CAMPY CVA AGAR

Cat. no. A40 Campy CVA Agar, 15x100mm Plate, 18ml 10 plates/bag

INTENDED USE

Hardy Diagnostics Campy CVA Agar Plates are recommended for the selective isolation of Campylobacter species from fecal specimens.

SUMMARY

Campylobacter species are microaerophilic organisms that inhabit the gastrointestinal tracts of various animals, including poultry, dogs, cats, sheep, and cattle. C. jejuni and C. coli are the most common Campylobacter species associated with gastrointestinal infection and are clinically indistinguishable. In fact, it is thought that approximately 5 to 10% of cases reported as being due to C. jejuni in the U.S. are probably due to C. coli . Campylobacter lari has also been recognized as a cause of gastroenteritis, but less frequently than C. jejuni . C. jejuni continues to be the most common enteric pathogen isolated form patients with diarrhea. Symptoms of C. jejuni or C. coli infection usually include fever, abdominal cramping, and diarrhea that lasts for several days to more than 1 week. Symptomatic infections, such as gastroenteritis, are usually self-limiting and do not require antibiotic therapy, although relapses may occur in 5 to 10% of untreated patients. Deaths attributed to C. jejuni infection are uncommon. (1,2,5,6)

Campylobacter infections are usually sporadic and tend to occur in the summer and early fall. Outbreaks are associated with ingestion of contaminated milk and water. Ingestion of improperly handled or under cooked food, primarily poultry products, raw milk, or contaminated water are common sources for human infections. It takes relatively few Campylobacter cells to cause illness and or symptoms of gastroenteritis in humans. It is thought that the infective dose of C. jejuni ranges from 500 - 10,000 cells, depending on the strain, damage to cells from environmental stresses, and the susceptibility of the host. (5,11) Infants and young children are the most susceptible. Travelers to developing countries are also at risk for Campylobacter infections. (1,2,4-6)

A number of selective media are recommended for the isolation of C. jejuni and C. coli ., including blood-free media, such as Charcoal-Cefoperazone-Deoxycholate Agar (CCDA), Charcoal-based Selective Medium (CSM). There are also blood containing media, such as Skirrow medium and Campy CVA medium. To achieve the highest yield of Campylobacter organisms from stool samples, a combination of media appears to be optimal, and may increase the recovery by as much as 10 to 15% over the use of a single medium. If only a single medium is used, Campy CVA is recommended. The use of cefoperazone-containing media, as opposed to cephalothin-containing media, is recommended for the primary isolation of Campylobacter from fecal samples (see "Limitations" section below). (1,6,8) 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. (12)

Hardy Diagnostics Campy CVA Agar contains peptamin which provides carbon, sulfur, and nitrogenous compounds required for growth. Yeast extract supplies B vitamins to the medium, and dextrose is incorporated as an energy source. Sheep blood supplements the medium with X-factor and other growth factor requirements. The addition of antimicrobials to the media 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. Vancomycin inhibits gram-positive microorganisms. Amphotericin B is incorporated to inhibit the growth of yeast.

FORMULA

Ingredients per liter of deionized water:*

Peptamin 20.0gm
Sodium Chloride 5.0gm
Yeast Extract 2.0gm
Dextrose 1.0gm
Sodium Bisulfite 0.1gm
Cefoperazone 20.0mg
Vancomycin 10.0mg
Amphotericin B 2.0mg
Sheep Blood, Defibrinated 50.0ml
Agar 17.5gm

Final pH 7.0 +/- 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. (6-8) 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.

Fecal specimens are the preferred sample for isolating Campylobacter species from patients with gastrointestinal infections; however rectal swabs are acceptable for cultures. A transport medium, such as Cary-Blair or Campy-Thio medium, should be used if there is a delay of more than 2 hours to the lab, and for transport of rectal swabs. Specimens received in transport medium should be processed immediately or stored at 4ºC. until processed. (1,2)

Several references suggest using the filtration method in conjunction with direct culturing on selective media. Please see listed references for procedure of the filtration method for recovering Campylobacter species.

