Campylobacter Blood Agar (CVA)- Composition, Principle, Preparation, Results, Uses

Campylobacter is a genus of bacteria that can cause various gastrointestinal infections in humans and animals. Campylobacter jejuni is the most common species associated with human campylobacteriosis, a diarrheal disease that can be severe and sometimes fatal. Campylobacteriosis is usually acquired through consumption of contaminated food, water, or milk, or through contact with infected animals or their feces.

Campylobacter Blood Agar (CVA) is a selective and enriched medium that contains the following ingredients per liter of distilled water:

• Peptone: 10.0 g
• Proteose peptone: 10.0 g
• Beef extract: 5.0 g
• Yeast extract: 5.0 g
• Sodium chloride: 5.0 g
• Dextrose: 1.0 g
• Agar: 15.0 g
• Sheep blood: 100.0 ml
• Antibiotic supplement: 10.0 ml

The antibiotic supplement consists of:

• Cephalothin: 15.0 mg
• Polymyxin B: 10,000 IU
• Trimethoprim: 5.0 mg
• Amphotericin B: 2.0 mg
• Vancomycin: 10.0 mg

The composition of CVA is designed to provide the optimal nutritional and environmental conditions for the growth of Campylobacter jejuni, while inhibiting the growth of other bacteria and fungi that may be present in the specimens.

The peptones and extracts provide a rich source of nitrogen, carbon, sulfur, and other essential nutrients for the bacteria. Yeast extract also supplies B vitamins that are required for bacterial metabolism.

Dextrose is a simple sugar that serves as an energy source for the bacteria. However, it is present in a low concentration to avoid acidification of the medium and interference with the oxidase test.

Sheep blood provides hemin, which is an iron-containing compound that is essential for the synthesis of cytochromes and catalase in Campylobacter jejuni. Sheep blood also provides other growth factors that may not be present in the peptones and extracts.

The antibiotic supplement contains five antimicrobial agents that selectively inhibit the growth of gram-positive bacteria, gram-negative bacilli, Proteus spp., yeasts, and molds.

Agar is a polysaccharide derived from seaweed that acts as a solidifying agent for the medium. It also helps to maintain the pH and osmotic balance of the medium.

The final pH of CVA is 7.0 +/- 0.2 at 25°C, which is suitable for the growth of Campylobacter jejuni. The medium should be stored at 2-8°C in a dark place and used within one month after preparation.

Campylobacter Blood Agar (CVA) is a selective and differential medium that allows the growth and identification of Campylobacter jejuni, a common cause of gastroenteritis in humans. C. jejuni is a gram-negative, microaerophilic, curved or spiral rod that can be isolated from fecal, food and environmental samples.

CVA works by providing a suitable environment for the growth of C. jejuni while inhibiting the growth of other bacteria that may be present in the samples. The medium contains several ingredients that have specific roles in achieving this goal:

• Peptones and extracts: These are sources of nitrogen, carbon, sulfur and other essential nutrients for the growth of C. jejuni. They also provide some B vitamins that are required by the bacteria.
• Yeast extract: This is another source of B vitamins as well as other growth factors for C. jejuni.
• Dextrose: This is a carbohydrate that serves as an energy source for C. jejuni. However, it also reduces the selectivity of the medium by allowing some oxidase-positive bacteria to grow. Therefore, it is recommended to subculture the isolates in a medium without dextrose and repeat the oxidase test for confirmation.
• Sheep blood: This is an important component that provides hemin, which is a cofactor for some enzymes involved in the metabolism of C. jejuni. It also supplies other growth factors that enhance the recovery of the bacteria. Sheep blood does not cause hemolysis by C. jejuni, which helps to differentiate it from other blood agar-hemolytic bacteria.
• Antimicrobial agents: These are substances that inhibit the growth of various bacteria that may interfere with the isolation of C. jejuni. The medium contains five antimicrobial agents: cephalothin, polymyxin B, trimethoprim, amphotericin B and vancomycin. Each of these agents has a specific spectrum of activity against different groups of bacteria.
• Agar: This is a polysaccharide that solidifies the medium and allows the formation of discrete colonies by C. jejuni.

In addition to these ingredients, CVA also requires special incubation conditions to optimize the growth and identification of C. jejuni. The medium should be incubated at 42°C in a microaerophilic atmosphere (5% O2, 10% CO2 and 85% N2) for 24 to 48 hours.

By following these principles, CVA can be used as an effective medium for the primary isolation and cultivation of C. jejuni from various samples.

To prepare Campylobacter Blood Agar (CVA), you will need the following ingredients and equipment:

• Peptone
• Proteose peptone
• Beef extract
• Yeast extract
• Sodium chloride
• Dextrose
• Agar
• Sheep blood
• Cephalothin
• Polymyxin B
• Trimethoprim
• Amphotericin B
• Vancomycin
• Distilled water
• Measuring cylinder
• Weighing balance
• Magnetic stirrer and bar
• pH meter and electrode
• Autoclave
• Water bath
• Sterile Petri dishes or tubes

The preparation of CVA involves two steps: preparing the antibiotic supplement and preparing the medium.

