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CAMPYLOBACTER BLOOD-FREE SELECTIVE MEDIUM (MODIFIED CCDA- PRESTON)

A medium, which when prepared from Campylobacter Blood-Free Selective Agar Base CM0739 and CCDA Selective Supplement SR0155, can be used for the isolation of Campylobacter jejuni, Campylobacter coli and Campylobacter laridis.

CAMPYLOBACTER BLOOD-FREE SELECTIVE AGAR BASE

Code: CM0739

Typical Formula*

gm/litre

Nutrient Broth No.2

25.0

Bacteriological charcoal

4.0

Casein hydrolysate

3.0

Sodium desoxycholate

1.0

Ferrous sulphate

0.25

Sodium pyruvate

0.25

Agar

12.0

pH 7.4 ± 0.2 @ 25°C

* Adjusted as required to meet performance standards

CCDA SELECTIVE SUPPLEMENT

Code: SR0155
An improved selective supplement for Campylobacter Blood-Free Selective Agar Base.

Vial contents (each vial is sufficient for 500ml of medium)

per vial
per litre

Cefoperazone

16mg

32mg

Amphotericin B

5mg

10mg

Directions
Suspend 22.75g of Campylobacter Blood-Free Selective Agar Base in 500ml of distilled water and bring to the boil to dissolve. Sterilise by autoclaving at 121°C for 15 minutes. Cool to 50°C. Aseptically add 1 vial of CCDA Selective Supplement SR0155 reconstituted as directed. Mix well and pour into sterile Petri dishes.

Description
Modified CCDA Medium is based on the original formulation described by Bolton et al.1 which was developed to replace blood with charcoal, ferrous sulphate and sodium pyruvate. Improved selectivity was achieved when cephazolin in the original formulation was replaced by cefoperazone as the selective agent 2. More recent work has shown an increased isolation rate can be achieved if the plates are incubated at 37°C rather than 42°C3.

Amphotericin B has been added to the formula to suppress the growth of yeast and fungal contaminants that may occur at 37°C.

Modified CCDA medium and Campy-BAP medium were equal in performance in a rapid colony-lift procedure for detection of thermophilic campylobacters in which membranes are blotted on agar cultures and then subjected to immunoassay 5.

In a study of healthy puppies and kittens for carriage of Campylobacter species6, modified CCDA medium was found to be a suitable medium and more productive for Campylobacter upsaliensis in this application than CAT medium. Modified CCDA medium has been confirmed as suitable for isolation of Campylobacter spp. from non-clinical samples following enrichment in Exeter broth7.

The use of Campylobacter Blood-Free Medium is specified by the U.K. Ministry of Agriculture, Fisheries and Food (MAFF) in a validated method for isolation of Campylobacter from foods4.

Technique
1. Prepare Campylobacter Blood-Free Selective Agar as described in the directions.
2. Emulsify approximately 0.5g of the specimen in 5ml of sterile 0.1% peptone water to form an approximate 1:10 dilution.
3. Inoculate onto the selective medium with cotton tipped swabs so that single isolated colonies are formed.
4. Incubate the plates in an atmosphere consisting of approximately 5-6% oxygen, 10% carbon dioxide and 84-85% nitrogen for 48 hours at 37°C.

This can best be achieved by using the Oxoid Gas Generating Kit for campylobacter (BR0056) in conjunction with the Oxoid Anaerobic Jar (HP0011) and an active catalyst (BR0042). For jars of smaller capacity AnaeroJar(2.5 litres) use the Oxoid Gas Generating Kit for Campylobacters (BR0060). Alternatively use CampyGen CN0025 or CN0035 which does not require the use of a catalyst or the addition of water.

The colonial morphology of campylobacters can be used as a guideline for identification to species level. Campylobacter jejuni strains produce grey, moist flat spreading colonies. Some strains may have a green hue or a dry appearance, with or without a metallic sheen. Campylobacter coli strains tend to be creamy-grey in colour, moist, slightly raised and often produce discrete colonies.

Colonies tend to swarm when initially isolated from clinical specimens.

Storage conditions and Shelf life
Store the dehydrated medium at 10-30°C and use before the expiry date on the label.
Store the selective supplement in the dark at 2-8°C and use before the expiry date on the label.
The prepared medium may be stored for up to 2 weeks at 2-8°C .

Appearance
Dehydrated medium: Black coloured, free-flowing powder
Prepared medium: Black coloured gel

Quality control.
Incubation at 37°C for 48 hours.

