ZentralbI. MikrobioI. 147 (1992), 41-44 Gustav Fischer Verlag lena [Institut fur Wasser-, Boden- und Lufthygiene des Bundesgesundheitsamtes, Forschungsstelle Bad Elster, Deutschland]
Comparison of two Media for the Isolation of Thermophilic Campylobacters from Waste Waters of Different Quality J. JACOB and W. STELZER Key words: Waste water, fecal pollution, Campylobacter
Summary Counts of thermophilic campylobacters from 31 different waste water samples were parallel estimated with two different cultivation media. The resulting increased isolation sale obtained with an modified CharcoalCefoperazone-Deoxycholat-Medium (MCCD-Medium), was statistically significant. An 15.8 fold increased isolation rate, compared with the standard medium, could be estimated with the help of the geometric mean.
Zusammenfassung Die Gehaite an thermophilen Campylobacter-Bakterien wurden aus 31 Abwasserproben mit Hilfe zweier unterschiedlicher Anzuchtverfahren bestimmt. Die mittels eines modifizierten Aktivkohle-Ccfopcrazon-Deoxycholat-Medium (MCCD-Medium) erhohte Campylobacter-Isolationsrate erwies sich als statistisch signifikant. Eine 15,8fach erhohte Nachweishaufigkeit, verglichen mit dem Standardmedium, konnte mit Hilfe des geometrischen Mitteis festgestellt werden.
Introduction The isolation of thermophilic campylobacters from a variety of waters (waste-, surface waters) has become an important tool in water microbiology with respect to the hygienic state of these waters (3, 10, 23). Various media are introduced in this works, they differed in their composition, for instance in several antibiotics and concentration of antibiotics. Outcoming from Prestonmedium (7), developed for the isolation of campylobacters from faeces, a further modification of this medium was used for studies on river and surface waters, described by BOLTON et al. (3) and RIBEIRO et al. (21). This medium showed a low selectivity for the isolation of campylobacters from waste water, described in one of our previous studies (23). Therefore, STELZER et al. (23) successfully introduced a new medium that contained also Vancomycin according to SKIRROW (22) and cephalothin similar to GILCHRIST et al. (9). The resulting advantage of a more selectivity was in contrast to the known susceptibilities of some campylobacter strains to various antibiotics, for instance against rifampicin (14), cephalothin (8), Polymyxin-B (11, 19). It seems possible, that Campylobacter strains susceptible to these antibiotics, can't be recovered with this medium. New inspirations for better campylobacter isolation media are given by BOLTON et al. (5) and GOOSENS et al. (12). With the introduction of cefoperazone as a selective agent by GOOSENS et al. (11), GUN-MUNRO et al. (13), and KARMALI et al. (15) resulted an increased recovery of campylobacters from faeces (6). The new MCCD-medium introduced seems to be useful for the isolation of thermophilic campylobacters from waste water, and to compare these results with those of earlier works (23). With
42
J . JACOB and W. STELZER
respect to the qualitative results of K ORH ONE N et al. (16) and M ARTI KAINEN et al. (17), only quantitative investigations are useful to estimate the eampylobaeter contamination of the environment and their hygienic significance.
Material and methods The sampling points at a chicken slaughterhouse were A) washing after evisceration and removal of lungs and B) effluent chilling tank. Waste water samples were taken from a sewage plant described by STELZERet al. (23) at the following sampling points : C) sewage plant effluent and D) raw sewage .
Isolation and characterization of campylobacters For the isolation of campylobacte rs, a modification of the enrichmen t method described by STELZER et al. (23) was used. The inoculation of the selective agar plate was done with the support of membrane filters . The filters (pore size = 0 ,45 urn, SARTORlUS) were placed on the surface of the selective agar plate. After them, the centrifugated enrichment broth (t = 10 min, 7,000 rpm) was subjected to the membrane filters and during two hours the campylobacters were migrati ng through the filter onto the surface of the agar. Finally the filters were removed and the agar plates were incubated 48 hours at 42 °C with the procedure for generati ng the microaeroph ilic milieu , described by PENNIE et al. (20). In this study wc compare d two different isolation media. The standard-medium was the same, as described by STELZER et al. (23). The MCCD-medium was modified prepared after KARMALI et al. (15). Basal components for this medium were per I: activated charcoal (4 g), nutrient broth No. I (SIFlN ), 25 g, 12 g Agar-Noble (DIFCO), 3 g proteose-peptone No.3 (DIFCO), bovine gale (I g), ferrous sulfate (0. 25 g), sodium pyruvat (0.25 g). The p I! was adjusted to 7.4 . As selective agents we used cefoperazone (32 ug/ml), Vancomycin (20 ug/rnl), Tr imethoprim (30 ug/rnl), Amphoteri cin B (2 Ilg/m1). The enrichme nt broth has the same composition, but without agar componen ts. The campylobacter count was estimated using an MPN-table.
