What is Charcoal Selective Medium?

• Charcoal Selective Medium, also known as Charcoal Cefoperazone Desoxycholate Agar (CCDA), is a culture medium used for the isolation of Campylobacter bacteria. It was first introduced in 1984 by Bolton et al., who discovered that charcoal could effectively replace blood in a culture medium for isolating Campylobacter.
• Further research and testing demonstrated that the addition of cefoperazone, an antibiotic, to the medium improved the recovery of Campylobacter compared to using cefazolin. This finding was reported by Bolton et al. in 1984. In 1986, Karmali enhanced the selectivity and recovery rate of Campylobacter spp. by adding vancomycin and cycloheximide to the medium, resulting in a higher isolation rate compared to Skirrow’s medium, another commonly used selective medium for Campylobacter isolation.
• Gun-Munro et al. conducted a study in which they evaluated six different selective media using human, animal, and avian fecal specimens. They found that CCDA demonstrated enhanced isolation of Campylobacter spp. and better suppression of commensal microbial flora compared to the other media tested.
• In 1988, Griffiths further evaluated CCDA and concluded that it was superior to other tested media in terms of recovering Campylobacter and reducing the growth of contaminating organisms.
• These findings were later confirmed by Endtz et al. in 1991, who reported a higher isolation rate for Campylobacter when using Charcoal Selective Medium.
• Overall, Charcoal Selective Medium, or CCDA, has proven to be an effective culture medium for isolating Campylobacter bacteria. Its composition, which includes charcoal, cefoperazone, desoxycholate, and other selective agents, allows for the enhanced recovery of Campylobacter while inhibiting the growth of other microbial flora.

Principle of Charcoal Selective Medium

The principle of Charcoal Selective Medium, or CCDA, lies in its composition and the specific ingredients used, which work together to create a favorable environment for the growth of Campylobacter while inhibiting the growth of other bacteria, yeast, and mold.

The medium contains beef extract, gelatin, and casein peptones, which provide a rich source of nitrogen, carbohydrates, vitamins, amino acids, and peptides. These nutrients are essential for the growth of Campylobacter bacteria.

Charcoal is included in the medium as a detoxifying agent. It reduces the oxygen tension and acts as a quenching agent for photochemically-produced toxic oxygen derivatives. This property improves the aerotolerance of Campylobacter species, allowing them to grow and survive in the culture medium.

Hematin, sodium pyruvate, and ferrous sulfate are additional supplements that enhance the aerotolerance of Campylobacter. They contribute to the stabilization of the bacteria in the medium, further promoting their growth.

Sodium chloride is included in the medium to provide essential electrolytes and maintain osmotic equilibrium. This helps to maintain the integrity of Campylobacter cells and supports their growth.

Sodium desoxycholate is a selective agent present in the medium. It inhibits the growth of certain bacteria, thereby selectively promoting the growth of Campylobacter species.

Cefoperazone, a cephalosporin antibiotic, is added to the medium. It suppresses the growth of gram-negative enteric bacilli and some gram-positive species, further enhancing the selectivity of the medium for Campylobacter.

Vancomycin, a glycopeptide antibiotic, is included to inhibit the growth of many species of gram-positive bacteria. This antibiotic contributes to the selective nature of the medium.

Cycloheximide, an antifungal agent, is added to the medium to inhibit the growth of yeast and mold contaminants that may be present in the sample.

By combining these ingredients, Charcoal Selective Medium provides a supportive environment for the growth of Campylobacter bacteria while inhibiting the growth of unwanted contaminants, facilitating the isolation and identification of Campylobacter in clinical and laboratory settings.

