anaeroGRO™

Campylobacter Selective Agar (CAMPY)

Cat. no. AG701 Campylobacter Selective Agar*, 15x100mm Plate, 18ml 1 plate/pouch

* All AnaeroGRO™ plated media is provided in standard 15x100mm monoplates or biplates. Each plate or set of plates is packaged in an oxygen-free gas flushed foil pouch containing a desiccant and an oxygen scavenger sachet.

INTENDED USE

Hardy Diagnostics AnaeroGRO™ Campylobacter Selective Agar is recommended for the selective isolation of Campylobacter jejuni subsp. jejuni from stool specimens. Growth of normal fecal flora is inhibited on this medium.

SUMMARY

Campylobacter species are microaerophilic organisms that inhabit the gastrointestinal tract of various animals, including poultry, dogs, cats, sheep, and cattle. Microaerophilic organisms require an atmosphere consisting of 85% nitrogen, 5% oxygen, and 10% carbon dioxide for optimal growth. 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 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 from 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 one 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.(1,6,10,11,13)

Campylobacteriosis is usually sporadic and tends 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 or symptoms of gastroenteritis in humans. It is thought that the infective dose of C. jejuni ranges from 500 - 10,000 cells, depending upon the strain, damage to cells from environmental stresses, and susceptibility of the host.(1,7) Infants and young children are the most susceptible. Travelers to developing countries are also at risk for Campylobacteriosis.(1,2,6,10,11)

Early research by Dekeyser et al. on acute gastroenteritis using a filtration technique and a blood-containing selective medium isolated Campylobacter jejuni as the etiological agent.(4) Skirrow and others reported similar outcomes using blood-based selective media containing antimicrobics to suppress the growth of normal fecal flora.(13) Later, Blaser et al. showed success in isolating C. jejuni using a Brucella Agar base supplemented with blood and selective agents.(3)

Peptones, yeast extract and other digests contained in these early formulations support the growth of Campylobacter spp. and supply a nutritious basal medium by providing nitrogenous compounds, carbon, sulfur, trace elements and complex B vitamins. The addition of blood, dextrose and other nutrients provides an energy source and further enhances the growth of cultures. The antimicrobial agents polymyxin B, trimethoprim, and vancomycin, along with incubation at 42ºC. in a microaerophilic environment, suppress the growth of normal fecal flora, thereby facilitating the isolation of C. jejuni from specimens.

AnaeroGRO™ Campylobacter Selective Agar is packaged in an oxygen-free, reduced state to prevent the formation of toxic oxidized by-products that may damage obligate anaerobes and inhibit the growth of more fastidious species. Culture media that is exposed to environmental oxygen leads to a build-up of reactive oxygen species (ROS) that initiate damaging free radical reactions, which inhibit the growth of anaerobic bacteria. Therefore, ingredients have been added to the AnaeroGRO™ media to neutralize the growth inhibiting toxic effects of peroxide and other reactive oxygen species (ROS) that may develop during the medium's brief exposure to oxygen after it is sterilized and before it is packaged in an oxygen-free environment.

FORMULA

Ingredients per liter of deionized water:*

Pancreatic Digest of Casein 10.0gm
Peptic Digest of Animal Tissue 10.0gm
Soy Peptone 3.0gm
Sodium Chloride 5.0gm
Yeast Extract 2.0gm
Reducing Agents/Peroxide Inhibitors 1.5gm
Dextrose 1.0gm
L-Cysteine 0.5gm
Sodium Bisulfite 0.1gm
Vancomycin 10.0mg
Polymyxin B 2500.0IU
Trimethoprim 5.0mg
Laked Horse Blood 70.0ml
Agar 15.0gm

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 15-30ºC. 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, moisture, and freezing.

PRECAUTIONS

PROCEDURE

Specimen Collection: Consult listed references for information on specimen collection.(1,2,5-11) Infectious material should be submitted directly to the laboratory without delay and protected from excessive heat, cold and oxygen exposure. If there is to be a delay in processing, it is recommended that the specimen be inoculated into an appropriate transport medium, such as Cary-Blair C&S Transport Vial (Cat. no. 28050510), and refrigerated until inoculation. Minimize specimen exposure to ambient oxygen levels in air.

Fecal specimens are the preferred sample for isolating Campylobacter species from patients with gastrointestinal infections; however rectal swabs are acceptable for culture. Cary-Blair (Cat. no. 4132BX) or Campy Thio Medium (Cat. no. K128) should be used as a transport medium 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 4ºCC. until processed.(6,11)

Method of Use: Open the AnaeroGRO™ pouch just prior to use and immediately apply rectal swabs or liquid fecal specimens directly to the agar surface in an area approximately 1 to 1.25 inches in diameter and streaked with a sterile inoculating loop to obtain isolated colonies. Incubate inoculated plates at 42 to 43ºC. in an anaerobic jar using a microaerophilic gas mixture (Cat. no. TN100) or CampyGen™ gas generating system (Cat. no. CN025A). For strain specific gas mixtures, consult appropriate reference materials for best practices. Note: Campylobacter jejuni is a microaerophile, not an obligate anaerobe.