Results We performed a T-Test with the logarithmic campylobaet er counts of 3 1 parallel investiga ted waste water samples (data not all listed in Tab. 2). The test indicates, that the increase of eampylobaeter recovery with the MCCD-medium was statistically significant (P < 0.005) . With the help of the geometric means (x g standard medium = 39.8 MPNII OOml, x g MCCD-medium = 63 1 MPNIIOO ml) we calculated an 15.8 fold higher recovery of thermophilic eampylobacters with the MCCD-med ium , compared with the standard-medium. With the use of the MCCD-medium now we estimated a drastica lly increase of campylobacter counts in raw sewage (A) and sewage plant effluent (B). As a result of these studies, we must consider earlier results (23) as strong underesti mating in view of the campylobaeter amount and in release of campylobaeters into the environment by sewage treatment processes. The estimated eampy/obaeter contamination of the chicken slaughterhouse is approx imately the same published by AHO et al. (I ), in Finland . But in contrast to this, in the chicken processing line we investigated in this study, exists no final chlorinated chilling tank. With respect to this fact we assu me, that the campylobacter contamination of the chicken carcasses must be very higher in the slaughtery we investigated in this study, in contrast to chicken slaughterhouses described elsewhere (l) .
Discussion The significant higher quality of the modified charcoal cefoperazone deoxycholate medium (MCCD) was of importance for the compar ison with earlier results, because it seems now clear that the campylo bacter counts from environmental sources were underestimated in some previous works (l, 2, 3 , 23) .
Comparison of two Media for the Isolation
43
Table I. Chemical pollution (arithmetic mean values, numbers of collected samples in parenthesis). Tabelle I. Chemische Belastung (arithmetische Mittel, Anzahl der gesammeiten Proben in Klammern). A COD (Mn) mg OzlI pH
NH4 + mg/l P04 3 - mglI suspended solids mg/I
158.7 7.0 2.6 14.7
B (4) (4) (4) (4)
19.8 7.0 1.6 5.7
C (5) (4) (5) (5)
2I.I 7.1 10. I 9.8
49.2 (5)
365.0 (4)
D (5) (5) (5) (5)
see ref. (24)
64.4 (5)
Table 2. Bacteriological pollution of different waste waters (Numbers of collected samples in parenthesis). Tabelle 2. Bakteriologische Belastung von verschiedenen Abwassern (Anzahl der gesammelten Proben in Klammern). Sampling site
Colony count/rnl') (20°C) Total cotiformsttnv; Thermophilic campylobacter ') MPN/100 ml Thermophilic campylobacter-t MPNflOO ml Range of campylobacter's MPNflOOml Range of campyiobacter'i MPNflOOml
A
B
C
D
5.6' 107 (9) 53.6 . 104 (8) 25.9· 103 (5)
2. I . 107 (8) 25. I . 104 (9) 5.0' 10' (6)
0.3 . 107 (9) 2.3 . 104 (12) 1.2 . IOz (8)
14.2 . 107 (8) 34. I . 104 (I I) 7.6' 102 (7)
56.2 . 103 (5)
2.4 . 103 (6)
I.7 . 103 (8)
6.9' 103 (7)
I.5 ·10' - 1.1 . 105 0.3 - 2.3 . 102 o - 9.3 . 102 (5) 2.1 . 101 (5)
(6)
-
2.4 . 105
o(6)
I.1 . 104
(8) 0-I.I·104 (8)
0.3 - 4.6 . 103 (7) 0- 2.4 . 104 (7)
I) Arithmetic mean, Standard-Medium; 2) Arithmetic mean, MCCD-Medium.
