Vol. 7, No. 5
JOL;RNAL OF CLINICAL MICROBIOLOGY, May 1978, p. 493-494 0095-1 137/78/0007-0493$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Rapid Determination of Corynebacterium diphtheriae Toxigenicity by Counterimmunoelectrophoresis NANCY L. THOMPSON AND PAUL D. ELLNER*
Department of Microbiology, Columbia University, College of Physicians & Surgeons, New York, New York 10032 Received for publication 12 January 1978 A method for detecting toxin production in Corynebacterium diphtheriae cultures by counterimmunoelectrophoresis is described. Precipitin lines were observed in toxigenic strains in 30 min.
The determination of the toxigenicity of isolates of Corynebacterium diphtheriae is a timeconsuming procedure. In vitro methods include passive agar gel diffusion (3, 5, 10, 13, 14) and tissue culture (11, 15). Since most clinical microbiology laboratories have limited access to animals or cell cultures, the passive agar gel method has been the most widely used. This procedure usually takes at least 48 h after the organism has been isolated, and interpretation is often difficult (1, 16). Counterimmunoelectrophoresis (CIE) not only increases the sensitivity 10- to 100-fold over passive gel diffusion, but decreases the reaction time as well by actively producing antigen-antibody contact in an electric field. CIE has been used to detect microbial antigens of N. meningitidis (2), S. pneumoniae (4), H. influenzae (9), S. aureus (12), and hepatitis virus (6) in body fluids and to identify cultures of group B streptococci (8). It occurred to us that CIE might appropriately be applied to the detection oftoxin in cultures of C. diphtheriae. Cultures of Park-Williams (ATCC 13812), Park 8 (ATCC 296), Gravis (ATCC 9059), Intermedius (ATCC 8032), Mitis (ATCC 8024), and an avirulent (ATCC 11913) strain of C. diphtheriae were used. In addition, C. hemolyticum and three unspeciated diphtheroid isolates were also tested. Modified Columbia broth, pH 7.3, and Frobisher Proteose peptone broth, pH 7.8 (5), were evaluated as basal media. Horse serum, rabbit serum, and the combination of Casamino Acids and Tween 80 (7) (K-L enrichment, Difco) were each evaluated as supplements at a concentration of 20%. Complete medium was dispensed in 10-ml amounts in 125-ml Erlenmeyer flasks. Cultures were incubated at 35°C under 7% CO2 for varying periods of time. The effect of shaking was compared with stationary incubation. CIE was performed on whole broth cultures with a kit obtained from Hyland Laboratories
which consisted of disposable electrophoresis base units with foil electrodes and sponge wicks, prepunched agar gel plates, barbital buffer solution (pH 8.6), and a power supply. Samples were run at room temperature for 30 min at 40 mA. Diphtheria antitoxin was obtained from Merrill National Laboratories. (This antitoxin is now produced by Connaught Laboratories and may be obtained from Elkins-Sinn, Richmond, Va.; the undiluted antitoxin has a 5-year shelf life at 2 to 8°C.) Varying dilutions of the antitoxin ranging from 1:30 to 1:2,000 were prepared with barbital buffer and contained from 1 to 64 U of antitoxin per ml. Plates were examined for precipitin lines immediately after the current was turned off. The effect of the basal medium, supplement, and length of incubations is shown in Table 1. Lines first appeared in proteose peptone broth supplemented with horse or rabbit serum at 16 h and became distinct by 24 h. No advantage was gained by shaking the cultures or by extending the incubation beyond 48 h. Better lines were obtained with proteose peptone broth than with Columbia broth, but this may have been due to differences in the pH of these media. Proteose peptone broth, pH 7.8, supplemented with 20% horse serum was adopted as the standard medium. Cultures were incubated under stationary conditions for 24 h prior to testing by CIE. The optimal dilution of antitoxin was found to be 1:125 and contained approximately 16 U of antitoxin per ml. The results of testing the various strains are shown in Fig. 1. Distinct lines were obtained with the Park-Williams, Park 8, Gravis, and Intermedius strains in 24-h cultures; lines were not detected with the Mitis strain until 48 h of incubation. We were subsequently advised by the American Type Culture Collection that this Mitis strain was "barely toxigenic." No lines occurred with the avirulent strain, C. hemolyti493
494
J. CI,IN. MICROBIOL,.
NOTES
TABLE 1. Effect of basal medium and supplements on CIE with Park- Williams strain' 2% Proteose peptone broth (pH 7.8)
Columbia broth (pH 7.3) Time (h)
Unsupplemented
Horse serum
Rabbit serum
None None None None None None None None None Faint Faint None 24 Faint Faint None 48 a Descriptions refer to absence or appearance 4 8 16
K-L
Unsupplemented
None None None None None None None None None None of precipitin lines.