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.(11)

INTERPRETATION OF RESULTS

Examine plates at 24, 48, and 72 hours for growth. Colonies of C. jejuni subsp. jejuni are usually detected at 24 hours and produce two colony types: (1) small, raised, grayish-brown, smooth, and glistening with an entire translucent edge; or (2) flat, mucoid, translucent, grayish, with an irregular edge. A small percentage of strains may appear tan or faintly pink.(11) Colonies may spread over the entire surface of the agar, especially when isolated from fresh clinical specimens.

When examining plates at 24 hours, examine quickly and return plates to a reduced oxygen atmosphere promptly to avoid damaging cultures during log phase growth.

An oxidase test may be used to screen suspect colonies, since C. jejuni subsp. jejuni is oxidase positive. Indoxyl Acetate (Cat. no. Z111) is another rapid test that is useful for the identification of Campylobacter.

LIMITATIONS

The plates must be inoculated immediately after opening the AnaeroGRO™ pouch. After inoculation, the plates must be placed immediately into a microaerophilic atmosphere (pouch or jar) to ensure optimal growth of microaerophilic bacteria.

C. jejuni is thermophilic and should be incubated at 42ºC. Otherwise, growth of colonies may be delayed.

Campylobacter species are not easily visualized with the safranin counterstain normally used in the Gram stain procedure; therefore, carbol fuchsin or 0.1% aqueous basic fuchsin (Cat. no. BF008) 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.(10,11)

Most Campylobacter species require a microaerobic atmosphere containing approximately 5% O2, 10% CO2, 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.(11)

Certain Campylobacter species, such as C. sputorum, C. concisus, C. mucosalis, etc., may require hydrogen for primary isolation and growth.(11)

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.

Polymyxin B may be inhibitory to some strains of C. jejuni and C. coli.(11) Therefore, 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.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, swabs, applicator sticks, other culture media, transport media (Cat. no. 28050510 or 4132BX), incubators, incinerators, microaerophilic culture materials, such as gas generators (Cat. no. CN025A), compact systems (Cat. no. CN020C), pouches, (Cat. no. AG020C), sealing clips (Cat. no. AN005C), chambers, and jars (Cat. no. 16000), etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Campylobacter jejuni subsp. jejuni
ATCC ® 33291***
A 24-48hr 42°C Microaerophilic** Growth
Campylobacter fetus subsp. fetus
ATCC ® 33246
A 48hr 42°C Microaerophilic** Growth
Staphylococcus
aureus

ATCC ® 25923
B 48hr 35°C Aerobic No growth; partial to complete inhibition
Enterococcus faecalis
ATCC ® 29212
B 48hr 35°C Aerobic No growth; partial to complete inhibition
Escherichia coli
ATCC ® 25922***
B 48hr 35°C Aerobic No growth; partial to complete inhibition
Proteus mirabilis
ATCC ® 12453
B 8hr 35°C Aerobic No growth; partial to complete inhibition

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

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

USER QUALITY CONTROL

Physical Appearance

AnaeroGRO™ Campylobacter Selective Agar should appear opaque to translucent, and medium red in color.

REFERENCES

1. APHA Technical Committee on Microbiological Methods for Foods. Compendium of Methods for the Microbiological Examination of Foods, APHA, Washington, D.C.

2. American Public Health Association. Standard Methods for the Examination of Dairy Products, APHA, Washington, D.C.

3. Blaser, M.J., J. Cravens, B. Powers, W.L. Wang. 1978. Campylobacter enteritis associated with canine infections. Lancet; 2:979-981.

4. Dekeyser, P, M. Gossuin-Detrain, J.P. Butzler, and J. Sternon. 1972. Acute enteritis due to related vibrio: first positive stool cultures. J. Infect. Dis.; 125:390-392.

5. Dowell, V.R., Jr. and T.M. Hawkins. 1987. Laboratory Methods in Anaerobic Bacteriology. In: CDC Laboratory Manual. DHEW Publication No. (CDC) 87-8272. U.S. Department of Health, Education and Welfare, Public Health Service. Center for Disease Control, Atlanta, GA.

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

7. 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.

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

9. Jousimies-Somer, H.R., S.P. Citron, D. Baron, E.J. Wexler, and H.M. Finegold. 2002. Wadsworth-KTL Anaerobic Bacteriology Manual, 6th ed. Star Publishing Company, New York, N.Y.

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

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

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

13. Skirrow, M.B. 1977. Campylobacter enteritis: a "new" disease. Br. Med. J. Vol. 2. No. 6078.


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