This is evident, because in this study we estimated significant higher campylobacter counts in direct comparison of the two media. The results we obtained with this study show first quantitative results about thermophilic campylobacters from waste waters, obtained with the MCCD-medium. The waste water sampling points we selected for this study are of hygienical importance, for instance, chilling tanks are described as possible cross-contamination sources for broiler carcasses by AHO et al. (1). In connection to the now observed campylobacter spread from the sewage treatment plant into the environment we need further studies about surface and river waters, performed with the MCCD-medium, because the occurrence of campylobacters in the environment may be greater that we estimated some works ago (23). Further results (STELZER et al., in preparation), show, that the greatest part of the earlier in campylobacter's recovery negative sampling points are now campylobacter positive ones, when we use the MCCDmedium.
References I. AHO, M., and J. HIRN: Prevalence of Campylobacter in the Finnish Broiler Chicken Chain from the Producer to the Consumer. Acta vet. scand. 29 (1988) 45 I -462.
44
J. JACOB and W. STELZER
2. ARIMI, S. M., C. R. FRICKER and R. W. A. PARK: Occurrence of thermophilic campylobacters in sewage and their removal by treatment processes. Epidem. Inf. 101 (1988) 279- 286. 3. BOLTON, F. J., D. COATES, D. N. HUTCHINSON and A. F. GODFREE. A study of thermophilic campylobacters in a river system. J. app!. Bact. 62 (1987) 167-176. 4. BOLTON, F. J., HOLT and D. N. HUTCHINSON: Campylobacter biotyping scheme of epidemiologic value. J. Clin. Pathol. 37 (1984) 677-681. 5. BOLTON, F. J., D. N. HUTCHINSON and D. COATES: Blood-free selective medium for isolation of Campylobacter jejuni from faeces. J. Clin. Microbiol. 19 (1984) 169-171. 6. BOLTON, F. J., D. N. HUTCHINSON and G. PARKER: Deassesment of Selective Agars and Filtration Techniques for Isolation of Campylobacter Species from faeces. Eur. J. Clin. Microbio!. Infect. Dis. 7
(1988) 155-160. 7. BOLTON, F. J., and L. ROBERTSON: A selective medium for isolating Campylobacter jejuni/coli. J. Clin. Pathol. 35 (1982) 462-467. 8. BROOKS, B. W., M. M. GARCIA, A. D. E. FRASER, H. LIOR, R. B. STEWART, A. M. LAMMERDlNG: Isolation and Characterization of Cephalothin - Susceptible Campylobacter coli from Slaughter Cattle. J. Clin. Microbiol. 24 (1986) 591-595. 9. GILCHRIST, M. J. R., C. M. GREWELL and J. A. WASHINGTON: Evaluation of media for Isolation of Campylobacter fetus subsp. jejuni from Fecal Specimens. J. Clin. Microbiol. 14 (1981) 393-395. 10. GONDROSEN, B.: Survival of thermotolerant campylobacters in water. Acta vet. scand. 27 (1986) 1-10. II. GOOSENS, H., M. DE BOECK, H. COIGNAN, L. KLAES, C. VAN DEN BORRE and J. P. BUTZLER: Modified Selective Medium for Isolation of Campylobacter spp. from Feces: Comparison with Preston Medium, a Blood-Free Medium, and a Filtration System. J. Clin. Microbiol. 24 (1986) 840-843. 12. GOOSENS, H., L. KLAES, J. GALAND, C. VAN DEN BORRE and J. O. BUTZLER: Semisolid Blood-Free Selective-Motility Medium for the Isolation of Campylobacter from Stool Specimens. J. Clin. Microbiol.
