BCYE Agar- Composition, Principle, Preparation, Results, Uses

BCYE agar stands for Buffered Charcoal Yeast Extract agar, which is a selective growth medium used to culture or grow certain types of bacteria, particularly the Gram-negative species Legionella pneumophila. Legionella pneumophila is the causative agent of Legionnaires` disease, a severe form of pneumonia, and Pontiac fever, a milder flu-like illness. Legionella bacteria are found in natural and artificial water sources, such as cooling towers, hot tubs, fountains, and plumbing systems. They can infect humans when they inhale aerosols or droplets containing the bacteria.

BCYE agar is a complex medium that contains various ingredients to support the growth of Legionella species. The main components of BCYE agar are:

• Buffered charcoal: Charcoal acts as a detoxifying agent that neutralizes toxic substances that may inhibit the growth of Legionella. It also helps to decompose hydrogen peroxide, a metabolic by-product that can harm the bacteria. Charcoal may also enhance the carbon dioxide concentration and modify the surface tension of the medium.
• Yeast extract: Yeast extract provides a rich source of vitamins, nitrogen, and carbon that are essential for the growth of Legionella. It also contains some amino acids that can be utilized by the bacteria as energy sources.
• L-cystine: L-cystine is an amino acid that is required for the growth of Legionella. It plays a vital role in the metabolism of the bacteria and stimulates the activity of oxygen-scavenging enzymes. L-cystine also helps to maintain the optimal pH of the medium.
• Ferric pyrophosphate: Ferric pyrophosphate is a source of iron that is needed for the growth of Legionella. Iron is involved in various enzymatic reactions and electron transport processes in the bacteria. Ferric pyrophosphate also enhances the activity of oxygen-scavenging enzymes along with L-cystine.
• α-ketoglutarate: α-ketoglutarate is another amino acid that stimulates the growth of Legionella. It acts as an intermediate in the Krebs cycle and provides energy for the bacteria. α-ketoglutarate also helps to maintain the optimal pH of the medium along with L-cystine.
• ACES buffer: ACES buffer is a buffering agent that helps to maintain the pH of the medium at 6.9±0.2. This pH range is favorable for the growth of Legionella species.
• Agar: Agar is a solidifying agent that gives the medium a gel-like consistency. Agar also helps to prevent the loss of moisture and nutrients from the medium.

BCYE agar is a selective and differential medium that supports the growth of Legionella species, which are the causative agents of Legionnaires` disease and Pontiac fever. Legionella species are fastidious bacteria that require special nutritional and environmental conditions for optimal growth. They do not grow on conventional media or blood agar, and they are difficult to isolate from clinical and environmental samples that may contain other microorganisms.

BCYE agar provides the essential nutrients and factors that Legionella species need for growth. The main components of BCYE agar are:

• Buffered charcoal: Charcoal acts as a detoxifying agent that neutralizes toxic substances that may inhibit the growth of Legionella. It also absorbs hydrogen peroxide, which is a metabolic by-product that can harm the bacteria. Charcoal may also enhance the carbon dioxide concentration and modify the surface tension of the medium.
• Yeast extract: Yeast extract is a rich source of vitamins, nitrogen, and carbon that support the growth of Legionella. It also provides some amino acids that are required by the bacteria.
• L-cystine: L-cystine is an essential amino acid that Legionella species cannot synthesize. It plays a crucial role in the metabolism and energy production of the bacteria. L-cystine also stimulates the activity of oxygen-scavenging enzymes that protect the bacteria from oxidative stress.
• Ferric pyrophosphate: Ferric pyrophosphate is a source of iron that Legionella species need for growth. Iron is involved in various enzymatic reactions and electron transport processes in the bacteria. Ferric pyrophosphate also enhances the activity of oxygen-scavenging enzymes.
• α-ketoglutarate: α-ketoglutarate is an intermediate of the Krebs cycle that provides energy and carbon skeletons for Legionella species. It also stimulates the activity of oxygen-scavenging enzymes.
• Agar: Agar is a solidifying agent that allows the formation of discrete colonies of Legionella on the surface of the medium.

To prepare BCYE Agar, you will need the following ingredients and equipment:

• BCYE Agar powder (20 g)
• Distilled water (500 ml)
• Legionella Supplement (1 vial)
• Autoclave
• Water bath
• Sterile Petri dishes
• Sterile pipettes
• Stirring rod

Follow these steps to prepare BCYE Agar:

1. Measure 500 ml of distilled water and pour it into a flask or beaker.
2. Add 20 g of BCYE Agar powder to the water and mix well until dissolved. You may need to heat the mixture slightly to help dissolve the powder.
3. Sterilize the mixture by autoclaving at 121°C and 15 psi for 15 minutes. Make sure the container is loosely capped to allow steam escape.
4. After autoclaving, cool the mixture to 50°C in a water bath. Do not let it solidify.
5. Aseptically add the contents of one vial of Legionella Supplement to the mixture and stir well to distribute evenly.
6. Pour the mixture into sterile Petri dishes with constant stirring to ensure that charcoal particles are evenly distributed. You can use a sterile pipette to transfer the mixture if needed.
7. Allow the agar to solidify at room temperature or in a refrigerator.
8. Store the prepared plates in a sealed plastic bag at 2-8°C until use. Do not use plates that show signs of contamination or deterioration.

