Campylobacter Blood Agar (CVA) is a type of culture medium used for the isolation and cultivation of Campylobacter species, which are gram-negative, microaerophilic bacteria that are commonly associated with food-borne illnesses such as campylobacteriosis. The medium is made by adding blood, typically sheep or horse blood, to a base agar medium. The blood in the medium provides a source of nutrients for the bacteria. The medium also contains a selective agent, such as cephalothin or vancomycin, which inhibit the growth of other types of bacteria and promote the growth of Campylobacter. CVA is typically incubated under microaerophilic conditions, which mimic the low oxygen environments where Campylobacter thrive. CVA can be used for the isolation, cultivation, and detection of Campylobacter from clinical specimens such as feces, as well as for antimicrobial susceptibility testing.
What is Campylobacter Blood Agar ?
Campylobacter Blood Agar is a type of agar medium used for the isolation and cultivation of Campylobacter species from clinical specimens. The medium is prepared by adding sterile defibrinated sheep or horse blood to a selective Campylobacter agar base. The agar base contains nutrients that support the growth of Campylobacter species, while the added blood provides essential growth factors and enhances the detection of hemolytic activity.
The selective Campylobacter agar base contains cefoperazone, amphotericin B, and teicoplanin, which suppress the growth of most other bacteria and fungi, allowing for the isolation of Campylobacter species. The medium is incubated in a microaerophilic atmosphere (5% oxygen, 10% carbon dioxide, and 85% nitrogen) at 42°C for 48 hours.
Campylobacter Blood Agar is a highly specific medium and is commonly used in clinical laboratories for the isolation and identification of Campylobacter species from fecal, rectal, and other clinical specimens. Campylobacter species are a common cause of bacterial gastroenteritis and are also associated with other infections such as septicemia and meningitis.
Principle of Campylobacter Blood Agar:
The medium contains a variety of peptones and extracts that supply all the necessary growth factors, including nitrogen compounds, carbon, sulfur, and trace ingredients for the recovery of Campylobacter species. Yeast extract is a source of B vitamins, while dextrose is used as an energy source. Sheep blood provides hemin and supplies other necessary additional growth factors. The addition of antimicrobial agents, cephalothin, polymyxin B, trimethoprim, amphotericin B, and vancomycin, suppresses the growth of normal microbial flora in fecal samples, facilitating the isolation of C. jejuni. Vancomycin is a glycopeptide that inhibits many species of gram-positive bacteria, and polymyxin B inhibits most gram-negative bacilli except Proteus. Trimethoprim is an inhibitor of Proteus spp. Amphotericin B is an antifungal agent that can inhibit a wide variety of yeasts and molds. Agar is the solidifying agent. Campylobacter jejuni are thermophilic, therefore inoculated plates should be incubated at 42°C to accelerate growth; the higher temperature also helps to inhibit any background flora that may be present.
Composition of Campylobacter Blood Agar:
The composition of Campylobacter Blood Agar may vary depending on the manufacturer, but generally, it contains the following components:
| Ingredients | Grams / Liter |
|---|---|
| Agar | 15.0gm |
| Trimethoprim | 5.0mg |
| Casein peptone | 15.0gm |
| Meat peptone | 5.0gm |
| Sodium Chloride | 5.0gm |
| Yeast Extract | 2.0gm |
| Dextrose | 1.0gm |
| Vancomycin | 10.0mg |
| Amphotericin B | 0.2mg |
| Polymyxin B | 2500 U |
| Sodium Bisulfite | 0.1gm |
| Cephalothin | 15.0mg |
| Sheep Blood | 100ml |
| Demineralized water | 1000.0 ml |
- Agar – a solidifying agent that forms the base of the medium.
- Trimethoprim – to make it selective for Campylobacter species
- Casein peptone – which provides essential nutrients for the growth of Campylobacter species.
- Meat peptone – which provides essential nutrients for the growth of Campylobacter species.
- Sodium Chloride – which helps to maintain the osmotic balance of the medium and enhance the growth of Campylobacter species.
- Yeast Extract – which provides essential nutrients for the growth of Campylobacter species.
- Dextrose – which provides the necessary energy source for the growth of Campylobacter species.
- Vancomycin – for the selective isolation of Campylobacter species from clinical specimens.
- Amphotericin B – uses in Campylobacter Blood Agar
- Polymyxin B – for the selective isolation of Campylobacter species from clinical specimens.
- Sodium Bisulfite – creates an anaerobic environment that favors the growth of Campylobacter species and inhibits the growth of most other microorganisms.
- Cephalothin – for the selective isolation of Campylobacter species from clinical specimens.
- Sheep Blood – for the cultivation and identification of Campylobacter species from clinical specimens.
- Demineralized water – Demineralized water is commonly used in laboratory experiments, scientific research, and medical procedures
Preparation and Procedure of Campylobacter Blood Agar:
The preparation of Campylobacter Blood Agar involves several steps, including sterilization and aseptic techniques. Here are the general steps for preparing Campylobacter Blood Agar:
Preparation of antibiotic supplement
- Add components to 10.0ml of distilled water and filter sterilize.