27 (1989) 1077-1080. 13. GUN-MUNRO, J., R. P. RENNIE, J. H. THORNLEY, H. L. RICHARDSON, D. HODGE and J. LYNCH: Laboratory and Clinical Evaluation of Isolation Media for Campylobacter jejuni. J. Clin. Microbiol. 25
(1987) 2274-2277. 14. Humphrey, T. J.: The synergistic inhibition of Campylobacter jejuni by rifampicin and hydrogen peroxide. Lett. Appl. Microbiol. 10 (1990) 97-100. 15. KERMALI, M. A., A. E. SIMOR, M. ROSCOE, P. C. FLEMING, S. S. SMITH, J. LANE: Evaluation of a BloodFree, Charcoal-Based, Selective Medium for the Isolation of Campylobacter Organisms from Feces. J. Clin. Microbiol. 23 (1986) 456-459. 16. KORHONEN, L. K. and P. J. MARTIKAINEN: Comparison of some enrichment broths and growth media for the isolation of thermophilic campylobacters from surface water samples. J. Appl. Baeteriol. 68 (1990)
593-599. 17. MARTIKAINEN, P. J., L. K. KORHONEN and T. U. KOSUNEN: Occurrence of thermophilic campylobacters in rural and urban surface waters in central Finland. Water Res. 24 (1990) 91-96. 18. MAWER, S. L.: Campylobacters in man and the environment in Hull and East Yorkshire. Epidem. Inf. 101 (1988) 287-294. 19. NG, L. K., D. E. TAYLOR and M. E. SciLES: Characterization of Freshly Isolated Campylobacter coli Strains and Suitability of Selective Media for Their Growth. J. Clin. Microbio!. 26 (1988) 518-523. 20. PENNIE, R. A., J. N. ZURINO, C. E. ROSE and R. L. GUERRANT: Economical simple method for production of the gaseous environment required for cultivation of Campylobacter jejuni. J. Clin. Microbiol. 20 (1984) 320-322. 21. RIBEIRO, C. D., and T. H. PRICE: The use of Preston enrichment broth for the isolation of "thermophilic" campylobacters from water. J. Hyg. 92 (1984) 45-51. 22. SKIRROW, M. B.: Campylobacter enteritis: a 'new' disease. Brit. Med. J. (1977) 9-1 J. 23. STELZER, W., H.-P. MOCHMANN, U. RICHTER and H.-J. DOBBERKAU: Characterization of Campylobacter jejuni and Campylobacter coli Isolated from Waste Water. Zbl. Bakt. Hyg. A 269 (1988) 188-196. Authors address: Dipf-Biol. JENS JACOB, Institut fur Wasser-, Boden und Lufthygiene des BGA HeinrichHeine-SIr. 12,0-9933 Bad Elster, Deutschland.
Comparison of two Media for the Isolation of Thermophilic Campylobacters from Waste Waters of Different Quality J. JACOB and W. STELZER Key words: Waste water, fecal pollution, Campylobacter
Summary Counts of thermophilic campylobacters from 31 different waste water samples were parallel estimated with two different cultivation media. The resulting increased isolation sale obtained with an modified CharcoalCefoperazone-Deoxycholat-Medium (MCCD-Medium), was statistically significant. An 15.8 fold increased isolation rate, compared with the standard medium, could be estimated with the help of the geometric mean.
Zusammenfassung Die Gehaite an thermophilen Campylobacter-Bakterien wurden aus 31 Abwasserproben mit Hilfe zweier unterschiedlicher Anzuchtverfahren bestimmt. Die mittels eines modifizierten Aktivkohle-Ccfopcrazon-Deoxycholat-Medium (MCCD-Medium) erhohte Campylobacter-Isolationsrate erwies sich als statistisch signifikant. Eine 15,8fach erhohte Nachweishaufigkeit, verglichen mit dem Standardmedium, konnte mit Hilfe des geometrischen Mitteis festgestellt werden.