Horse serum
Rabbit serum
K-L
None None Faint Distinct Distinct
None None Faint Distinct Distinct
None None None None None
studies on toxigenic and nontoxigenic clinical isolates are certainly indicated. LITERATURE CITED
FIG. 1. CIE of various cultures against diphtheria antitoxin (16 U/ml). Wells no. 1 to 3, diphtheroids; well no. 4, C. hemolyticum; well no. 5, avirulent C. diphtheriae (ATCC 11913); well no. 6, Park- Williams (ATCC 13812); well no. 7, Park 8 (ATCC 296); well no. 8, Gravis (ATCC 9059); weU no. 9, Intermedius (ATCC 8032); well no. 10, Mitis (ATCC 8024).
the three diphtheroid isolates. We have demonstrated that the toxigenicity of C. diphtheriae cultures may be rapidly determined by CIE using materials readily available to clinical microbiology laboratories. Further cum, or
1. Bickman, S. T., and W. L. Jones. 1972. Problems in the use of the in vitro toxigenicity test for Corynebacterium diphtheriae. Am. J. Clin. Pathol. 57:244-246. 2. Edwards, E. A. 1971. Immunologic investigations of meningococcal disease. J. Immunol. 106:314-317. 3. Elek, S. D. 1949. The plate virulence test for diphtheria. J. Clin. Pathol. 2:250-258. 4. Fossieck, B., R. Craig, and P. Y. Paterson. 1973. Counterelectroimmunophoresis for rapid diagnosis of meningitis due to Diplococcus pneumoniae. J. Infect. Dis. 127:106-109. 5. Frobisher, M., E. 0. King, and E. I. Parsons. 1951. A test in vitro for virulence of Corynebacterium diphtheriae. Am. J. Clin. Pathol. 21:282-285. 6. Gocke, D. J., and C. Howe. 1970. Rapid detection of Australia antigen by counterimmunoelectrophoresis. J. Immunol. 104:1031-1032. 7. Hermann, G. J., M. S. Moore, and E. I. Parsons. 1958. A substitute for serum in the diphtheria in vitro toxigenicity test. Am. J. Clin. Pathol. 29:181-183. 8. Hill, H. R., et al. 1975. Rapid identification of group B streptococci by counterimmunoelectrophoresis. J. Clin. Microbiol. 1:188-191. 9. Ingram, D. L., P. Anderson, and D. H. Smith. 1972. Countercurrent immunoelectrophoresis in the diagnosis of systemic diseases caused by Hemophilus influenzae type b. J. Pediatr. 81:1156-1159. 10. King, E. O., M. Frobisher, and E. I. Parsons. 1949. The in vitro test for virulence of Corynebacterium diphtheriae. Am. J. Public Health 39:1314-1320. 11. Laird, W., and N. Groman. 1973. Rapid direct tissue culture test for toxigenicity of Corynebacterium diphtheriae. Appl. Microbiol. 25:709-712. 12. Nagel, J. G., et al. 1975. Teichoic acid serologic diagnosis of staphylococcal endocarditis. Ann. Intern. Med. 82:13-17. 13. Ouchterloney, 0. 1949. An in vitro test of the toxinproducing capacity of Corynebacterium diphtheriae. Lancet i:346-353. 14. Ouchterloney, 0. 1949. In vitro method for testing the toxin-producing capacity of diphtheria bacteria. Acta Pathol. Microbiol. Scand. 26:516-524. 15. Schubert, J. H., S. T. Biskham, and G. L. Wiggins. 1968. Tissue culture method for toxigenicity testing of Corynebacterium diphtheriae. Appl. Microbiol. 16:1748-1752. 16. Young, P. S., and D. D. Smith. 1976. The detection of toxin production by Corynebacterium diphtheriae in diagnostic laboratories. Pathology 8:101-104.
