b~tenlational Jountal of Fotni Microbiology, 15 (1992) 377-382 © 1992 Elsevier Science Publishers B.V. All rights reserved I)108-1605/92/$05.0(I


FOOD 00472

Short communication

Simple and economical culture of Campylobacter ]e]uni and Campflobacter coii in CO 2 in moist air A . D . E . Fraser 1, V. Chandan 2, H. Yamazaki 2 B.W. Brooks ~ and M.M. Garcia i t Agriculture Canada, Animal Diseases Research lnstitutt: NEPE,,IN, Nepean, Ontario and 2 blstitute of Biochonistry. Carleton Unh'ersity. Otmu'a. Ontario, Canada (Received 21 November 1991;accepted 15 February 1992)

Strains of Campylobacter jejuni and C. coil representing the 18 serogroups (Lior) most commonly isolated from humans in Canada were grown on solid media in an atmosphere of 10% CO, in moist air, 99% relative humidity. When the growth of all 18 serogroups on Mueller Hinton agar in a mieroaerobic atmosphere (5% O 2, I1}%CO 2 and 85% N2) was compared with the growth of all 18 serogroups on the same media in 10% CO 2 in moist air, colony sizes were significantly larger (p < 0.05) for strains grown in 10% CO 2 in moist air. No significant difference in colony numbers was seen between the two atmospheres. The addition of blood to the media significantlyenhanced the growth of the campylobaeters in both types of atmospheres (p < 0.05). This simple CO, atmosphere permitted the use of a common CO, incubator thereby reducing the cost and difficulty of culturing these organisms. Key words: Campylobacter; Simplified culture; Economic culture

Introduction Previous studies have shown that Campylobacter jejuni and C. coli grow well in an atmosphere consisting of 5% 0 2, 10% C O 2 and 85% N z (Griffiths and Park, 1990; Luechtefeid et ai., 1982; Skirrow et al., 1982). This atmosphere is widely used in laboratories throughout the world for the isolation o f thermophilic campylobacters. The special atmospheric requirement has, however, necessitated the use o f expensive gas mixtures and gassing jars which have a limited capacity for holding sample~ tGriffiths and Park, 1990; Luechtefeld et al., 1982; Skirrow et al., 1982). Eliminating the need for these would economize and facilitate campylobacter diagno,:is by standard culture methods. Here we show that an atmosphere of 10% C O 2 in moist air supported the growth o f reference strains of 18 different serogroups (l,~or) of C. jejuni "and C. coll. The 18 serogroups represent those most commonly isolated from humans in

Co~respomlence address: A.D.E. Fraser, Agriculture Canada, Animal Diseases Research Institute, NEPEAN, P.O. Box 11300, Station H, Nepean, Ontario, Canada K2H 8P9.

378 TABLE 1 I {st of Camnvlobacter slrains used in thi,~study " Serogroup h LIOI LIO2 LIO4 LIO5 LIOo LIO7 LIO8 L109 LIO I 1 L10! 7 LIO18 L1020 LIO21 LIO28 LIO29 L1036 L1044 LIO53

Species C. jejuni C. jejuni C jejuni C. jejuni C. jejuni C. jejuni C. coli C. jejuni C. jejuni C. jejuni (~:jejuni C. coli C. coli C. jt~ttni C: coli C. jejuni C. coli C. jejuni

Biotype c ! Ii ! i I!! I i I ! I !!! I I I II !1 !1 I

Source Human Human Human Human Human Human Human Human Human Chicken Chicken Swine Chicken Human Human Human Human Human

" Strains provided by H. Lior, National Reference Center for Campylobacters, LCDC, Ottawa, Ontario, Canada. b Lior et a1.(1982). Lior(1984). C a n a d a (H. Lior, personal communication). Use of this atmosphere greatly simplified and economized the culture of these organisms. Preliminary aspects o f this work were presented at the Vth International Workshop on Campylobacter Infections, Puerto Vallarta, Mexico, 1989.

