Campylobacter blood agar (CVA), is a selective medium that allows for the primary isolation from stool specimens of Campylobacter Jejuni. Dekeyser et al. Dekeyser et al. reported that Campylobacter jejuni was isolated from patients suffering from diarrhea and acute gastroenteritis using a filtration method and a selective media with antimicrobials. Skirrow, however, reported that a select medium containing three antimicrobics was used for isolation. Blaser and colleagues reported that they were able to isolate C. jejuni from feces using a selective medium that contained four antimicrobials: amphotericin (vancomycin), polymyxin B and trimethoprim. Reller et al. in 1983 also introduced a better selective medium containing cefoperazone and Vancomycin. This combination of antimicrobials allowed for better suppression of normal fecal bacteria, thereby allowing for better isolation of C.jejuni from the fecal specimen.
Composition of Campylobacter Blood Agar (CVA)
|Polymyxin B||2500 U|
|Demineralized water||1000.0 ml|
Final pH 7.0 +/- 0.2 at 25ºC.
Principle of Campylobacter Blood Agar (CVA)
The medium is enriched with a range of peptones, extracts, and trace elements that provide all necessary growth factors, including carbon, nitrogen, sulfur, and trace components for the recovery of Campylobacter species. Dextrose and yeast extract are both sources of B vitamins. Hemin is provided by sheep blood, which also supplies additional growth factors and hemin. Cephalothin, trimethoprim, amphotericin, vancomycin and polymyxin B are all antimicrobial agents that suppress normal microbial growth in fecal specimens. This allows for the isolation of C. jejuni. Vancomycin, a glycopeptide, inhibits many species gram-positive bacteria. Polymyxin B injures most gram negative bacilli with the exception of Proteus. Trimethoprim can inhibit Proteus species. Amphotericin A is an antifungal agent which can be used to inhibit many yeasts and molds. Agar is the solidifying agent. To accelerate the growth of Campylobacter Jejuni, inoculated plates should remain at 42°C. This temperature will also help to suppress any background flora.
Preparation of Campylobacter Blood Agar (CVA)
Preparation of antibiotic supplement
Filter sterilize and add components to 10.0ml of distilled.
Preparation of medium
- Mix all components except for sheep blood and antibiotic solution in distilled water until the volume is 890.0ml.
- Bring to boil by heating gently.
- Autoclave for 15 minutes at 15 lbs pressure and 121degC
- Cool at 45degC-50degC. Aseptically, add 100.0ml sterile sheep blood to the mixture and 10.0ml sterile antibiotic solution.
- Mix well and place in sterile Petri dishes.
Results Interpretation on Campylobacter Blood Agar (CVA)
- Campylobacter Jejuni is typically small and grayish in color, flat with irregular edges, and non-hemolytic mucoid colonies at 24-48 hours.
- It is necessary to incubate the isolates for 48 hours. Some isolates may not be visible after 24 hours.
- Some colonies can appear as convex, whole and glistening colonies that are 1-2mm in diameter.
- For isolates taken from fresh clinical specimens, spreading and swarming is common.
Uses of Campylobacter Blood Agar (CVA)
- It is an enriched, selective blood agar which will support good growth for Campylobacter Jejuni. It can also be used to isolate and cultivate Campylobacter from fecal and food specimens.
Limitations of Campylobacter Blood Agar (CVA)
- For complete identification, it is recommended that colonies grown from pure culture be subject to biochemical, immunological and molecular testing.
- C. jejuni has a thermophilic nature so incubating plates at 42oC will accelerate growth. The higher temperature will also help to suppress any background flora. Lower temperatures can slow down growth and reduce the selectivity of medium.
- Because of the presence of dextrose, some Campylobacter isolates can produce weakly positive oxygenase reactions when grown in Campylobacter Blood Agar. It is recommended to subculture such isolates in the medium, without dextrose, and repeat the oxidase testing.
- Cephalothin inhibits Campylobacter fetus and Campylobacter lari as well as Campylobacter hyointestinalis and Campylobacter upsaliensis.