JOL;RNAL OF CLINICAL MICROBIOLOGY, May 1978, p. 493-494 0095-1 137/78/0007-0493$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Rapid Determination of Corynebacterium diphtheriae Toxigenicity by Counterimmunoelectrophoresis NANCY L. THOMPSON AND PAUL D. ELLNER*
Department of Microbiology, Columbia University, College of Physicians & Surgeons, New York, New York 10032 Received for publication 12 January 1978 A method for detecting toxin production in Corynebacterium diphtheriae cultures by counterimmunoelectrophoresis is described. Precipitin lines were observed in toxigenic strains in 30 min.
The determination of the toxigenicity of isolates of Corynebacterium diphtheriae is a timeconsuming procedure. In vitro methods include passive agar gel diffusion (3, 5, 10, 13, 14) and tissue culture (11, 15). Since most clinical microbiology laboratories have limited access to animals or cell cultures, the passive agar gel method has been the most widely used. This procedure usually takes at least 48 h after the organism has been isolated, and interpretation is often difficult (1, 16). Counterimmunoelectrophoresis (CIE) not only increases the sensitivity 10- to 100-fold over passive gel diffusion, but decreases the reaction time as well by actively producing antigen-antibody contact in an electric field. CIE has been used to detect microbial antigens of N. meningitidis (2), S. pneumoniae (4), H. influenzae (9), S. aureus (12), and hepatitis virus (6) in body fluids and to identify cultures of group B streptococci (8). It occurred to us that CIE might appropriately be applied to the detection oftoxin in cultures of C. diphtheriae. Cultures of Park-Williams (ATCC 13812), Park 8 (ATCC 296), Gravis (ATCC 9059), Intermedius (ATCC 8032), Mitis (ATCC 8024), and an avirulent (ATCC 11913) strain of C. diphtheriae were used. In addition, C. hemolyticum and three unspeciated diphtheroid isolates were also tested. Modified Columbia broth, pH 7.3, and Frobisher Proteose peptone broth, pH 7.8 (5), were evaluated as basal media. Horse serum, rabbit serum, and the combination of Casamino Acids and Tween 80 (7) (K-L enrichment, Difco) were each evaluated as supplements at a concentration of 20%. Complete medium was dispensed in 10-ml amounts in 125-ml Erlenmeyer flasks. Cultures were incubated at 35°C under 7% CO2 for varying periods of time. The effect of shaking was compared with stationary incubation. CIE was performed on whole broth cultures with a kit obtained from Hyland Laboratories
which consisted of disposable electrophoresis base units with foil electrodes and sponge wicks, prepunched agar gel plates, barbital buffer solution (pH 8.6), and a power supply. Samples were run at room temperature for 30 min at 40 mA. Diphtheria antitoxin was obtained from Merrill National Laboratories. (This antitoxin is now produced by Connaught Laboratories and may be obtained from Elkins-Sinn, Richmond, Va.; the undiluted antitoxin has a 5-year shelf life at 2 to 8°C.) Varying dilutions of the antitoxin ranging from 1:30 to 1:2,000 were prepared with barbital buffer and contained from 1 to 64 U of antitoxin per ml. Plates were examined for precipitin lines immediately after the current was turned off. The effect of the basal medium, supplement, and length of incubations is shown in Table 1. Lines first appeared in proteose peptone broth supplemented with horse or rabbit serum at 16 h and became distinct by 24 h. No advantage was gained by shaking the cultures or by extending the incubation beyond 48 h. Better lines were obtained with proteose peptone broth than with Columbia broth, but this may have been due to differences in the pH of these media. Proteose peptone broth, pH 7.8, supplemented with 20% horse serum was adopted as the standard medium. Cultures were incubated under stationary conditions for 24 h prior to testing by CIE. The optimal dilution of antitoxin was found to be 1:125 and contained approximately 16 U of antitoxin per ml. The results of testing the various strains are shown in Fig. 1. Distinct lines were obtained with the Park-Williams, Park 8, Gravis, and Intermedius strains in 24-h cultures; lines were not detected with the Mitis strain until 48 h of incubation. We were subsequently advised by the American Type Culture Collection that this Mitis strain was "barely toxigenic." No lines occurred with the avirulent strain, C. hemolyti493
494
J. CI,IN. MICROBIOL,.
NOTES
TABLE 1. Effect of basal medium and supplements on CIE with Park- Williams strain' 2% Proteose peptone broth (pH 7.8)
Columbia broth (pH 7.3) Time (h)
Unsupplemented
Horse serum
Rabbit serum
None None None None None None None None None Faint Faint None 24 Faint Faint None 48 a Descriptions refer to absence or appearance 4 8 16
K-L
Unsupplemented
None None None None None None None None None None of precipitin lines.