Materials and Methods C a m p y l o b a c t e r strains a n d culture condaions. The C. j e j u n i and C. coli strains used in the present study are listed in Table 1. These serogroup reference strains are from the collection of the National Reference Service for Campylobacters, Laboratory Centre for Disease Control, Ottawa, C a n a d a and were kindly provided by H. Lior. They were also serotyped (Lior et al., 1982) and biotyped (Lior, 1984) by H. Lior. Campylobacter stocks were originally maintained on Mueller Hinton agar plus 10% sheep blood at 37°C in 5% 0 2, 10% C O 2 and 85% N z (microaerobic atmosphere) (Brooks et al., 1986). 48-h cultures were harvested into sterile saline (0.85% NaCl) to be used as inocula and the concentration estimated spectrophotometrically in a Bausch and L o m b Speetronic 21 spectrophotometer. Saline suspensions with an absorbance of 1.0 at 600 nm contained approximately 1.0 x 109 colony forming units ( C F U ) per ml. This value was determined by

379 removing aliquots of the saline suspensions, diluting the aliquots and plating the dilutions on Mueller Hinton agar with 10% sheep blood. After 48 h microaerobic incubation at 37°C, colonies were counted. A 0.1 mi inoculum (containing approximately l03 CFU per ml) of each strain was plated on either: (l) Mueller Hinton agm with (MHAB) or without (MHA) 10% sheep blood, (2) Brucella agar (BA), or (3) Campyiobacter blood-free selective agar (CBFA). All media were obtained from Oxoid Canada, Inc. Plates were incubated for 24, 48 or 72 h at 37°C in (l) gassing jars containing 5% 0 2, 10% CO,, and 85% N 2 (microaerobic atmosphere) (Luechtefeld et al., 1982) which were placed upright in an incubator large encugh to contain the jars, or (2) a CO 2 incubator (National CO 2 Incubator, Weinicke Co.) containing 10% CO 2 (remainder air). As little as 1-2% CO 2 in air was also used. However, this lower level of CO 2 was difficult to regulate accurately with the incubator used here so a level of 10% CO: was chosen for this study. The gassing jars were never filled to more than half their capacity with plates or tubes (Skirrow et ai., 1982). The moisture level in the CO 2 incubator was maintained at approximately 95% relative humidity (RH) by filling the bottom of the incubator with water or at approximately 99% RH by bubbling the gas mixture of 10% CO 2 through this water, both as per manufacturer's instruction. For this study, the incubator was routinely kept at a 99% RH moisture level. To minimize potential contamination problems inherent to such a moist environment, plates were placed in large plastic autoclave bags which had been laid open on their sides. The bags used were large enough to line the entire area of each shelf of the incubator. The flow rate of the gas mixture was maintained at 5-10 liters per min. The extent of growth was determined by colony count (number of CFU) per plate (averaged over 5 plates) and by colony size in mm (averaged over l0 colonies per plate). To determine the significant differences in growth between the two atmospheres, if any, the Student's t-test was used. Combined data for colony numbers of all 18 strains were compared between the two atmospheres as were combined data for colony sizes. For the latter calculations a value of O.1 mm in size was assigned to pinpoint colonies. Colony numbers and colony sizes of all 18 strains were also compared between media with and without 10% sheep blood.

Results and Discussion Each of the 18 serogroup reference strains was plated on BA, CBFA, MHA and MHAB and incubated at 37°C either in a microaerobic atmosphere (5% 0 2, 10% CO 2 and 85% N 2) or in 10% CO 2 in moist air. The growth in terms of colony ,ombers of all 18 strains on any given medium was essentially the same between the two atmospheres (data not included). MHA and MHAB media, however, supported the best growth of the 18 strains as compared to each of the other media (P < 0.05) (data not included). The results of growth after 72 h on MHA and MHAB in the microaerobic atmosphere and in 10% CO 2 in moist air are shown in Table II. There was no significant difference in sample means of the

38O TABLE !1 Growth of Campylobacter strains on Mueller Hinton agar with (MHAB) or without (MHA) sheep blood in a microaerobic atmosphere or 10% CO: (in moist air) Strain

Atmosphere Microaerobic

10% CO 2 in moist air







Colony size b


Colony size


Colony size

44-+ 3 ~" ng 60-+ 4 30-+ 2 34-+ 7 30± 3 39-+ 8 36-+ 6 ng ng 156,+ 3 57+ ! 40-+ 4 36-_:, 5 50-+ 5 37-+ 2 57-+ 4 27-+ 4

pp ng 1.0_+I¢ pp pp pp pp pp ng ng pp pp pp pp 0.5,+10 !.0+_ 1 pp i.0_+ I

126.+ 3 74-+ 6 150,+ 2 60,+10 30-+ 6 120,+ 8 51,+ 6 40-+ 5 90-+ 4 40_+ 3 198,+ I 120+ 5 56-+ 5 290_+ 4 64_+ 4 56-+ 3 147_+ 6 50_+ 6

pp l.O+lO 1.0+ 3 1.5,+ 5 2.0-+ 2 1.0+ 2 1.0+ i 1.5,+ 6 pp pp pp 1.0+ 6 1.0_+10 1.0_+ 5 1.0+ 1 1.0+ 2 0.5-+ 1 1.0_+ !