- Preparation of medium
- Add components, except sheep blood and antibiotic solution, to distilled water and bring the volume to 890.0ml and mix thoroughly.
- Autoclave the Campylobacter Blood Agar Base at15 lbs pressure at 121°C for 15 minutes to sterilize it.
- After the autoclaving, Cool at 45°C-50°C. Aseptically add 100.0ml of sterile sheep blood and 10.0ml of sterile antibiotic solution.
- Mix thoroughly and pour into sterile Petri dishes or distribute into sterile tubes.
- Allow the medium to solidify and dry the surface.
- Store the Campylobacter Blood Agar plates in a cool, dry place until ready to use.
Note: All the steps should be performed under aseptic conditions using proper sterilization techniques to avoid contamination.
In summary, the preparation of Campylobacter Blood Agar involves the addition of Campylobacter selective supplement, optional trimethoprim, and sheep blood to a prepared Campylobacter Blood Agar Base. The medium is then dispensed into sterile petri dishes and allowed to solidify before use.
Result Interpretation of Campylobacter Blood Agar:
Campylobacter Blood Agar is a selective and differential medium used for the isolation and identification of Campylobacter species. Here is how to interpret the results obtained from Campylobacter Blood Agar:
- Selectivity: The selective agents cephalothin, polymyxin B, vancomycin, and amphotericin B inhibit the growth of many bacteria, except for Campylobacter species. Therefore, if there is growth on the plate, it is most likely Campylobacter species.
- Morphology: Campylobacter colonies on Campylobacter Blood Agar are usually small, gray, flat, and translucent. They have a distinct “seagull-wing” shape due to their characteristic darting motility.
- Hemolysis: Campylobacter Blood Agar is enriched with sheep blood, which allows for the differentiation of Campylobacter species based on their hemolytic activity. Campylobacter jejuni is usually beta-hemolytic, while Campylobacter coli and Campylobacter fetus are usually non-hemolytic.
- Confirmation: To confirm that the colonies are indeed Campylobacter species, various biochemical tests can be performed. The most common confirmatory tests include oxidase test, catalase test, and hippurate hydrolysis test.
- Antimicrobial susceptibility testing: Campylobacter species are commonly resistant to many antibiotics. Therefore, antimicrobial susceptibility testing is crucial to determine the appropriate treatment for Campylobacter infections.
In summary, the interpretation of results from Campylobacter Blood Agar involves observing the morphology of the colonies, their hemolytic activity, and performing confirmatory tests to identify Campylobacter species. Antimicrobial susceptibility testing is also crucial to determine the appropriate treatment for Campylobacter infections.
- Campylobacter jejuni typically appears as small, grayish, flat, mucoid colonies with irregular borders, nonhemolytic at 24 and 48 hours.
- A full 48 hour incubation is required, as some isolates may be barely visible after only 24 hours of incubation.
- Some colonies may appear as round colonies 1-2 mm in diameter that are convex, entire, and shiny.
- Propagation and swarming are common for isolates from fresh clinical specimens.
Uses of Campylobacter Blood Agar
Here is a list of the uses of Campylobacter Blood Agar:
- Isolation and identification of Campylobacter species: Campylobacter Blood Agar is the preferred medium for the isolation and identification of Campylobacter species from clinical specimens, food, and environmental samples.
- Differentiation of Campylobacter species: Campylobacter Blood Agar allows for the differentiation of Campylobacter species based on their hemolytic activity. Campylobacter jejuni is usually beta-hemolytic, while Campylobacter coli and Campylobacter fetus are usually non-hemolytic.
- Antimicrobial susceptibility testing: Campylobacter species are commonly resistant to many antibiotics. Therefore, antimicrobial susceptibility testing is crucial to determine the appropriate treatment for Campylobacter infections.
- Research: Campylobacter Blood Agar is commonly used in research to study the pathogenesis, virulence, and antimicrobial resistance of Campylobacter species.
- Quality control: Campylobacter Blood Agar is used in quality control laboratories to assess the performance of diagnostic tests for Campylobacter species.
In summary, Campylobacter Blood Agar is primarily used for the isolation, identification, and differentiation of Campylobacter species, as well as for antimicrobial susceptibility testing and research purposes.
Limitations:
Although Campylobacter Blood Agar is a useful medium for the isolation and identification of Campylobacter species, it has certain limitations that should be considered:
- Selectivity: Although Campylobacter Blood Agar is selective for Campylobacter species, some other bacteria such as Arcobacter, Helicobacter, and some other Gram-negative bacteria may also grow on the medium, leading to false-positive results.
- Sensitivity: The sensitivity of Campylobacter Blood Agar varies depending on the type of sample being tested. For example, samples with low numbers of Campylobacter species may not be detected by Campylobacter Blood Agar.
- Time-consuming: Campylobacter Blood Agar is a time-consuming medium that requires incubation at 42°C in a microaerophilic environment, which may take up to 48 hours for the isolation and identification of Campylobacter species.
