Charcoal Selective Medium- Composition, Principle, Preparation, Results, Uses

Charcoal selective medium (CSM) is a complex and selective medium that contains the following ingredients and their quantities per liter of distilled water:

• Beef extract: 10 g
• Gelatin peptone: 10 g
• Casein peptone: 10 g
• Sodium chloride: 5 g
• Sodium desoxycholate: 0.1 g
• Charcoal: 10 g
• Hematin: 0.005 g
• Sodium pyruvate: 0.1 g
• Ferrous sulfate: 0.05 g
• Cefoperazone: 0.032 g
• Vancomycin: 0.01 g
• Cycloheximide: 0.002 g
• Agar: 15 g

The final pH of the medium is adjusted to 7.4 ± 0.2 at 25°C.

Each ingredient has a specific role in the medium, as explained in the principle section. The medium is prepared by dissolving the components in water, heating to boiling, autoclaving, cooling, and pouring into sterile plates or tubes.

The medium is dark gray to black in color due to the presence of charcoal. The medium should be stored at 2-8°C and used within one month of preparation. The medium should not be used if there are signs of deterioration, such as discoloration, contamination, or changes in physical appearance.

The medium can be supplemented with other antibiotics or substances to enhance its selectivity or specificity for certain Campylobacter species. For example, trimethoprim can be added to inhibit Proteus and Escherichia coli; polymyxin B can be added to inhibit Pseudomonas and Aeromonas; and cefixime can be added to inhibit Salmonella and Shigella.

The composition of charcoal selective medium may vary slightly depending on the manufacturer or the source of the recipe. However, the basic ingredients and their functions remain the same. The medium should be used according to the instructions provided by the manufacturer or the reference method.

Charcoal selective medium is a type of agar-based medium that is designed to selectively isolate and identify Campylobacter species from various sources, such as food and human fecal specimens. Campylobacter species are gram-negative, microaerophilic, spiral-shaped bacteria that can cause gastroenteritis, enteritis, and other infections in humans and animals.

The principle of charcoal selective medium is based on the following aspects:

• The medium contains charcoal, which acts as a detoxifying agent and reduces the oxygen tension in the medium. This creates a microaerobic environment that is favorable for the growth of Campylobacter species, which are sensitive to high levels of oxygen.
• The medium also contains hematin, sodium pyruvate, and ferrous sulfate, which improve the aerotolerance of Campylobacter species. These supplements are thought to act as quenching agents of photochemically-produced toxic oxygen derivatives, such as hydrogen peroxide and superoxide radicals, that can damage the bacterial cells.
• The medium provides nutrients for the growth of Campylobacter species, such as beef extract, gelatin peptone, and casein peptone, which supply nitrogen, carbohydrates, vitamins, and other essential elements. Sodium chloride maintains the osmotic balance of the medium and the cells.
• The medium incorporates selective agents, such as sodium desoxycholate, cefoperazone, vancomycin, and cycloheximide, which inhibit the growth of other bacteria and fungi that may contaminate the specimens. Sodium desoxycholate is a bile salt that inhibits some gram-negative and gram-positive bacteria. Cefoperazone is a cephalosporin antibiotic that suppresses the growth of gram-negative enteric bacilli and some gram-positive species. Vancomycin is a glycopeptide antibiotic that inhibits many species of gram-positive bacteria. Cycloheximide is an antifungal agent that inhibits yeast and mold.

By using charcoal selective medium, one can selectively isolate and presumptively identify Campylobacter species based on their colony morphology and biochemical characteristics. The medium can also be used in conjunction with other methods, such as immunological, molecular, or mass spectrometry testing, for complete identification and confirmation of Campylobacter species.

To prepare the charcoal selective medium, you will need the following ingredients and equipment:

• Beef extract: 3 g
• Gelatin peptone: 10 g
• Casein peptone: 10 g
• Sodium chloride: 5 g
• Sodium desoxycholate: 0.1 g
• Charcoal: 10 g
• Hematin: 0.005 g
• Sodium pyruvate: 0.1 g
• Ferrous sulfate: 0.05 g
• Cefoperazone: 0.032 g
• Vancomycin: 0.01 g
• Cycloheximide: 0.002 g
• Distilled or deionized water: 1000 ml
• pH indicator (phenol red or bromothymol blue): optional
• Autoclave
• Balance
• Measuring cylinder
• Flask or bottle
• Magnetic stirrer and bar
• Sterile Petri dishes or tubes

Follow these steps to prepare the medium:

1. Weigh the beef extract, gelatin peptone, casein peptone, sodium chloride, sodium desoxycholate, charcoal, hematin, sodium pyruvate, ferrous sulfate, cefoperazone, vancomycin, and cycloheximide and transfer them to a flask or bottle.
2. Add distilled or deionized water to make up the volume to 1000 ml. Mix thoroughly using a magnetic stirrer and bar.
3. Optionally, you can add a few drops of pH indicator to check the pH of the medium. It should be around 7.4 ± 0.2 at 25°C. If needed, adjust the pH by adding small amounts of hydrochloric acid or sodium hydroxide.
4. Gently heat the medium and bring it to boiling with frequent stirring. Be careful not to boil over or burn the medium.
5. Autoclave the medium for 15 minutes at 15 psi pressure at 121°C.
6. Cool the medium to 45-50°C in a water bath or on a benchtop.
7. Pour the medium into sterile Petri dishes or distribute it into tubes as desired. Shake the flask or bottle while dispensing to keep the charcoal in suspension.
8. Allow the medium to solidify and store it in a refrigerator until use.