49-+ 3 ng 73-+ 3 24-+ 2 12,+ 2 21,+ 4 24+ 5 20-+ 5 ng ng 141_+ 8 59+ 7 40_+ 7 29-+ 6 36_+ 4 39_+ 4 14+ 4 10_+ 2

pp 118+ 3 ng 60-+ 3 2 . 0 , + 1 200-+ 4 pp 50-+ 4 pp 34+ 4 pp 170,+ 4 pp 30-+ 3 pp 28-+ 2 ng 90-+ 4 ng 20-+ 2 1 . 0 , + 8 183_+ 2 1.0_+8 66_+ 2 1.0_+7 64_+ 4 1.0_+1 260_+ 3 1.0_+2 120_+ 3 1.0-+3 56_+ 6 pp 127,+ 5 1.5_+3 30_+ 3

Mean -+SE a 41,+33

0.3-+ 0.4

98+_65 0.9+ 0.2


Colony size 2.0+ 3 2.0-+ 2 3.0-+ ! 1.5+ 1 2.0-+ 4 1.0-+!0 3.0-+ 2 2.5-+ 2 1.0+ 3 pp 2.0+ 2 2.5_+ 4 2.0_+ 5 2.0-+ 3 1.0_+ 3 2.0-+ 2 1.5-+ I 2.5_+ 6

33-+34 0.6-+0.5 9 5 - + 6 9 !.9+0.7

Colony forming units (CFU). h Colony size in mm. ¢ Mean + % standard error. d Sample mean + standard error. pp, Pinpoint (for calculating sample mean pp was assigned a value of 0.1 ram). ng, No growth (for calculating sample mean ng was assigned a value of 0).

colony n u m b e r s of the 18 s t r a i n s b e t w e e n the two a t m o s p h e r e s . However, s a m p l e m e a n s of colony sizes w e r e significantly d i f f e r e n t ( P < 0.05), that is, colony sizes w e r e g r e a t e r for the 18 s t r a i n s g r o w n in 10% C O 2 in moist air as c o m p a r e d to the m i c r o a e r o b i c a t m o s p h e r e . T h e p r e s e n c e o f b l o o d in the m e d i a significantly improved the growth in t e r m s of b o t h colony n u m b e r a n d size o f all strains r e g a r d l e s s of a t m o s p h e r e c o m p a r e d to m e d i a w i t h o u t b l o o d ( P < 0.05). It was i m p o r t a n t to m a i n t a i n a h u m i d a t m o s p h e r e in the i n c u b a t o r since n o growth o n solid m e d i a was o b s e r v e d in a dry i n c u b a t o r ( d a t a n o t shown). A h u m i d e n v i r o n m e n t a l o n e , however, with n o r m a l a t m o s p h e r i c levels of 0 2 a n d CO2, was n o t a b l e to s u p p o r t growth of the strains ( d a t a n o t shown). It is possible t h a t the p r e s e n c e of m o i s t u r e e n h a n c e d gas e x c h a n g e b e t w e e n the c a m p y l o b a c t e r cells a n d t h e i r e n v i r o n m e n t .

381 When comparing the growth of the 18 reference strains in liquid media (Mueller Hinton Broth or Brucella Broth with or without sheep blood), the growth rates or doubling times of the 18 strains were approximately 18 h in the broths without blood and approximately 120 rain in the broths with blood in both atmospheres (data not shown). Strains could be kept up to 4 weeks without subculture on a single MHAB plate in 10% CO2 in moist air at 37°C and still produce good growth on subculture in terms of colony number and size (results not included). In addition, the serotype of each strain was unaffected b~' growth in 10% CO 2 in moist air and strains which had been subcuitured more than 20 times in 10% CO, in moist air had the same serotvpe as strains subcultured similarly in the microaerobic atmosphere (unpublished data). The electrophoretic protein profiles (SDS-PAGE) of whole cell preparations of the 18 strains were also unaffected by growth in 10% CO, in moist air and they were similar for both atmosplleres (unpublished results). Use of the 10% CO 2 in moist air atmosphere greatly reduced the cost and difficulty of culturing these campylobacters. When the CO 2 level was adjusted to between 1 and 2%, the 18 strains were able to grow to the same extent as they had at a level of 10% CO 2 (data not shown). The presence of CO 2 may reduce the level of superoxide radicals and may, therefore, serve to enhance the aerotolerance of these strains (BoRon and Coates, 1983; BoRon et al., 1984; Griffiths and Park, 1990; Skirrow et al., 1982). However, since the level of O 2 used in the present study was greater than 18% this would suggest that campyiobacter strains are not truly microaerophilic. Campylobacters would more appropriately be termed capnophilic organisms. The 10% CO 2 in moist air atmosphere has been successfully used to isolate Campylobacter spp. from field specimens.