Positive control:

Expected results

Campylobacter jejuni ATCC® 33291 *

Good growth; grey coloured colonies

Negative control:

 

Escherichia coli ATCC® 25922 *

No growth

* This organism is available as a Culti-Loop®

References
1. Bolton, F.J., Hutchinson, D.N. and Coates, D. (1984) J. Clin. Microbiol. 19, 169-171.
2. Hutchinson, D.N. and Bolton, F.J. (1984) J. Clin. Path. 34, 956-957
3. Bolton, F.J., Hutchinson, D.N. and Parker, G. (1988) Eur. J. Clin. Microbiol. Infect. Dis. 7. 155-160.
4. MAFF Validated Methods for the Analysis of Foodstuffs: Method for the detection of thermotolerant Campylobacter in Foods (v30) J. Assoc. Publ. Analysts (1993) 29. 253-262.
5. Rice, B.E., Chinta Lamichhane, Joseph, S.W. and Rollins, D.M. (1996) Clin. Diag. Lab. Immunol. 3, 669-677.
6. Hald, B. and Madsen, M. (1997) J. Clin. Microbiol. 35, 3351-3352.
7. Humphrey, T.J., Martin, K.W. and Mason, M.J. (1997) PHLS Microbiology Digest 13 (2), 86-88.

CEFOPERAZONE, AMPHOTERICIN B, TEICOPLANIN SUPPLEMENT (CAT)

Code: SR0174

A selective supplement for the isolation of thermophilic Campylobacter spp. and improved recovery of Campylobacter upsaliensis from faeces.

Vial contents(each vial is sufficient to supplement 500 ml of medium).

per vial

per litre

Cefoperazone

8.0mg

16.0mg

Teicoplanin

4.0mg

8.0mg

Amphotericin B

10.0mg

20.0mg

Directions
Prepare 500ml of sterile Blood-Free Campylobacter Agar Base as directed. Cool to 50°C and aseptically add one vial of SR0174 reconstituted as directed.
Mix well and pour the resulting CAT medium into sterile Petri dishes. Incubate cultures at 37°C for 48-72 hours in a microaerobic atmosphere.

Description
Because of the sensitivity of Campylobacter upsaliensis to a wide range of antibiotics, isolation of the organism from faeces using selective media has hitherto been difficult. The recommended isolation method uses a membrane filter culture technique on non-selective agar. This does not give good recovery from faeces containing less than 105 CFU/g5, and is a technically demanding method which is relatively slow to perform.
CAT Supplement SR0174 is based on the formulation described by Aspinall et al.7. When added to Blood-Free Campylobacter Agar Base which contains charcoal, it gives good isolation of thermophilic Campylobacter spp. The isolation of Campylobacter upsaliensis on a selective medium is possible because CAT Supplement contains reduced levels of cefoperazone compared to other campylobacter supplements. This inhibits most Enterobacteriaceae, but not enterococci. Teicoplanin is included to inhibit enterococci. Amphotericin B is added as an antifungal agent.
Further work confirmed the effectiveness of CAT medium as an alternative to membrane filtration culture for selective isolation of thermophilic campylobacters including Campylobacter upsaliensis8.

Atabay, Corry and On9 isolated a previously unknown catalase-negative, urease-positive Campylobacter from cattle faeces using CAT medium. This organism could not be cultured on blood-free Campylobacter medium (CCDA).

A study in which the productivity of CAT medium, blood-free media and semi-solid medium were compared, showed that CAT medium, used in parallel with membrane filtration on non-selective blood agar, is likely to be the most productive method for recovery of the greatest number of Campylobacter and Arcobacter species10.

Storage conditions and Shelf life
CAT Supplement SR0174 should be stored at 2-8°C in the dark. When stored as directed, the reagents remain stable until the stated expiry date shown on the packaging.

Quality control

Positive controls:

Expected results

Campylobacter upsaliensis ATCC® 43954 *

Good growth; pale colonies

Campylobacter jejuni ATCC® 33291 *

Good growth; grey coloured colonies

Negative control:

 

Enterococcus faecalis ATCC® 33186

Inhibited

* This organism is available as a Culti-Loop®

References
1. Atabay, I., Corry, J.E.L., Post, D. 8th International Campylobacter Workshop 1995.
2. Sandstedt, K., Ursing, J., Walder, M. (1983). Curr. Microbiol. 8: 209-213.
3. Sandstedt, K. and Ursing, J. (1991). Sys. Appl. Microbiol. 14: 39-45.
4. Patton, C.M., Shaffer, N., Edmonds, P. et al. (1989). J. Clin. Microbiol. 27: 66-73.
5. Goosens, H., Vlaes, L., Butzler, J.P. et al. (1991). Lancet. 337: 1486-7.
6. Bolton, F.J., Hutchinson, D.N., Parker, G. (1987). J. Clin. Pathol. 40: 702-3.
7. Aspinall, S.T., Wareing, D.R.A., Hayward, P.G. and Hutchinson, D.N. (1993). J. Clin. Pathol. 46: 829-831.
8. Aspinall, S.T., Wareing, D.R.A., Hayward, P.G. and Hutchinson, D.N. (1996). J. Appl. Bact. 80: 667-672.
9. Atabay, H.I., Corry, J.E.L. and On, S.L.W. (1997). Lett. Appl. Microbiol. 24: 59-64.
10. Atabay, H.I., Corry, J.E.L. and Post, D.E. (1996). Campylobacters, Helicobacters and Related Organisms. Newell, D.G., Ketley, J.M. and Feldman, R. A. (eds) Part 1-5. Plenum Press, New York.

 
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