Composition of Charcoal Selective Medium

Ingredients	Gm/Liter
Beef extract	10.0g
Gelatin Peptone	10.0g
Sodium Chloride	5.0g
Casein Peptone	3.0g
Charcoal	4.0g
Sodium desoxycholate	1.0g
Ferrous sulfate	0.25g
Sodium Pyruvate	0.25g
Cycloheximide	100.0mg
Cefoperazone	32.0mg
Hematin	32.0mg
Vancomycin	20.0mg
Agar	12.0g

pH 7.4± 0.2 (at 25°C)

Preparation of Charcoal Selective Medium

To prepare Charcoal Selective Medium, the following steps can be followed:

1. Start with distilled or deionized water and measure the required volume to make 1000 ml of the medium.
2. Add the components of the medium to the water. These components typically include beef extract, gelatin, casein peptones, charcoal, hematin, sodium pyruvate, ferrous sulfate, sodium chloride, sodium desoxycholate, cefoperazone, vancomycin, and cycloheximide. Make sure to follow the specified amounts for each component as per the formulation.
3. Mix the contents thoroughly to ensure even distribution of the components in the water. Stir gently to avoid foaming.
4. Heat the mixture while stirring until it reaches boiling point. Continue stirring frequently to prevent any scorching or sticking.
5. Once the mixture has reached boiling point, maintain the heat and allow it to boil for a few minutes while continuing to stir. This helps to ensure proper dissolution of the components and sterilization of the medium.
6. After boiling, remove the mixture from heat and let it cool down to approximately 45-50°C. Cooling can be expedited by placing the container in a water bath or using other suitable methods.
7. Once the temperature has cooled to the desired range, the medium is ready for further processing. It is important to work under aseptic conditions to prevent contamination.
8. The medium can be poured into sterile Petri dishes or distributed into tubes, depending on the preferred format for use.
9. While dispensing the medium into the final containers, it is advisable to shake the flask gently to keep the charcoal particles in suspension. This ensures an even distribution of charcoal throughout the medium.
10. Once the medium has been poured or distributed, allow it to solidify or cool completely before sealing the containers. This prevents any external contamination from entering the medium.

By following these steps, the Charcoal Selective Medium can be prepared and utilized for the isolation and cultivation of Campylobacter bacteria in a laboratory or clinical setting.

Procedure on Charcoal Selective Medium

The procedure for using Charcoal Selective Medium (CCDA) for the isolation of Campylobacter species can be summarized as follows:

1. Inoculation and Streaking:

• As soon as the specimen is received in the laboratory, it should be inoculated and streaked onto the medium.
• Rectal swabs and liquid stools can be directly inoculated onto Charcoal Selective Medium.
• For formed stool specimens, emulsify the stool in sterile saline (0.85%) before inoculation.
• Place 1 or 2 drops of liquid or formed stool suspension onto the agar surface and streak it for isolation.

2. Incubation:

• Incubate the inoculated plates in a microaerophilic environment, which is a mixture of 5% oxygen, 10% carbon dioxide, and 85% nitrogen.
• Maintain the plates in this environment for 48-72 hours.
• The incubation temperature should be set at 40-42°C, as Campylobacter jejuni is a thermophilic organism. Additionally, setting duplicate plates to incubate at 33-37°C can allow for the growth of certain Campylobacter species.

3. Colony Observation:

• After the appropriate incubation period, observe the plates for the characteristic colonies of Campylobacter.
• Fresh medium may show flat, irregular, or spreading colonies. Some strains may appear as a thin film on the agar or form colonies that trail along the line of streaking.
• On less fresh medium, the colonies are typically 1 to 2 mm in diameter, round, convex, and glistening.
• The color of the colonies can vary, ranging from yellowish to gray or pinkish.
• It is important to note that Campylobacter colonies on Charcoal Selective Medium are nonhemolytic, meaning they do not cause the breakdown of red blood cells.

By following this procedure, the selective and supportive properties of Charcoal Selective Medium can be utilized for the isolation and observation of characteristic Campylobacter colonies, aiding in the detection and identification of Campylobacter species.