Introduction The isolation of thermophilic campylobacters from a variety of waters (waste-, surface waters) has become an important tool in water microbiology with respect to the hygienic state of these waters (3, 10, 23). Various media are introduced in this works, they differed in their composition, for instance in several antibiotics and concentration of antibiotics. Outcoming from Prestonmedium (7), developed for the isolation of campylobacters from faeces, a further modification of this medium was used for studies on river and surface waters, described by BOLTON et al. (3) and RIBEIRO et al. (21). This medium showed a low selectivity for the isolation of campylobacters from waste water, described in one of our previous studies (23). Therefore, STELZER et al. (23) successfully introduced a new medium that contained also Vancomycin according to SKIRROW (22) and cephalothin similar to GILCHRIST et al. (9). The resulting advantage of a more selectivity was in contrast to the known susceptibilities of some campylobacter strains to various antibiotics, for instance against rifampicin (14), cephalothin (8), Polymyxin-B (11, 19). It seems possible, that Campylobacter strains susceptible to these antibiotics, can't be recovered with this medium. New inspirations for better campylobacter isolation media are given by BOLTON et al. (5) and GOOSENS et al. (12). With the introduction of cefoperazone as a selective agent by GOOSENS et al. (11), GUN-MUNRO et al. (13), and KARMALI et al. (15) resulted an increased recovery of campylobacters from faeces (6). The new MCCD-medium introduced seems to be useful for the isolation of thermophilic campylobacters from waste water, and to compare these results with those of earlier works (23). With
42
J . JACOB and W. STELZER
respect to the qualitative results of K ORH ONE N et al. (16) and M ARTI KAINEN et al. (17), only quantitative investigations are useful to estimate the eampylobaeter contamination of the environment and their hygienic significance.
Material and methods The sampling points at a chicken slaughterhouse were A) washing after evisceration and removal of lungs and B) effluent chilling tank. Waste water samples were taken from a sewage plant described by STELZERet al. (23) at the following sampling points : C) sewage plant effluent and D) raw sewage .
Isolation and characterization of campylobacters For the isolation of campylobacte rs, a modification of the enrichmen t method described by STELZER et al. (23) was used. The inoculation of the selective agar plate was done with the support of membrane filters . The filters (pore size = 0 ,45 urn, SARTORlUS) were placed on the surface of the selective agar plate. After them, the centrifugated enrichment broth (t = 10 min, 7,000 rpm) was subjected to the membrane filters and during two hours the campylobacters were migrati ng through the filter onto the surface of the agar. Finally the filters were removed and the agar plates were incubated 48 hours at 42 °C with the procedure for generati ng the microaeroph ilic milieu , described by PENNIE et al. (20). In this study wc compare d two different isolation media. The standard-medium was the same, as described by STELZER et al. (23). The MCCD-medium was modified prepared after KARMALI et al. (15). Basal components for this medium were per I: activated charcoal (4 g), nutrient broth No. I (SIFlN ), 25 g, 12 g Agar-Noble (DIFCO), 3 g proteose-peptone No.3 (DIFCO), bovine gale (I g), ferrous sulfate (0. 25 g), sodium pyruvat (0.25 g). The p I! was adjusted to 7.4 . As selective agents we used cefoperazone (32 ug/ml), Vancomycin (20 ug/rnl), Tr imethoprim (30 ug/rnl), Amphoteri cin B (2 Ilg/m1). The enrichme nt broth has the same composition, but without agar componen ts. The campylobacter count was estimated using an MPN-table.
Results We performed a T-Test with the logarithmic campylobaet er counts of 3 1 parallel investiga ted waste water samples (data not all listed in Tab. 2). The test indicates, that the increase of eampylobaeter recovery with the MCCD-medium was statistically significant (P < 0.005) . With the help of the geometric means (x g standard medium = 39.8 MPNII OOml, x g MCCD-medium = 63 1 MPNIIOO ml) we calculated an 15.8 fold higher recovery of thermophilic eampylobacters with the MCCD-med ium , compared with the standard-medium. With the use of the MCCD-medium now we estimated a drastica lly increase of campylobacter counts in raw sewage (A) and sewage plant effluent (B). As a result of these studies, we must consider earlier results (23) as strong underesti mating in view of the campylobaeter amount and in release of campylobaeters into the environment by sewage treatment processes. The estimated eampy/obaeter contamination of the chicken slaughterhouse is approx imately the same published by AHO et al. (I ), in Finland . But in contrast to this, in the chicken processing line we investigated in this study, exists no final chlorinated chilling tank. With respect to this fact we assu me, that the campylobacter contamination of the chicken carcasses must be very higher in the slaughtery we investigated in this study, in contrast to chicken slaughterhouses described elsewhere (l) .
Discussion The significant higher quality of the modified charcoal cefoperazone deoxycholate medium (MCCD) was of importance for the compar ison with earlier results, because it seems now clear that the campylo bacter counts from environmental sources were underestimated in some previous works (l, 2, 3 , 23) .
