shows that in the case of NCTC 8798 soluble starches in general resulted in higher levels of heat-resistant spores and enterotoxin than potato, corn, or arrowroot starches. The inclusion of soluble starch (ICN Pharmaceuticals) in Duncan and Strong medium resulted in the highest level of toxin and among the highest level of heat-resistant spores. Rice starch (Matheson, Coleman and Bell) also yielded a high number of spores and the second highest level of toxin; however, another rice starch (ICN Pharmaceuticals) was much poorer in both respects. A high number of heat-resistant spores was also produced using wheat starch, but toxin yields were poor, an unexpected result. Omitting starch altogether resulted in a fairly high amount of toxin. In fact in only four experiments using starch was the level of toxin higher than in the no-starch control. In some cases starch actually decreased enterotoxin specific activity. The relatively high level of enter-
456
APPL. ENVIRON. MICROBIOL.
NOTES
TABLE 1. Influence of starch source on heat-resistant spore formation and enterotoxin formation by C. perfringens NCTC 8798 NCTC 10239 Type
Manufacturer
Heat-resistant spores/ml spores/mi
Sp act of toxin (jAgI mg)
Sp act of Heat-resistant toxin (ILgl soe/l soe/img)
104 105 105 104
6.6 10.8 7.2 3.9
39.8 40.0
1.5 x 105 1.2 x 105 6.3 x 104 8.2 x 104
8.5 6.1 7.1 7.9
1.7 x 106 6.1 x 106 2.4 x 10'
18.5 34.1 18.3
7.1 x 104 8.4 x 104 6.7 x 104
3.5 3.3 3.6
MC/B Leco
4.3 x 106 8.4 x 106
30.9 30.9
8.0 x 104 9.6 x 104
7.2 5.7
Rice
MC/B ICN
1.3 x 107 6.3 x 106
78.5 28.2
1.3 x 105 2.1 x 105
5.5 4.2
Wheat
Sigma
1.4 x 107
15.5
2.2 x 105
7.2
5.3 x 104
48.9
1.0 x 105
6.9
Soluble
BA
Fisher ICN Pfanstiehl
4.7 x 4.8 x 1.3 x 1.1 x
MC/B Fisher ICN Baker
1.2 3.2 3.5 2.6
Corn
106
54.1
106
107 107
31.0 106.9 55.4
106 106
32.3 46.5
106 106
MC/B Fisher Corn Products
Arrowroot
Potato
No starch
x x x x
5.0 x 1.8 x 3.1 x 9.4 x
Abbreviations: BA, Baker and Adamson; ICN, ICN Pharmaceuticals; MC/B, Matheson, Coleman and Bell.
otoxin in the no-starch control may have been due to depressed growth of the nonsporulating population (7) which would serve to increase specific activity. The low number of spores produced without starch by NCTC 8798 was expected and has been reported (7). In the case of NCTC 8798 all starches were effective in increasing the heat-resistant spore level above the no-starch control. The soluble starches were easier to use than all other types. The poor solubility of the latter resulted in the requirement for prolonged sonication to disrupt sporangium. If harvesting of mature spores had been the object of the study, such weakly solu-
ble starches, which sediment along with the spores, would have made cleaning of spores additionally tedious. Results obtained using NCTC 10239 were less definitive. The requirement for starch for heatresistant spore formation by this strain is questionable. This may be a reflection of the low sporulating ability of this strain. Unlike the case of NCTC 8798, the components of Duncan and Strong medium without starch may provide the strain with sufficient energy for sporulation by the low sporulating population. In any
event these results indicate that strain to strain variation may be as important as the starch source with regard to sporulation by C. perfringens. This research was supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wis., by research grant FD-00203-05 from the Food and Drug Administration, by Public Health Service research grant AI-11865-05 from the National Institute of Allergy and Infectious Diseases, and by contributions from the Food Research Institute by member industries. C. L. Duncan is a recipient of Public Health Service Research Career Development Award AI-70721-02 from the National Institute of Allergy and Infectious Diseases.
LITERATURE CITED 1. Duncan, C., R. Labbe, and R. Reich. 1972. Germination of heat- and alkali-altered spores of Clostridium perfringens type A by lysozyme and an intiation protein. J. Bacteriol. 109:550-559. 2. Duncan, C., and E. Somers. 1972. Quantitation of Clostridium perfringens type A enterotoxin by electroimmunodiffusion. Appl. Microbiol. 24:801-804. 3. Duncan, C., and D. Strong. 1968. Improved medium for sporulation of Clostridium perfringens. Appl. Microbiol. 16:82-89. 4. Duncan, C., and D. Strong. 1969. Ileal loop fluid accumulation and production of diarrhea in rabbits by cell-free products of Clostridium perfringens. J. Bacteriol. 100:86-94.
VOL. 31, 1976 5. Duncan, C., D. Strong, and M. Sebald. 1972. Sporulation and enterotoxin production by mutants of Clostridium perfringens. J. Bacteriol. 110:378-391. 6. Labbe, R., and C. Duncan. 1974. Sporulation and enterotoxin production by Clostridium perfringens under conditions of controlled pH and temperature. Can. J. Microbiol. 20:1493-1501. 7. Labbe, R., and C. Duncan. 1975. Influence of carbohy-
NOTES
457
drates on growth and sporulation of Clostridium perfringens type A. Appl. Microbiol. 29:345-351. 8. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 9. Skjelkvale, R., and C. Duncan. 1975. Enterotoxin formation by different toxigenic types of Clostridium perfringens. Infect. Immun. 11:563-575.