Preparing the antibiotic supplement

The antibiotic supplement contains cephalothin, polymyxin B, trimethoprim, amphotericin B, and vancomycin. These antibiotics are added to inhibit the growth of the normal microbial flora in fecal specimens, thereby facilitating isolation of C. jejuni.

To prepare the antibiotic supplement, follow these steps:

1. Weigh out the required amounts of each antibiotic according to the table below:

2. Add the antibiotics to 10.0 ml of distilled water in a sterile container and mix well.
3. Filter sterilize the solution using a 0.22 µm membrane filter and a syringe.
4. Label the container with the name, date, and concentration of the antibiotic supplement and store at 2-8°C until use.

Preparing the medium

The medium contains a variety of peptones and extracts that supplies all the necessary growth factors for recovery of Campylobacter species. Yeast extract is a source of B vitamins while dextrose is utilized as an energy source. Sheep blood provides hemin and supplies other necessary additional growth factors. Agar is the solidifying agent.

To prepare the medium, follow these steps:

1. Weigh out the required amounts of each ingredient according to the table below:

2. Add the ingredients, except sheep blood and antibiotic solution, to distilled water and bring the volume to 890.0 ml in a measuring cylinder. Mix thoroughly using a magnetic stirrer and bar.
3. Adjust the pH to 7.0 +/- 0.2 using a pH meter and electrode. If necessary, add 1N sodium hydroxide or hydrochloric acid to adjust the pH.
4. Transfer the medium to a suitable container and label it with the name, date, and pH of the medium.
5. Gently heat and bring to boiling in a water bath or on a hot plate.
6. Autoclave for 15 min at 15 lbs pressure at 121°C.
7. Cool at 45°C-50°C in a water bath.
8. Aseptically add 100.0 ml of sterile sheep blood and 10.0 ml of sterile antibiotic solution to the medium and mix thoroughly.
9. Pour into sterile Petri dishes or distribute into sterile tubes as desired.
10. Label the plates or tubes with the name, date, and batch number of the medium and store at 2-8°C until use.

You have now prepared Campylobacter Blood Agar (CVA) for primary isolation and cultivation of C. jejuni from fecal, food and environmental specimens.

Campylobacter jejuni is the most common species of Campylobacter that causes gastroenteritis in humans. It can be identified by its characteristic morphology and motility on CVA. The following are some of the features to look for when examining CVA plates for C. jejuni:

• Size and shape: C. jejuni colonies are usually small (0.5-2 mm in diameter), flat, and irregular in shape. They may have a moist or mucoid appearance and a grayish color. Some colonies may be round, convex, and glistening, especially if they are isolated from fresh specimens.
• Hemolysis: C. jejuni colonies are usually non-hemolytic on CVA, meaning they do not cause any change in the color or appearance of the sheep blood agar around them. However, some strains may produce weak or partial hemolysis, which can be seen as a faint greenish zone around the colonies.
• Growth pattern: C. jejuni colonies tend to spread and swarm on CVA, forming confluent growth that covers the entire surface of the plate. This is more common for isolates from fresh clinical specimens than from frozen or refrigerated ones. Swarming can be reduced by using a lower inoculum or by streaking the plate with a sterile loop or needle.
• Motility: C. jejuni is a highly motile bacterium that has a corkscrew-like movement. This can be observed under a phase-contrast microscope or by using a wet mount preparation of a colony suspended in saline. The motility of C. jejuni can also be inferred from its swarming behavior on CVA.

Other species of Campylobacter may also grow on CVA, but they have different characteristics that can help to differentiate them from C. jejuni. For example:

• Campylobacter coli is similar to C. jejuni in morphology and hemolysis, but it is less motile and less likely to swarm on CVA.
• Campylobacter fetus is inhibited by cephalothin, one of the antibiotics in CVA, and does not grow on this medium.
• Campylobacter lari is also inhibited by cephalothin and does not grow on CVA.
• Campylobacter hyointestinalis and Campylobacter upsaliensis are also inhibited by cephalothin and do not grow on CVA.

It is important to note that CVA is not a definitive medium for identifying Campylobacter species, and that further tests are required to confirm the identity of the isolates. Some of these tests include:

• Oxidase test: Campylobacter species are oxidase-positive, meaning they produce a dark purple color when exposed to an oxidizing agent such as tetramethyl-p-phenylenediamine (TMPD). However, some strains may produce weakly positive reactions when grown on CVA due to the presence of dextrose in the medium. Therefore, it is recommended to subculture the isolates on a medium without dextrose and repeat the oxidase test.
• Catalase test: Campylobacter species are catalase-positive, meaning they produce bubbles when exposed to hydrogen peroxide. This test can help to differentiate Campylobacter from Helicobacter, another genus of curved gram-negative rods that are catalase-negative.
• Gram stain: Campylobacter species are gram-negative rods that have a curved or spiral shape. They may appear as single cells or in pairs or chains. They may also show bipolar staining, meaning they have darker ends than the middle part of the cell.
• Biochemical tests: Campylobacter species can be further differentiated by their ability to ferment carbohydrates such as glucose, lactose, sucrose, and maltose; to hydrolyze urea; to reduce nitrate; and to produce indole and hippurate. These tests can be performed using commercial kits or media such as API Campy strips or Skirrow`s agar.
• Immunological tests: Campylobacter species can be identified by their antigenic properties using specific antibodies or antigens. These tests can be performed using methods such as latex agglutination, enzyme immunoassay (EIA), immunofluorescence (IF), or polymerase chain reaction (PCR).