Enrichment broths are used to enhance the recovery of Campylobacter from stool samples, such as Preston enrichment and Campy-Thio. Enrichment cultures may be beneficial when low numbers of the organisms are expected. Use of enrichment cultures as part of routine stool culture setup is probably not necessary. (1,3,7)

Method of Use:

Direct Inoculation:

1. Swab: Inoculate a Campy CVA Agar plate using the four quadrant streak technique for maximum isolation.

2. Diarrheal Stool: Inoculate a Campy CVA Agar 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 CVA Agar 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 CVA Agar plate and streak for isolation.

Incubate Campy CVA Agar 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. They may also appear as round, convex, entire, glistening colonies 1-2mm in diameter. Certain strains of C. jejuni may appear lightly pink or tan in color.

Consult the listed references for more information regarding the identification and further testing of Campylobacter species. (1-3,7)

LIMITATIONS

Campylobacter species are not easily visualized with the safranin counterstain normally used in the Gram stain procedure; therefore carbolfuchsin or 0.1% aqueous basic fuchsin can be used as the counterstain, or extending the staining time of the safranin to at least 10 minutes can improve the intensity of the stain. (1,6)

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. (1)

Certain Campylobacter species, such as C. sputorum , C. concisus , C. mucosalis , etc., may require hydrogen for primary isolation and growth (1)

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 antimicrobial agents that are present in some Campylobacter Selective medias, such as cephalothin, colistin, and polymyxin B, may be inhibitory to some strains of C. jejuni and C. coli, and are inhibitory to C. fetus . (1) 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 CVA Agar 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
Escherichia coli
ATCC ® 25922
B 48hr 35°C Aerobic No growth; partial to complete inhibition

** Atmosphere of incubation is enriched with 5% O2, 10% CO2, and 85% N2.

User Quality Control

PHYSICAL APPEARANCE

Campy CVA Agar should appear opaque, and cherry red in color.

C. jejuni growing on Campy CVA Agar

Campylobacter jejuni (ATCC ® 33291) colonies growing on Campy CVA Agar (Cat. no. A40). Incubated under microaerophilic conditions for 48 hours at 35ºC.

E. coli inhibited on Campy CVA Agar

Escherichia coli (ATCC ® 25922) growth inhibited on Campy CVA Agar (Cat. no. A40). Incubated aerobically for 24 hours at 35ºC.

REFERENCES

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

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

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

4. Marshall, R.T. Standard Methods for the Examination of Dairy Products, APHA, Washington, D.C.

5. Vanderzant, C. Compendium of Methods for the Microbiological Examination of Foods, APHA, Washington, D.C.

6. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology, J.B. Lippincott Company, Philadelphia, PA.

7. Isenberg, H.D. 1998. Essential Procedures for Clinical Microbiology. American Society for Microbiology, Washington, D.C.

8. Gun-Munro, J., R.P. Rennie, J.H. Thornley, H.L. Richardson, D. Hodge, and J. Lynch. 1987. Laboratory and clinical evaluation of isolation media for Campylobacter jejuni. J. Clin. Microbiol. 25: 2274-2277.

9. Karmali, M.A., A.E. Simor, M. Roscoe, P.C. Fleming, S.S. Smith, and J. Lane. 1986. Evaluation of a blood-free, charcoal-based, selective medium for the isolation of Campylobacterorganisms from feces. J. Clin. Microbiol. 23:456-459.

10. Centers for Disease Control and Prevention. 2001. Campylobacter infections. www.cdc.gov/ncidod/dbmd/diseaseinfo/campylobacter/.

11. Hunt, J.M., C. Abeyta and T. Tran. 1998. Chap. 7 Campylobacter. Bacteriological Analytical Manual, 8th ed., Rev. A.vm.cfsan.fda.gov/-ebam/bam-7.

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