The charcoal selective medium is designed to isolate and identify Campylobacter species, which are gram-negative, microaerophilic, spiral-shaped bacteria that cause gastroenteritis in humans and animals. Campylobacter species are fastidious and require special conditions for growth, such as low oxygen tension, high humidity, and elevated temperature. The charcoal selective medium provides these conditions and also inhibits the growth of other bacteria and fungi by the addition of antibiotics and antifungal agents.

The most common Campylobacter species isolated from human fecal specimens are Campylobacter jejuni and Campylobacter coli. These species can be differentiated from other bacteria by their characteristic morphology and motility on the charcoal selective medium. Campylobacter jejuni produces two types of colonies on the medium:

• One is small, raised, grayish-brown, smooth and glistening with an entire translucent edge. This type of colony is typical of most strains of C. jejuni and indicates a high level of aerotolerance.
• The other colony type is flat, mucoid, translucent, grayish and has an irregular edge. This type of colony is typical of some strains of C. jejuni that have low aerotolerance and produce more exopolysaccharide.

Campylobacter coli produces colonies that are similar to the flat type of C. jejuni, but they are usually smaller and more opaque. They may also have a pinkish hue due to the production of porphyrins.

Other Campylobacter species, such as Campylobacter fetus and Campylobacter lari, may also grow on the charcoal selective medium, but they are less common and have different colony characteristics. For example, C. fetus produces small, convex, white to cream-colored colonies that may be surrounded by a clear zone of hemolysis. C. lari produces small, flat, grayish-white colonies that may have a metallic sheen.

To confirm the identity of the Campylobacter species isolated from the charcoal selective medium, further tests are required, such as biochemical tests (e.g., catalase, oxidase, hippurate hydrolysis), immunological tests (e.g., latex agglutination), molecular tests (e.g., PCR), or mass spectrometry (e.g., MALDI-TOF). These tests can also help to differentiate between subspecies and strains of Campylobacter that may have different clinical significance and epidemiology.

Charcoal selective medium is a useful tool for the detection and identification of Campylobacter species, which are gram-negative, microaerophilic, spiral-shaped bacteria that cause gastroenteritis in humans and animals. Campylobacter species are among the most common causes of bacterial diarrhea worldwide, and can also cause systemic infections, such as bacteremia, meningitis, endocarditis, and abortion. Campylobacter infections are usually acquired through the consumption of contaminated food, water, or milk, or through contact with infected animals or their feces.

Charcoal selective medium is designed to enhance the growth and recovery of Campylobacter species from various specimens, such as food and human fecal samples. The medium contains charcoal, hematin, sodium pyruvate, and ferrous sulfate, which act as oxygen scavengers and improve the aerotolerance of Campylobacter species. The medium also contains sodium desoxycholate, cefoperazone, vancomycin, and cycloheximide, which inhibit the growth of other bacteria, fungi, and yeasts that may interfere with the isolation of Campylobacter species.

Charcoal selective medium allows for the presumptive identification of Campylobacter species based on their characteristic colony morphology and motility. Campylobacter jejuni, the most common species isolated from human infections, produces two types of colonies: one is small, raised, grayish-brown, smooth and glistening with an entire translucent edge; the other is flat, mucoid, translucent, grayish and has an irregular edge. Campylobacter coli, another common species associated with human infections, produces similar colonies but may be slightly larger and more opaque. Other Campylobacter species may produce different colony types or may not grow well on charcoal selective medium.

Charcoal selective medium is not sufficient for the definitive identification of Campylobacter species. Further tests, such as biochemical reactions, immunological assays, molecular methods, or mass spectrometry analysis are required to confirm the identity and speciation of the isolates. Additionally, charcoal selective medium may not be able to detect all Campylobacter species or strains that may be present in a specimen. Therefore, it is recommended that specimens cultured on charcoal selective medium should also be cultured on nonselective media to ensure the recovery of potential pathogens.

Charcoal selective medium is a valuable diagnostic tool for the detection and identification of Campylobacter species from food and human fecal specimens. It provides a selective and differential environment that enhances the growth and recovery of Campylobacter species while inhibiting other microorganisms. It also allows for the presumptive identification of Campylobacter species based on their colony morphology and motility. However, charcoal selective medium has some limitations and should be used in conjunction with other methods for the complete identification and characterization of Campylobacter species.

• Charcoal Selective Medium is not sufficient for the definitive identification of Campylobacter species. It is recommended that biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for complete identification.
• Extending incubation to 72 hours may increase the isolation rate of Campylobacter, but it may also increase the risk of overgrowth by other microorganisms.
• Campylobacter coli, Campylobacter fetus and some strains of Campylobacter jejuni are inhibited by cefoperazone, which is one of the selective agents in the medium.
• Incubation of Campylobacter jejuni should be carried out at 42°C as it is a thermophilic organism. Higher temperature imparts selectivity by inhibiting accompanying microflora and promotes the growth of Campylobacter jejuni. However, some strains of Campylobacter may not grow well at this temperature and may require lower temperatures or longer incubation times.
• The agents in a selective medium may inhibit some strains of the desired species or permit growth of a species it was designed to inhibit, especially if the species is present in large numbers in the specimens. Specimens cultured on selective media should, therefore, should also be cultured on nonselective media to obtain additional information and help ensure recovery of potential pathogens.
• Charcoal Selective Medium may not be suitable for isolating Campylobacter from environmental or veterinary samples, as it may not be able to suppress the growth of other bacteria that are present in these sources.

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