Campylobacter Blood Agar (CVA) is an enriched selective blood agar that will support good growth of Campylobacter jejuni and is used for the primary isolation and cultivation of this organism from fecal, food and environmental specimens. Campylobacter jejuni is one of the most common causes of bacterial gastroenteritis in humans and animals worldwide. It can also cause invasive infections such as bacteremia, meningitis, endocarditis, and abortion. Therefore, it is important to detect and identify this pathogen accurately and rapidly from clinical and non-clinical samples.

CVA has several advantages over other media for the isolation of Campylobacter jejuni. First, it contains a variety of peptones and extracts that provide all the essential nutrients for the growth of this fastidious organism. Second, it contains sheep blood that supplies hemin and other growth factors that enhance the growth and colony morphology of Campylobacter jejuni. Third, it contains a combination of antibiotics that suppress the growth of most normal flora in the specimens, especially gram-positive bacteria, gram-negative bacilli, Proteus spp., and fungi. Fourth, it allows for the incubation of the plates at 42°C, which is the optimal temperature for the growth of Campylobacter jejuni and also inhibits the growth of other bacteria that may grow at lower temperatures.

CVA can be used to isolate Campylobacter jejuni from various types of specimens, such as feces, rectal swabs, stool cultures, food products (such as poultry, meat, milk, cheese, eggs), water samples, and environmental samples (such as soil, animal bedding, animal feed). The specimens should be inoculated onto CVA plates as soon as possible after collection or transport in appropriate media. The plates should be incubated in a microaerophilic atmosphere (5% O2, 10% CO2, 85% N2) at 42°C for 24 to 48 hours. The plates should be examined for typical colonies of Campylobacter jejuni that are small, grayish, flat with irregular edges, non-hemolytic, mucoid, and oxidase positive. The colonies can be confirmed by Gram staining (showing curved or spiral-shaped gram-negative rods), catalase test (positive), hippurate hydrolysis test (positive), and motility test (showing darting or corkscrew-like motility). The colonies can also be further identified by using biochemical tests (such as API Campy), serological tests (such as latex agglutination), molecular tests (such as PCR or MALDI-TOF MS), or antimicrobial susceptibility testing.

CVA is a useful medium for the primary isolation and cultivation of Campylobacter jejuni from various specimens. It provides a selective and differential environment that enhances the recovery and identification of this important pathogen. It can also be used in conjunction with other media (such as Skirrow`s agar or charcoal cefoperazone deoxycholate agar) to increase the isolation rate and diversity of Campylobacter species. CVA is recommended by several guidelines and standards for the detection of Campylobacter jejuni in clinical and non-clinical samples.

• CVA is a selective medium that inhibits most of the normal flora in fecal specimens, but it may not be able to suppress all the potential contaminants. Therefore, it is important to examine the plates carefully for any atypical colonies that may resemble Campylobacter spp. and confirm them by further tests.
• CVA is designed for the isolation and cultivation of C. jejuni, which is the most common species of Campylobacter associated with human gastroenteritis. However, there are other species of Campylobacter that may cause infections in humans or animals, such as C. coli, C. upsaliensis, C. lari, C. fetus, and C. hyointestinalis. Some of these species may not grow well or at all on CVA due to the presence of cephalothin or other antibiotics in the medium. Therefore, it is recommended to use other media or methods for the detection and identification of these species, such as charcoal-based selective media, PCR, or MALDI-TOF MS.
• CVA does not provide any differential characteristics for Campylobacter spp., such as hemolysis, motility, or pigment production. Therefore, it is necessary to perform additional tests to confirm the identity of the isolates, such as Gram stain, oxidase test, catalase test, hippurate hydrolysis test, and serotyping. However, some of these tests may be affected by the composition of CVA or the incubation conditions. For example, some Campylobacter isolates may produce weakly positive oxidase reactions when grown on CVA due to the presence of dextrose in the medium. In such cases, it is advisable to subculture the isolates on a medium without dextrose and repeat the oxidase test. Similarly, some Campylobacter isolates may show reduced motility or hemolysis when grown on CVA due to the high temperature or low oxygen tension. Therefore, it is important to use optimal conditions for these tests and interpret the results with caution.
• CVA is a qualitative medium that does not provide any quantitative information on the number or proportion of Campylobacter spp. in a specimen. Therefore, it is not suitable for epidemiological studies or surveillance purposes that require enumeration or estimation of Campylobacter load in fecal samples or food products. For such applications, other methods such as direct plating on non-selective media, membrane filtration, or most probable number (MPN) techniques may be more appropriate.

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