CAMPY, BLOOD-FREE AGAR

Cat. no. G06 Campy, Blood-Free Agar, 15x100mm Plate, 18ml 10 plates/bag

INTENDED USE

Hardy Diagnostics Campy, Blood-Free Agar is a selective medium recommended for the isolation and cultivation of Campylobacter jejuni , C. coli, and C. laridis from feces. (1)

SUMMARY

Campylobacter are a major enteric pathogen and a variety of culture media have been developed to isolate these thermophilic enteropathogenic microorganisms. There are a number of selective media for the isolation of C. jejuni and C. coli ., including blood-free and blood containing formulations; however, formulations containing blood are disadvantagious for developing countries where supplies for sterile blood are not optimal.

To achieve the highest yield of Campylobacter organisms from stool, a combination of media may increase recovery rates by as much as 10 to 15% over the use of a single formulation. (5,7) In addition, the use of cefoperazone-containing media, as opposed to cephalothin-containing media, is recommended for the primary isolation of Campylobacter from fecal samples. (2,5,7) Specimens cultured on selective media should also be cultured on non-selective media to obtain additional information and to help ensure recovery of potential pathogens.

Campy, Blood-Free Agar is a charcoal based media supplemented with hematin and pyruvic acid, and made selective by the addition of cefoperazone, vancomycin and amphotericin B. The combination of a charcoal base, which neutralizes toxic oxygen derivatives, and the combined selectivity of the antimicrobics provides for an excellent media for the recovery of C. jejuni , C. coli , and C. laridis from feces.

FORMULA

Ingredients per liter of deionized water:*

Pancreatic Digest of Casein 12.0gm
Peptic Digest of Animal Tissue 5.0gm
Sodium Chloride 5.0gm
Activated Charcoal 4.0gm
Yeast Extract 3.0gm
Beef Extract 3.0gm
Corn Starch 1.0gm
Pyruvic Acid 100.0mg
Hematin 32.0mg
Cefoperazone 20.0mg
Vancomycin 20.0mg
Amphotericin B 2.0mg
Agar 13.5gm

Final pH 7.2 +/- 0.2 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8ºC. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat and freezing.

PRECAUTIONS

PROCEDURE

Specimen Collection: Specimens should be submitted directly to the laboratory without delay and protected from excessive heat and cold. If there is to be a delay in processing, specimens should be inoculated into an appropriate transport media and refrigerated until inoculation.

Method of Use: Bring media to room temperature before use. Inoculate media with stool specimen using a sterile swab. Alternatively, drop 1-2 drops of liquid specimen using a sterile pipet. Streak to obtain isolated colonies. Incubate at 42ºC. in a microaerophilic atmosphere containing 5% O 2 , 10% CO 2 , and 85% N 2 for a minimum of 48 hours. Examine for growth and screen suspect colonies.

INTERPRETATION OF RESULTS

C. jejuni grows as a small colony, usually grayish and flat with irregular edges. A small percentage of strains may appear tan or slightly pinkish. Colonies tend to spread or swarm, especially when initially isolated from fresh clinical specimens.

LIMITATIONS

Additional tests including gram stain and biochemical testing should be performed on pure cultures for complete identification. For more information see appropriate references. (2-4)

Most Campylobacter species require a microaerobic atmosphere containing approximately 5% O 2 , 10% CO 2 , and 85% N 2 for optimal recovery. The concentration of oxygen generated in candle jars is not optimal for the isolation of Campylobacter spp. and should not be used. (2)

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, other culture media, swabs, applicator sticks, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Campylobacter jejuni
ATCC ® 33291***
A 24-48hr 35°C Microaerophilic** Growth
Escherichia coli
ATCC ® 25922***
B 24hr 35°C Aerobic Partial to complete inhibition
Proteus mirabilis
ATCC ® 12453
B 24hr 35°C Aerobic Inhibited

** Atmosphere of incubation is enriched with 5% O 2 , 10% CO 2 , and 85% N 2 .

*** Recommended QC strains for User Quality Control according to the CLSI document M22 when applicable.

User Quality Control

PHYSICAL APPEARANCE

Campy, Blood-Free Agar should appear opaque, with slight traces of precipitate, and black in color.

C. jejuni growing on Campy, Blood-Free Agar

Campylobacter jejuni (ATCC ® 33291) colonies on Campy, Blood-Free Agar (Cat. no. G06). Incubated under microaerophilic conditions for 48 hours at 35ºC.

E. coli inhibited on Campy, Blood-Free Agar

Escherichia coli (ATCC ® 25922) growth inhibited on Campy, Blood-Free Agar (Cat no. G06). Incubated aerobically for 24 hours at 35ºC.

REFERENCES

1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

2. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

3. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

4. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

5. Karmali, M.A., et al. 1986. Evaluation of a blood-free, charcoal-based medium for the isolation of Campylobacter organisms from feces. J. Clin. Mircrobiol.; 23:456-459.

6. MacFaddin, J.F. Biochemical Tests for Identification of Medical Bacteria,, Lipincott Williams & Wilkins, Philadelphia, PA.

7. Oyarzabal, O.A., K.S. Macklin, J.M. Barbaree, and R.S. Miller. 2005. Evaluation of Agar Plates for Direct Enumeration of Camplyobacter spp. from Poultry Carcass Rinses. Appl. and Environ. Microbio. 71(6):3351-3354.


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121415hh