Acknowledgements We would like to thank Dawn Martin, Deborah McCabe, Ed Riche and Ruth Robertson for excellent technical assistance. We would also like to acknowledge the contributions of Robert Cormack who initiated this work as part of his Fourth Year Honours Thesis at Carleton University, Ottawa, Ontario, Canada and Gordon Furzer, Jenny Ho, Elli Nakamichi, Tracy Mills and Ranu Sharma of Confederation High School, Nepean, Ontario, Canada who worked in our laboratory as part of the Carleton Board of Education Co-operative Education Program. Thanks are also due to H. Lior, Chief of the National Reference Center for Campylobacters, L.C.D.C., Ottawa, Ontario, Canada for providing the strains used in this study and for providing the serotyping and biotyping services during the course of our investigations.

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382 Bolton. F.J., Coates. D. and Hutchinson, D.N. (1984) The ability of campylobacter media supplements to neutralize photochemically induced toxicity and hydrogen peroxide. J. Applied Bacteriol. 56. 151-157. Brtn~ks, B.W., Garcia M.M.. Fraser. A.D,E.. Lion', It., Stewart, R.B. and Lammerding. A.M. (1986) l~flation and characterization of cephalothin-susceptible Campyh,lnwu'r colt from slaughtered cattle. J. Clin. Microbiol. 24, 591-595. Butzler, J.P. and Skirrow, M.P. (1979) CamFffh~bacter enteritis. Clin. Gastroenterol. 8, 737-765. Fraser. A.D.E., Cormack. R.S.. Yamazaki, H.. Brooks. B.W. and Garcia. M.M. (1989) Are C. jejuni and C: colt capnophilic aerobes? The Vth International Workshop on Campylobacter Infections. Programs and Abstracts. J3 p. 84. Puerto Vallarta. Mexico. Griffiths, P.L. and Park. R.W.A, (1990) Campylobacters associated with bacterial disease. J. Applied Bacteriol. 69. 281-301. Korolik, V., Coloe, P.J. and Krishnapillai. V. (1988) A specific DNA probe for the identification of Campylobacter jejuni. J. Gen. Microbiol. 134, 521-529. Luechtefeld, N.W., Relier, L.B., Blaser. M.J. and Wang, W-L.L. (1982) Comparison of atmospheres of incubation for primary isolation of Campylobacter fi, tus subsp, jt,juni from animal specimens: 5q~ oxygen versus candle jar. J. Clin. MicrobioL 15, 53-57. Lior. H. (1984) New exten0ed biotyping scheme of C~mzpylohacter jt'jzozL Ca,.nl~ylobacter colt and "Campylobaeter laridis'. J. Clin. Microbiol. 20, 636-640. Lior. H., Wt~dward. D.L., Edgar. J.A., Laroche. L.J. and Gill, P. (1982) Serotyping of Campylobacter jt'juni by slide agglutination based on heat labile antigenic factors. J. Clin. Microbiol. 15, 761-768. Rashtchian. A. (1985) Detection of Campyh~baclt,r species in clinical specimens using nucleic acid probes. In" Pearson. A.D., Skirrow. M.B.. Lior, H. and Rowe. B. (Eds.), Campylobacter Ill. P.H.LS.. London, po. 71-73. Skirrow, M.B., Benjamin. J., Razi, M.tt.tt. and Waterman, S. (1982) Isolalion, cultivation and identification of Campyh,bacwrjcjuni and C. coll. In: Corry, J.E.L., Roberts, D. and Skinner, F.A. (Eds,). Isolation and Identification Methods for Food Poisoning Organisms, Academic Press, New York, pp. 313-328. Stanek, G., ltirschl, A. and Roller, M. (1983) ELISA in the serological diagnosis of campylobacter infections. In: Pearson, A.D., Skirrow, M.B., Rowe, B., Davies, J.R, and Jones, D.M. (Eds.), Campylobacter II, P.H.L.S., London, p. 73. Tompkins, L.S., Mickelsen, P. and McClure, J. (1983) Use of a DNA probe to detect Campylobacter jejuni in fecal specimens. In: Pearson, A.D., Skirrow. M.B.. Rowe, B., Davies, J.R. and Jones, D.M. (Eds.), Campylobacter II, P.H.L.S., London, pp. 50-51.

Simple and economical culture of Campylobacter jejuni and Campylobacter coli in CO2 in moist air.

Strains of Campylobacter jejuni and C. coli representing the 18 serogroups (Lior) most commonly isolated from humans in Canada were grown on solid med...
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