Horse serum
Rabbit serum
K-L
None None Faint Distinct Distinct
None None Faint Distinct Distinct
None None None None None
studies on toxigenic and nontoxigenic clinical isolates are certainly indicated. LITERATURE CITED
FIG. 1. CIE of various cultures against diphtheria antitoxin (16 U/ml). Wells no. 1 to 3, diphtheroids; well no. 4, C. hemolyticum; well no. 5, avirulent C. diphtheriae (ATCC 11913); well no. 6, Park- Williams (ATCC 13812); well no. 7, Park 8 (ATCC 296); well no. 8, Gravis (ATCC 9059); weU no. 9, Intermedius (ATCC 8032); well no. 10, Mitis (ATCC 8024).
the three diphtheroid isolates. We have demonstrated that the toxigenicity of C. diphtheriae cultures may be rapidly determined by CIE using materials readily available to clinical microbiology laboratories. Further cum, or
1. Bickman, S. T., and W. L. Jones. 1972. Problems in the use of the in vitro toxigenicity test for Corynebacterium diphtheriae. Am. J. Clin. Pathol. 57:244-246. 2. Edwards, E. A. 1971. Immunologic investigations of meningococcal disease. J. Immunol. 106:314-317. 3. Elek, S. D. 1949. The plate virulence test for diphtheria. J. Clin. Pathol. 2:250-258. 4. Fossieck, B., R. Craig, and P. Y. Paterson. 1973. Counterelectroimmunophoresis for rapid diagnosis of meningitis due to Diplococcus pneumoniae. J. Infect. Dis. 127:106-109. 5. Frobisher, M., E. 0. King, and E. I. Parsons. 1951. A test in vitro for virulence of Corynebacterium diphtheriae. Am. J. Clin. Pathol. 21:282-285. 6. Gocke, D. J., and C. Howe. 1970. Rapid detection of Australia antigen by counterimmunoelectrophoresis. J. Immunol. 104:1031-1032. 7. Hermann, G. J., M. S. Moore, and E. I. Parsons. 1958. A substitute for serum in the diphtheria in vitro toxigenicity test. Am. J. Clin. Pathol. 29:181-183. 8. Hill, H. R., et al. 1975. Rapid identification of group B streptococci by counterimmunoelectrophoresis. J. Clin. Microbiol. 1:188-191. 9. Ingram, D. L., P. Anderson, and D. H. Smith. 1972. Countercurrent immunoelectrophoresis in the diagnosis of systemic diseases caused by Hemophilus influenzae type b. J. Pediatr. 81:1156-1159. 10. King, E. O., M. Frobisher, and E. I. Parsons. 1949. The in vitro test for virulence of Corynebacterium diphtheriae. Am. J. Public Health 39:1314-1320. 11. Laird, W., and N. Groman. 1973. Rapid direct tissue culture test for toxigenicity of Corynebacterium diphtheriae. Appl. Microbiol. 25:709-712. 12. Nagel, J. G., et al. 1975. Teichoic acid serologic diagnosis of staphylococcal endocarditis. Ann. Intern. Med. 82:13-17. 13. Ouchterloney, 0. 1949. An in vitro test of the toxinproducing capacity of Corynebacterium diphtheriae. Lancet i:346-353. 14. Ouchterloney, 0. 1949. In vitro method for testing the toxin-producing capacity of diphtheria bacteria. Acta Pathol. Microbiol. Scand. 26:516-524. 15. Schubert, J. H., S. T. Biskham, and G. L. Wiggins. 1968. Tissue culture method for toxigenicity testing of Corynebacterium diphtheriae. Appl. Microbiol. 16:1748-1752. 16. Young, P. S., and D. D. Smith. 1976. The detection of toxin production by Corynebacterium diphtheriae in diagnostic laboratories. Pathology 8:101-104.