Comparison of two Media for the Isolation
43
Table I. Chemical pollution (arithmetic mean values, numbers of collected samples in parenthesis). Tabelle I. Chemische Belastung (arithmetische Mittel, Anzahl der gesammeiten Proben in Klammern). A COD (Mn) mg OzlI pH
NH4 + mg/l P04 3 - mglI suspended solids mg/I
158.7 7.0 2.6 14.7
B (4) (4) (4) (4)
19.8 7.0 1.6 5.7
C (5) (4) (5) (5)
2I.I 7.1 10. I 9.8
49.2 (5)
365.0 (4)
D (5) (5) (5) (5)
see ref. (24)
64.4 (5)
Table 2. Bacteriological pollution of different waste waters (Numbers of collected samples in parenthesis). Tabelle 2. Bakteriologische Belastung von verschiedenen Abwassern (Anzahl der gesammelten Proben in Klammern). Sampling site
Colony count/rnl') (20°C) Total cotiformsttnv; Thermophilic campylobacter ') MPN/100 ml Thermophilic campylobacter-t MPNflOO ml Range of campylobacter's MPNflOOml Range of campyiobacter'i MPNflOOml
A
B
C
D
5.6' 107 (9) 53.6 . 104 (8) 25.9· 103 (5)
2. I . 107 (8) 25. I . 104 (9) 5.0' 10' (6)
0.3 . 107 (9) 2.3 . 104 (12) 1.2 . IOz (8)
14.2 . 107 (8) 34. I . 104 (I I) 7.6' 102 (7)
56.2 . 103 (5)
2.4 . 103 (6)
I.7 . 103 (8)
6.9' 103 (7)
I.5 ·10' - 1.1 . 105 0.3 - 2.3 . 102 o - 9.3 . 102 (5) 2.1 . 101 (5)
(6)
-
2.4 . 105
o(6)
I.1 . 104
(8) 0-I.I·104 (8)
0.3 - 4.6 . 103 (7) 0- 2.4 . 104 (7)
I) Arithmetic mean, Standard-Medium; 2) Arithmetic mean, MCCD-Medium.
This is evident, because in this study we estimated significant higher campylobacter counts in direct comparison of the two media. The results we obtained with this study show first quantitative results about thermophilic campylobacters from waste waters, obtained with the MCCD-medium. The waste water sampling points we selected for this study are of hygienical importance, for instance, chilling tanks are described as possible cross-contamination sources for broiler carcasses by AHO et al. (1). In connection to the now observed campylobacter spread from the sewage treatment plant into the environment we need further studies about surface and river waters, performed with the MCCD-medium, because the occurrence of campylobacters in the environment may be greater that we estimated some works ago (23). Further results (STELZER et al., in preparation), show, that the greatest part of the earlier in campylobacter's recovery negative sampling points are now campylobacter positive ones, when we use the MCCDmedium.
References I. AHO, M., and J. HIRN: Prevalence of Campylobacter in the Finnish Broiler Chicken Chain from the Producer to the Consumer. Acta vet. scand. 29 (1988) 45 I -462.