Quality Control

The quality control of Charcoal Selective Medium (CCDA) is crucial to ensure its reliability and accuracy in detecting and isolating Campylobacter species. Here is the information regarding the quality control of Charcoal Selective Medium:

1. Lot Testing: All lot numbers of Charcoal Selective Medium have undergone testing using specific quality control organisms. These organisms are used to assess the performance of the medium and ensure its acceptability. The testing conducted meets or exceeds the standards set by the Clinical and Laboratory Standards Institute (CLSI).
2. Control Organisms: The following control organisms are used for quality control testing:

• Campylobacter coli ATCC® 33559: This organism is used to evaluate the growth of Campylobacter coli on the medium. It should show good growth under microaerophilic conditions, with incubation up to 48 hours at 40-42°C.
• Campylobacter jejuni ATCC® 33291: This organism is utilized to assess the growth of Campylobacter jejuni on the medium. Similar to C. coli, it should exhibit good growth under microaerophilic conditions, with incubation up to 48 hours at 40-42°C.
• Cryptococcus neoformans ATCC® 34877: This control organism is used to test the medium’s ability to inhibit the growth of Cryptococcus neoformans. The expected result is partial to complete inhibition of this organism, as it is not the target pathogen for Charcoal Selective Medium.
• Escherichia coli ATCC® 25922, Proteus mirabilis ATCC® 12453, Pseudomonas aeruginosa ATCC® 27853, and Staphylococcus aureus ATCC® 25923: These control organisms are employed to evaluate the medium’s ability to inhibit the growth of common non-Campylobacter bacterial contaminants. The expected result is partial to complete inhibition of these organisms, as they are not the target pathogens for Charcoal Selective Medium.

3. Interpretation of Results: Quality control organisms are incubated under specific conditions as indicated, and the growth or inhibition of each organism is observed and recorded. Aberrant results, such as unexpected growth or lack of inhibition, may indicate a problem with the quality of the medium. In such cases, patient results should not be reported until the issue is resolved.

By conducting quality control testing with specific organisms and monitoring the expected growth or inhibition patterns, the quality and performance of Charcoal Selective Medium can be ensured, providing reliable and accurate results in the isolation and identification of Campylobacter species.

Result on Charcoal Selective Medium

Interpreting the results on Charcoal Selective Medium for Campylobacter can be done by observing the characteristics of the colonies formed. The medium typically supports the growth of Campylobacter jejuni, which produces two distinct types of colonies.

1. Small, raised, grayish-brown colonies with a smooth and glistening appearance, and an entire translucent edge: These colonies are typically well-defined and have a characteristic appearance. They are small in size and raised above the agar surface. The color is usually grayish-brown, and the colonies have a smooth and shiny texture. The edges of these colonies are translucent and have a uniform appearance.
2. Flat, mucoid, translucent colonies with a grayish color and irregular edge: Another colony type observed on Charcoal Selective Medium is flat in shape and appears mucoid or slimy. These colonies are translucent, meaning they allow light to pass through, and have a grayish color. The edges of these colonies are irregular in shape.

Fresh medium may exhibit colonies with a flat, irregular, or spreading morphology, and some strains may appear as thin films on the agar or form colonies that trail along the line of the streaking.

On less fresh medium, colonies tend to be slightly larger, ranging from 1-2 mm in diameter. They appear round, convex, and have a glistening appearance. The color of these colonies can vary, ranging from yellowish to grey or pinkish. It’s important to note that Campylobacter colonies on Charcoal Selective Medium are typically non-hemolytic, meaning they do not cause the breakdown of red blood cells.

By observing these colony characteristics, such as size, shape, color, texture, and edge morphology, it is possible to identify Campylobacter strains on Charcoal Selective Medium. Additional confirmatory tests may be required to definitively identify the specific species or subtypes within the Campylobacter genus.

Uses of Charcoal Selective Medium

Charcoal Selective Medium, or CCDA, has several important uses in microbiology, particularly for the selective isolation and presumptive identification of Campylobacter species. Here are the key applications of Charcoal Selective Medium:

1. Selective Isolation: Charcoal Selective Medium is primarily employed for the selective isolation of Campylobacter bacteria from various sources, including food and human fecal specimens. The medium contains selective agents such as cefoperazone, vancomycin, and sodium desoxycholate, which inhibit the growth of unwanted bacteria and promote the growth of Campylobacter. This selective nature allows for the isolation and enrichment of Campylobacter species present in complex samples.
2. Presumptive Identification: Charcoal Selective Medium can also aid in the presumptive identification of Campylobacter species. The characteristic colony morphology exhibited by Campylobacter on this medium, such as small, raised, grayish-brown colonies with a translucent edge or flat, mucoid, and irregular colonies, can provide initial clues for the identification of Campylobacter. However, it is important to note that further confirmatory tests and techniques are necessary for accurate identification at the species or subtype level.
3. Food Microbiology: Charcoal Selective Medium finds particular application in food microbiology, where it is used for the detection and isolation of Campylobacter species from food samples. Campylobacter is a common bacterial pathogen associated with foodborne illnesses, and the use of Charcoal Selective Medium enables the targeted recovery and identification of these bacteria from food matrices, aiding in the assessment of food safety and quality.
4. Clinical Microbiology: In clinical settings, Charcoal Selective Medium is employed to detect and isolate Campylobacter species from human fecal specimens. Campylobacter infections, often caused by Campylobacter jejuni or Campylobacter coli, are a leading cause of bacterial gastroenteritis worldwide. The use of Charcoal Selective Medium allows for the isolation and identification of Campylobacter in clinical samples, aiding in the diagnosis and management of Campylobacter-associated infections.

In summary, Charcoal Selective Medium is a valuable tool in microbiology laboratories, specifically for the selective isolation and presumptive identification of Campylobacter species from food and human fecal specimens. Its applications extend to areas such as food microbiology and clinical microbiology, contributing to the detection, surveillance, and control of Campylobacter infections.

Limitations of Charcoal Selective Medium

While Charcoal Selective Medium (CCDA) is a useful tool for the isolation and presumptive identification of Campylobacter species, it has certain limitations that should be considered. Here are some key limitations of Charcoal Selective Medium:

1. Complete Identification: While Charcoal Selective Medium provides initial clues for the identification of Campylobacter, it is recommended to perform additional tests such as biochemical, immunological, molecular, or mass spectrometry testing on colonies from pure culture for complete and accurate identification of Campylobacter species. These confirmatory tests are necessary to differentiate between different species or subtypes within the Campylobacter genus.
2. Incubation Time: The recommended incubation time for Charcoal Selective Medium is typically 48 hours. However, extending the incubation period to 72 hours may improve the isolation rate of Campylobacter. Some Campylobacter strains may exhibit slower growth, requiring a longer incubation period for their detection.
3. Inhibition of Certain Campylobacter Strains: Charcoal Selective Medium contains cephalosporins such as cefoperazone, which can inhibit certain strains of Campylobacter coli, Campylobacter fetus, and some strains of Campylobacter jejuni. This inhibition may lead to false-negative results or incomplete recovery of certain Campylobacter species. Alternative media or testing methods may be required to overcome this limitation.
4. Optimal Incubation Temperature: Campylobacter jejuni is a thermophilic organism and exhibits optimal growth at 42°C. Incubating Charcoal Selective Medium at this higher temperature provides selectivity by inhibiting accompanying microflora and promoting the growth of Campylobacter jejuni. Failure to incubate at the appropriate temperature may affect the recovery of Campylobacter species.
5. Selectivity Limitations: While Charcoal Selective Medium is designed to selectively inhibit unwanted bacteria and promote the growth of Campylobacter, there is a possibility that the selective agents may inhibit some strains of the desired species or allow the growth of a species it was intended to inhibit. This is especially relevant if the target species is present in large numbers in the specimens. It is important to consider culturing specimens on nonselective media as well to obtain additional information and ensure the recovery of potential pathogens.

In summary, while Charcoal Selective Medium is a valuable tool for the isolation and preliminary identification of Campylobacter species, it is essential to be aware of its limitations. Additional confirmatory tests, extended incubation periods, consideration of optimal incubation temperature, and the use of nonselective media may be necessary to overcome these limitations and obtain accurate and reliable results.

FAQ

References

1. https://exodocientifica.com.br/_technical-data/M1845.pdf
2. https://assets.fishersci.com/TFS-Assets/LSG/manuals/IFU1294.pdf
3. https://www.woah.org/fileadmin/Home/fr/Health_standards/tahm/3.09.03_CAMPYLO.pdf