The colonies of Legionella species on BCYE agar can be identified by their morphology, color, and fluorescence under longwave ultraviolet light. The most common species that causes human disease is Legionella pneumophila, which produces small to large, smooth, colorless to pale, blue-grey, slightly mucoid colonies becoming whiter and more filamentous with age. These colonies also fluoresce yellow-green under longwave UV light.

Other species of Legionella may have different characteristics on BCYE agar. For example, Legionella micdadei produces small, rough, white to tan colonies that do not fluoresce. Legionella bozemanii produces small, smooth, white to grey colonies that fluoresce blue-white. Legionella dumoffii produces small, smooth, white to yellow colonies that fluoresce blue.

The identification of Legionella species on BCYE agar should be confirmed by biochemical, immunological, molecular, or mass spectrometry testing on pure cultures. Some species may require additional supplements or growth conditions for optimal isolation. BCYE agar should be used in conjunction with other media and tests for the diagnosis of Legionnaires` disease.

Buffered Charcoal Yeast Extract (BCYE) Agar is a specialized medium that is used for the isolation and presumptive identification of Legionella species. Legionella are gram-negative bacteria that can cause serious respiratory infections such as Legionnaires` disease and Pontiac fever. These bacteria are ubiquitous in natural and artificial water sources, such as cooling towers, hot tubs, fountains, and plumbing systems. They can also contaminate medical devices, such as humidifiers, nebulizers, and respiratory therapy equipment.

BCYE Agar is designed to provide optimal conditions for the growth of Legionella, which have complex nutritional requirements and are sensitive to environmental factors. The medium contains amino acids, especially L-cystine, which are essential for the energy metabolism of Legionella. It also contains ferric pyrophosphate and α-ketoglutarate, which stimulate the growth of Legionella by activating oxygen-scavenging enzymes. The medium also contains charcoal, which acts as a detoxifying agent and neutralizes hydrogen peroxide, a toxic by-product of Legionella metabolism. Charcoal also helps to maintain the pH and surface tension of the medium. Yeast extract provides vitamins, nitrogen, and carbon sources for the bacteria. Agar is the solidifying agent that allows the formation of discrete colonies.

BCYE Agar can be used as a base medium for the isolation of Legionella from clinical and environmental samples. Clinical samples include sputum, bronchoalveolar lavage fluid, lung tissue, pleural fluid, blood, urine, and wound exudates. Environmental samples include water, biofilm, swabs, and filters. The samples are inoculated onto BCYE Agar plates and incubated at 35-37°C for 3-10 days in a humidified atmosphere with or without 2.5% CO2. The plates are examined daily for the presence of characteristic colonies of Legionella.

BCYE Agar can also be supplemented with selective agents to inhibit the growth of other bacteria and fungi that may interfere with the isolation of Legionella. Some of the commonly used selective agents are polymyxin B, anisomycin, vancomycin, cefamandole, trimethoprim lactate, and cycloheximide. These agents can be added individually or in combination to the medium before or after sterilization. The selective BCYE Agar plates are inoculated and incubated in the same manner as the non-selective ones.

BCYE Agar is a useful tool for the detection and identification of Legionella species in various samples. However, it is not sufficient to confirm the diagnosis of Legionella infections or to differentiate between different species or serogroups of Legionella. Therefore, it is recommended that additional tests, such as biochemical, immunological, molecular, or mass spectrometry methods, be performed on pure cultures of suspected colonies for complete identification and characterization.

• BCYE Agar is not a definitive medium for the identification of Legionella species. It is recommended that biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for complete identification.
• BCYE Agar may not support the growth of all Legionella species or strains. It is recommended that more than one type of medium is used for isolating Legionella species and that non-selective and selective BCYE Agar plates be inoculated in parallel.
• BCYE Agar may be contaminated by other gram-negative bacilli that grow on the medium. Selective agents such as antibiotics can be added to the medium to inhibit the growth of unwanted flora, but they may also affect the growth of some Legionella strains.
• BCYE Agar may require different incubation conditions depending on the source and type of sample. The Centers for Disease Control and Prevention (CDC) recommend incubation of environmental samples with 2.5% CO2; however, L. gormanii is the only known species with enhanced growth under this condition.
• BCYE Agar may produce variable colony morphology depending on the background color of the medium or filter. Colonies of Legionella that develop on white membrane filters may have a different appearance to those that develop against a black or dark background filter.
• BCYE Agar may pose a risk of aerosol formation when handling Legionella species. It is important to avoid creating aerosols and to follow appropriate biosafety precautions when working with this medium.

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