44
J. JACOB and W. STELZER
2. ARIMI, S. M., C. R. FRICKER and R. W. A. PARK: Occurrence of thermophilic campylobacters in sewage and their removal by treatment processes. Epidem. Inf. 101 (1988) 279- 286. 3. BOLTON, F. J., D. COATES, D. N. HUTCHINSON and A. F. GODFREE. A study of thermophilic campylobacters in a river system. J. app!. Bact. 62 (1987) 167-176. 4. BOLTON, F. J., HOLT and D. N. HUTCHINSON: Campylobacter biotyping scheme of epidemiologic value. J. Clin. Pathol. 37 (1984) 677-681. 5. BOLTON, F. J., D. N. HUTCHINSON and D. COATES: Blood-free selective medium for isolation of Campylobacter jejuni from faeces. J. Clin. Microbiol. 19 (1984) 169-171. 6. BOLTON, F. J., D. N. HUTCHINSON and G. PARKER: Deassesment of Selective Agars and Filtration Techniques for Isolation of Campylobacter Species from faeces. Eur. J. Clin. Microbio!. Infect. Dis. 7
(1988) 155-160. 7. BOLTON, F. J., and L. ROBERTSON: A selective medium for isolating Campylobacter jejuni/coli. J. Clin. Pathol. 35 (1982) 462-467. 8. BROOKS, B. W., M. M. GARCIA, A. D. E. FRASER, H. LIOR, R. B. STEWART, A. M. LAMMERDlNG: Isolation and Characterization of Cephalothin - Susceptible Campylobacter coli from Slaughter Cattle. J. Clin. Microbiol. 24 (1986) 591-595. 9. GILCHRIST, M. J. R., C. M. GREWELL and J. A. WASHINGTON: Evaluation of media for Isolation of Campylobacter fetus subsp. jejuni from Fecal Specimens. J. Clin. Microbiol. 14 (1981) 393-395. 10. GONDROSEN, B.: Survival of thermotolerant campylobacters in water. Acta vet. scand. 27 (1986) 1-10. II. GOOSENS, H., M. DE BOECK, H. COIGNAN, L. KLAES, C. VAN DEN BORRE and J. P. BUTZLER: Modified Selective Medium for Isolation of Campylobacter spp. from Feces: Comparison with Preston Medium, a Blood-Free Medium, and a Filtration System. J. Clin. Microbiol. 24 (1986) 840-843. 12. GOOSENS, H., L. KLAES, J. GALAND, C. VAN DEN BORRE and J. O. BUTZLER: Semisolid Blood-Free Selective-Motility Medium for the Isolation of Campylobacter from Stool Specimens. J. Clin. Microbiol.
27 (1989) 1077-1080. 13. GUN-MUNRO, J., R. P. RENNIE, J. H. THORNLEY, H. L. RICHARDSON, D. HODGE and J. LYNCH: Laboratory and Clinical Evaluation of Isolation Media for Campylobacter jejuni. J. Clin. Microbiol. 25
(1987) 2274-2277. 14. Humphrey, T. J.: The synergistic inhibition of Campylobacter jejuni by rifampicin and hydrogen peroxide. Lett. Appl. Microbiol. 10 (1990) 97-100. 15. KERMALI, M. A., A. E. SIMOR, M. ROSCOE, P. C. FLEMING, S. S. SMITH, J. LANE: Evaluation of a BloodFree, Charcoal-Based, Selective Medium for the Isolation of Campylobacter Organisms from Feces. J. Clin. Microbiol. 23 (1986) 456-459. 16. KORHONEN, L. K. and P. J. MARTIKAINEN: Comparison of some enrichment broths and growth media for the isolation of thermophilic campylobacters from surface water samples. J. Appl. Baeteriol. 68 (1990)
593-599. 17. MARTIKAINEN, P. J., L. K. KORHONEN and T. U. KOSUNEN: Occurrence of thermophilic campylobacters in rural and urban surface waters in central Finland. Water Res. 24 (1990) 91-96. 18. MAWER, S. L.: Campylobacters in man and the environment in Hull and East Yorkshire. Epidem. Inf. 101 (1988) 287-294. 19. NG, L. K., D. E. TAYLOR and M. E. SciLES: Characterization of Freshly Isolated Campylobacter coli Strains and Suitability of Selective Media for Their Growth. J. Clin. Microbio!. 26 (1988) 518-523. 20. PENNIE, R. A., J. N. ZURINO, C. E. ROSE and R. L. GUERRANT: Economical simple method for production of the gaseous environment required for cultivation of Campylobacter jejuni. J. Clin. Microbiol. 20 (1984) 320-322. 21. RIBEIRO, C. D., and T. H. PRICE: The use of Preston enrichment broth for the isolation of "thermophilic" campylobacters from water. J. Hyg. 92 (1984) 45-51. 22. SKIRROW, M. B.: Campylobacter enteritis: a 'new' disease. Brit. Med. J. (1977) 9-1 J. 23. STELZER, W., H.-P. MOCHMANN, U. RICHTER and H.-J. DOBBERKAU: Characterization of Campylobacter jejuni and Campylobacter coli Isolated from Waste Water. Zbl. Bakt. Hyg. A 269 (1988) 188-196. Authors address: Dipf-Biol. JENS JACOB, Institut fur Wasser-, Boden und Lufthygiene des BGA HeinrichHeine-SIr. 12,0-9933 Bad Elster, Deutschland.
