Campylobacter Blood Agar (CVA): Introduction, Composition, Principle, Preparation, Test Procedure, Result-Interpretation, and Keynotes

Campylobacter Blood Agar (CVA): Introduction, Composition, Principle, Preparation, Test Procedure, Result-Interpretation, and Keynotes

Introduction of Campylobacter Blood Agar (CVA)

Campylobacter Blood Agar (CVA) is a selective medium used for the primary isolation of Campylobacter jejuni from the clinical specimen, feces/stool.
The  recognized etiological agents of the gastrointestinal tract (GIT)
infections are Campylobacter jejuni and Campylobacter coli.  Blaser et al used a selective medium containing four antibiotics i.e. amphotericin, vancomycin, polymyxin B, and trimethoprim, and became a success in the isolation of Campylobacter jejuni in  1978. Later it was further modified by   Reller et al. to the selective medium containing only three antimicrobial agents like cefoperazone, vancomycin, and amphotericin for the isolation of the same bacterium, C. jejuni. This combination of antimicrobials provided better suppression of normal fecal flora and therefore it allowed better isolation of C. jejuni than previously developed selective blood agars.

Composition of Campylobacter Blood Agar (CVA)

  • Pancreatic Digest of Casein: 10.0 g
  • Peptic Digest of Animal Tissue (PDAT): 10.0 g
  • Dextrose: 1.0 g
  • Yeast Extract:  2.0 g
  • Sodium Chloride: 5.0 g
  • Sodium Bisulfite:  0.1 g
  • Agar: 15.0 g
  • Demineralized/distilled water: 1000 ml

Extra ingredients

  • Cefoperazone:  20.0 mg
  • Vancomycin:  10.0 mg
  • Amphotericin B:  2.0 mg
  • Defibrinated Sheep Blood: 50 mL

pH 7.2 ± 0.2 at 25ºC.

Principle of Campylobacter Blood Agar (CVA)

The ingredients of the medium include a variety of peptones and extracts that provide all the necessary growth factors like nitrogen compounds, carbon, sulfur, and trace ingredients for the recovery of Campylobacter species. Yeast extract is a water-soluble ingredient of the medium that contributes to vitamins, carbohydrates, nitrogen, and salts while dextrose is an energy source. The presence of sodium chloride in nutrient agar maintains a salt concentration in the medium that is similar to the cytoplasm of the microorganisms. Agar acts as the solidifying agent.  Water is an essential ingredient for the growth and reproduction of organisms and also serves as a transport medium for the agar’s various substances.

In the corporation of sheep, blood provides hemin and supplies other necessary additional growth factors.  The incorporation of various antibiotics in the medium inhibits a variety of microorganisms. Cefoperazone is a cephalosporin drug that suppresses the growth of gram-negative bacilli and some gram-positive bacteria also. Vancomycin is another antimicrobial agent of the glycopeptides group that inhibits many species of gram-positive bacteria while amphotericin B is an antifungal agent and thus it can inhibit a wide variety of fungi including yeasts and molds. C.  jejuni is a thermophile and therefore inoculated plates should be incubated at 42 °C to accelerate growth.  Another advantage of the higher temperature supports to inhibit of any background flora that may be present.

Preparation of Campylobacter Blood Agar (CVA)

 Antibiotic supplement Preparation

  • Add antibiotics to 10.0ml of demineralized/distilled water,
  • Mix it properly and filter it for sterilization.

Medium Preparation 

  1. Add all the ingredients, except sheep blood and antimicrobial solution, to distilled water and bring the final volume to 940 ml.
  2. Mix the solution thoroughly.
  3. Heat to boiling to dissolve the medium completely.
  4. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
  5. After autoclaving,  leave for cooling to 45-50°C.
  6. Aseptically add 50 ml of sterile sheep blood and 10.0 ml of sterile antimicrobial solution.
  7. Mix thoroughly and pour into sterile Petri dishes or distribute into sterile tubes.

Storage and Shelf life 

  • Store at 2-8ºC  and away from direct light.
  • Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), or contamination.
  • The product is light and temperature sensitive; protects from light, excessive heat, moisture, and freezing.

Test Requirements

Test Procedure of Campylobacter Blood Agar (CVA)- specimen/organism inoculation

  1. Allow the plates to warm at 37°C or to room temperature, and the agar surface to dry before inoculating.
  2. Inoculate and streak the specimen as soon as possible after collection.
  3. If the specimen to be cultured is on a swab, roll the swab over a small area of the agar surface.
  4. Streak for isolation with a sterile loop.
  5. Incubate plates aerobically at  42ºC for  48 hours in a CO2 incubator.
  6. Examine colonial characteristics.

Colony Morphology of Campylobacter Blood Agar (CVA)

Control strains

  • Escherichia coli ATCC 25922: No growth
  • Campylobacter jejuni ATCC 33291: Growth and colonies are small, grayish in coloration, flat with irregular edges, non-hemolytic, and mucoid. Some colonies of this isolate may appear as round colonies 1-2mm in diameter that are convex, entire, and glistening. Spreading and swarming are common in agar plates for isolates that are from fresh clinical specimens.
  •  Candida albicans ATCC 10231: No growth

Limitations

  1. Campylobacter Blood Agar (CVA) is recommended that biochemical, immunological, molecular, or mass spectrometry assaying be performed on colonies from pure culture for final identification.
  2. Campylobacter jejuni is thermophilic thus it is important to incubate the agar plates at 42ºC to accelerate growth.  The higher temperature also supports inhibiting any background flora that may be present.  If it is incubated at lower temperatures,  growth may be delayed and the selectivity of the medium is lowered.
  3. The presence of sugar (dextrose) in the medium causes some Campylobacter isolates to produce weakly positive oxidase reactions. Therefore, subculturing such isolates in the medium without dextrose and repetition of the oxidase test is recommended.

Keynotes on Campylobacter Blood Agar (CVA)

  • Campylobacter Blood Agar (CVA) is an enriched selective blood agar that will support good growth of C.  jejuni and is used for the primary isolation and cultivation of C.  jejuni from fecal, food, and also environmental samples.
  • The antimicrobial agent, cephalothin inhibits Campylobacter fetus, Campylobacter lari, Campylobacter hyointestinalis, and Campylobacter upsaliensis.
  • A full 48-hour incubation is required for some isolates since they may barely be visible after only 24 hours of incubation.

Related images of Campylobacter Blood Agar (CVA)

Campylobacter fetus growth on chocolate agar after 48 hours of incubation at 37°C 5% CO2 incubator

Campylobacter fetus growth on chocolate agar after 48 hours incubation at 37°C 5% CO2 incubator
Fig. Campylobacter fetus growth on chocolate agar after 48 hours of incubation at 37°C 5% CO2 incubator

Campylobacter in basic fuchsin staining showing curved bacteria

 

Campylobacter in basic fuchsin staining showing curved bacteria
Fig. Campylobacter in basic fuchsin staining showing curved bacteria

Further Reading

  • https://www.dalynn.com/dyn/ck_assets/files/tech/PC24.pdf
  • Ronald M. Atlas and James W. Snyder (2014). Handbook of media for clinical and public health microbiology. CRC Press. Taylor & Francis Group, LLC. Page no.94-95
  • https://assets.fishersci.com/TFS-Assets/LSG/manuals/IFU1280.pdf
  • https://pubmed.ncbi.nlm.nih.gov/2246143/
  • https://himedialabs.com/TD/M887.pdf
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