- Cost: Campylobacter Blood Agar is a relatively expensive medium compared to other diagnostic media.
- Interpretation: Interpretation of Campylobacter Blood Agar results can be challenging due to the variability in the morphology of Campylobacter colonies and the presence of other bacterial growth on the medium.
In summary, although Campylobacter Blood Agar is a useful diagnostic medium for the isolation and identification of Campylobacter species, it has certain limitations that should be taken into consideration when interpreting the results.
FAQs (Frequently Asked Questions):
What is Campylobacter Blood Agar?
A: Campylobacter Blood Agar is a special type of agar used in microbiology to cultivate Campylobacter bacteria.
What is Campylobacter?
A: Campylobacter is a type of bacteria commonly found in the intestines of animals, including poultry, cattle, and sheep. It can cause illness in humans if ingested, typically through contaminated food or water.
How is Campylobacter Blood Agar prepared?
A: Campylobacter Blood Agar is prepared by mixing blood from a sheep or horse with a nutrient-rich agar and then sterilizing the mixture.
What are the ingredients of Campylobacter Blood Agar?
A: The main ingredients of Campylobacter Blood Agar are blood from a sheep or horse, peptone, yeast extract, and agar.
What is the purpose of using Campylobacter Blood Agar?
A: The purpose of using Campylobacter Blood Agar is to create a suitable environment for the growth of Campylobacter bacteria.
How is Campylobacter Blood Agar used in microbiology?
A: In microbiology, Campylobacter Blood Agar is used to isolate and identify Campylobacter bacteria from samples, such as food or clinical specimens.
What are the characteristics of Campylobacter Blood Agar?
A: Campylobacter Blood Agar is a chocolate-colored agar that is selective and differential for Campylobacter bacteria.
What is the selective agent in Campylobacter Blood Agar?
A: The selective agent in Campylobacter Blood Agar is antibiotics, which inhibit the growth of other bacteria and allow for the growth of Campylobacter bacteria.
What is the differential agent in Campylobacter Blood Agar?
A: The differential agent in Campylobacter Blood Agar is a pH indicator, which allows for the differentiation of Campylobacter species based on their ability to produce acid.
How long does it take to see growth on Campylobacter Blood Agar?
A: It typically takes 48-72 hours to see growth of Campylobacter bacteria on Campylobacter Blood Agar.
What are the colony characteristics of Campylobacter on Campylobacter Blood Agar?
A: Campylobacter colonies on Campylobacter Blood Agar are usually small, gray-white, and translucent with a slightly raised center.
What are the limitations of using Campylobacter Blood Agar?
A: The limitations of using Campylobacter Blood Agar include the need for specialized equipment and expertise, as well as the potential for contamination and false negatives.
What are the clinical symptoms of Campylobacter infection?
A: The clinical symptoms of Campylobacter infection can include diarrhea, fever, abdominal pain, and nausea.
How is Campylobacter infection treated?
A: Campylobacter infection is typically treated with antibiotics, although in mild cases, the infection may resolve on its own.
How can Campylobacter infection be prevented?
A: Campylobacter infection can be prevented by practicing good food hygiene, such as washing hands and cooking food thoroughly, and avoiding consumption of raw or undercooked meat.
How common is Campylobacter infection?
A: Campylobacter infection is one of the most common causes of foodborne illness worldwide.
How is Campylobacter diagnosed?
A: Campylobacter infection is typically diagnosed by culture of the organism from clinical specimens such as stool or blood.
Conclusion:
In conclusion, Campylobacter Blood Agar is a specialized agar used in microbiology to cultivate Campylobacter bacteria. It is prepared by mixing blood from a sheep or horse with a nutrient-rich agar and then sterilizing the mixture. The purpose of using Campylobacter Blood Agar is to create a suitable environment for the growth of Campylobacter bacteria. In microbiology, it is used to isolate and identify Campylobacter bacteria from samples such as food or clinical specimens. Campylobacter infection is one of the most common causes of foodborne illness worldwide, and it can be prevented by practicing good food hygiene, such as washing hands and cooking food thoroughly, and avoiding consumption of raw or undercooked meat.
References:
- “Campylobacter Blood-Free Selective Medium”. CDC. Accessed March 30, 2023. https://www.cdc.gov/narms/lab-training/campylobacter-blood-free-selective-medium.html.
- “Campylobacter Blood Agar”. Hardy Diagnostics. Accessed March 30, 2023. https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/CampyBloodAgar.html.
- “Campylobacter Blood Agar”. Neogen. Accessed March 30, 2023. https://www.neogen.com/categories/microbiology/campylobacter-blood-agar/.
- “Campylobacter Blood-Free Selective Medium”. Food Safety and Inspection Service. Accessed March 30, 2023. https://www.fsis.usda.gov/wps/wcm/connect/ee89584f-70e1-43c9-9df7-76d8c2b301f4/MLG-41.pdf?MOD=AJPERES.
- “Campylobacter Blood Agar”. Thermo Fisher Scientific. Accessed March 30, 2023. https://www.thermofisher.com/order/catalog/product/CM0739B.
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