CAMPY CEFEX AGAR, MODIFIED

Cat. no. A122 Campy Cefex Agar, Modified, 15x100mm Plate, 18ml 10 plates/bag

INTENDED USE

Hardy Diagnostics Campy Cefex Agar, Modified plates are recommended for the selective isolation of cephalothin-resistant Campylobacter species such as C. jejuni , C. coli , and C. lari from human, animal, or food samples.

SUMMARY

Campylobacter species have been recognized as a major cause of diarrheal disease in children and adults. Originally these organisms were only associated with a variety of veterinary diseases. Campylobacter spp. has been characterized as among the top bacterial agents of human foodborne gastroenteritis. The organisms may also be transmitted by contaminated food or water. Poultry is a primary reservoir of Campylobacter spp. and studies show that prevalence may be greater than 80% in commercial chicken carcasses. 95% of human illnessess are associated with Campylobacter jejuni , followed by C. coli at 4%. Other species are involved in only 1% of infections.

Brucella Agar is a highly nutritious base and the addition of horse blood supplements the medium with X-factor (hemin) and other growth factor requirements. The addition of antimicrobials to the medium is required to suppress the growth of normal fecal flora. Cefoperazone is added to inhibit many gram-positive and gram-negative organisms, both aerobic and anaerobic. Amphotericin B is added to inhibit the growth of fungi.

Cephalothin-sensitive Campylobacter spp. such as C. fetus and C. upsaliensis may not be recovered on Campy Cefex Agar, Modified because it contains cefoperazone. (2)

FORMULA

Ingredients per 950ml deionized water:*

Brucella Agar 43.0gm
Ferrous Sulfate 0.5gm
Sodium Pyruvate 0.5gm
Sodium Bisulfite 0.2gm
Amphotericin B 0.02gm
Cefoperazone 33.0mg
Lysed Horse Blood 50.0ml

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), hemolysis, 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: Consult listed references for information on specimen collection. (1-3,5,9) Infectious material 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, the specimen should be inoculated onto an appropriate transport media and refrigerated until inoculation.

Method of Use:

Direct Inoculation:
1. Swab: Inoculate a Campy Cefex Agar, Modified plate using the four quadrant streak technique for maximum isolation.

2. Diarrheal Stool: Inoculate a Campy Cefex Agar, Modified plate with three drops of stool and streak for isolation. At the same time, make a direct smear and look for small curved gram-negative bacilli and fecal leukocytes.

3. Solid Stool: Prepare a 1:10 suspension of stool by placing pea-sized amount into 5ml of physiological saline (0.85%). Vortex the sample. Inoculate a Campy Cefex Agar, Modified plate with three drops of this suspension, and streak for isolation.

Indirect Inoculation:
1. Swab: Place the swab into an appropriate transport medium and refrigerate overnight.

2. Diarrheal Stool: Place five drops of the specimen approximately one centimeter below the surface of an appropriate broth medium (Cat. no. K128). Refrigerate overnight.

3. Solid Stool: Prepare a 1:10 suspension of stool by placing pea-sized amount into 5ml of physiological saline (0.85%). Vortex the sample. Place five drops of the specimen approximately one centimeter below the surface of an appropriate broth medium (Cat. no. K128). Refrigerate overnight.

4. Subculturing appropriate broth medium (Cat. no. K128): Place a pasteur pipet one inch below the surface of the broth medium and withdraw a large aliquot towards the surface. Place three drops onto a Campy Cefex Agar, Modified plate and streak for isolation.

Incubate Campy Cefex Agar, Modified plates at 42ºC. for 48-72 hours in a microaerophilic atmosphere of 85% nitrogen, 10% carbon dioxide, and 5% oxygen. In addition, media may be set up in duplicate, with the second set incubated at 35-37ºC. to allow for the growth of certain Campylobacter species.

INTERPRETATION OF RESULTS

Campylobacter jejuni colony morphology may appear as small, mucoid, grayish, flat colonies with irregular edges and no hemolytic patterns at 24-48 hours. Colonies may also appear pink or yellowish-gray with some colonies exhibiting a tailing effect along the streak line. (3) They may also appear as round, convex, entire, glistening colonies 1-2mm in diameter.

Consult the listed references for more information regarding the identification and further testing of Campylobacter species. (1-3,5)

LIMITATIONS

Cephalothin sensitive Campylobacter spp. such as C. fetus and C. upsaliensis may not be recovered on Campy Cefex Agar, Modified because it contains cefoperazone. (2)

Due to the presence of dextrose in the medium, some weak oxidase reactions may occur. Testing should be performed on growth taken from a medium without dextrose, if this phenomenon occurs.

The agents in selective media may inhibit some strains of desired species or permit the growth of species they were designed to inhibit. Therefore, specimens cultured on selective media should also be cultured on non-selective media to obtain additional information and to help insure recovery of potential pathogens.

The reactions observed with Campy Cefex Agar, Modified are not sufficient to speciate organisms.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, other culture media, slides, staining reagents, pasteur pipettes, microaerophilic atmosphere packets, incubation jars, catalysts, 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 48hr 35°C Microaerophilic** Growth
Proteus mirabilis
ATCC ® 12453
B 48hr 35°C Aerobic Partial to complete inhibition
Escherichia coli
ATCC ® 25922
B 48hr 35°C Aerobic Partial to complete inhibition
Candida albicans
ATCC ® 10231
B 48hr 35°C Aerobic Partial to complete inhibition

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

User Quality Control

PHYSICAL APPEARANCE

Campy Cefex Agar, Modified should appear clear, and dark red in color.

C. jejuni growing on Capmy Cefex Agar, Modified

Campylobacter jejuni (ATCC ® 33291) growing on Campy Cefex Agar, Modified (Cat. no. A122). Incubated microaerophilically for 48 hours at 35ºC.

P. mirabilis inhibited on Capmy Cefex Agar, Modified

Proteus mirabilis (ATCC ® 12453) growth inhibited on Campy Cefex Agar, Modified (Cat. no. A122). Incubated aerobically for 48 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. Blaser, M.J., et al. 1979. Campylobacter enteritis: clinical and epidemiologic features. Annals of Intern. Med.; 91:179-185.

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

6. MacFaddin, J.F. 1985. Media for Isolation, Cultivation, Identification, Maintenance of Bacteria, Vol. I. Williams & Wilkins, Baltimore, MD.

7. Quality Assurance for Commercially Prepared Microbiological Culture Media, M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.

8. Reller, L.B., et al. 1983. Controlled evaluation of an improved selective medium for the isolation of Campylobacter jejuni from human feces, Abstract, ASM Annual Meeting.

9. Oyarzabal, O.A, et al., "Evaluation of Agar Plates for Direct Enumeration of Campylobacter spp. from Poultry Carcass Rinses". Applied and Environmental Microbiology: June 2005, p. 3351-3354.


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