What is BCYE Agar?

• BCYE Agar, also known as Buffered Charcoal Yeast Extract Agar, is a specialized culture medium used for the isolation and cultivation of Legionella species. It was initially developed by Feeley et al in 1978 as Charcoal Yeast Extract (CYE) Agar, but later modifications were made to enhance its effectiveness.
• The original CYE Agar consisted of yeast extract, casein hydrolysate, beef extract, and starch. However, further improvements were introduced by Pasculle et al in 1980. They substituted activated charcoal for starch and added ACES (N-2-acetamido-2-aminoethane sulfonic acid) buffer to maintain the proper pH for optimal growth. This modified version was named BCYE Agar, which stands for Buffered Charcoal Yeast Extract.
• In 1981, Edelstein et al made another modification to BCYE Agar by incorporating the potassium salt of alpha-ketoglutaric acid. This addition increased the sensitivity of the medium, making it even more effective in isolating Legionella spp.
• BCYE Agar is primarily used for the cultivation and primary isolation of Legionella species from various samples suspected of harboring these bacteria. Legionella is a type of bacteria that can cause a severe respiratory infection known as Legionnaires’ disease. It is commonly found in water sources, such as cooling towers, hot tubs, and plumbing systems.
• The composition of BCYE Agar provides an optimal environment for the growth of Legionella. The yeast extract and beef extract supply essential nutrients, while the activated charcoal acts as a selective agent, absorbing unwanted substances and inhibiting the growth of competing microorganisms. The addition of ACES buffer helps maintain the pH within the suitable range for Legionella growth.
• The potassium salt of alpha-ketoglutaric acid serves as a metabolic enhancer, improving the sensitivity of the medium to detect and support the growth of Legionella bacteria.
• BCYE Agar is widely used in clinical and environmental laboratories for the detection and isolation of Legionella species. It allows researchers and diagnosticians to identify and study these bacteria, aiding in the prevention and control of Legionnaires’ disease outbreaks.
• In summary, BCYE Agar is a specialized culture medium designed for the isolation and cultivation of Legionella species. It incorporates various modifications to enhance its effectiveness, including the substitution of yeast extract, casein hydrolysate, and beef extract for the original components, as well as the addition of activated charcoal, ACES buffer, and the potassium salt of alpha-ketoglutaric acid. BCYE Agar is an essential tool in diagnosing and researching Legionella infections, particularly in water and other samples suspected of containing the bacteria.

Composition of BCYE Agar

Ingredients	Gm/L
Yeast Extract	10.0gm
ACES Buffer	10.0gm
Activated Charcoal	2.0gm
Potassium Hydroxide	2.8gm
Alpha-Ketoglutarate	1.0gm
L-Cysteine	0.4gm
Ferric Pyrophosphate	0.25gm
Agar	12.0gm

Final pH (at 25°C) 6.9±0.2

Principle of BCYE Agar

The principle of BCYE Agar lies in its ability to provide a specialized growth environment for the isolation and cultivation of Legionella species. Legionella bacteria are non-spore-forming, gram-negative rods that can cause pneumonia (Legionnaires’ disease) or Pontiac fever. These bacteria have specific growth requirements that are not met by conventional media or sheep blood agar.

BCYE Agar is designed to enhance the growth of Legionella by incorporating specific components. Amino acids serve as the major energy source for Legionella, and L-cystine is an essential amino acid that plays a crucial role in the growth metabolism of these bacteria. The presence of L-cystine, along with ferric pyrophosphate, promotes the growth of Legionella on BCYE Agar.

The media also includes charcoal, which serves as a detoxicant. Activated charcoal within BCYE Agar decomposes hydrogen peroxide, a metabolic byproduct that is toxic to Legionella species. Charcoal can also collect carbon dioxide and modify the surface tension of the medium, providing a favorable growth environment for Legionella.

Yeast extract is another key component of BCYE Agar. It acts as a rich source of vitamins, nitrogen, and carbon, providing essential nutrients necessary for the growth of Legionella bacteria.

The addition of ACES buffer to BCYE Agar helps maintain the optimal pH for the growth of Legionella. This buffer ensures that the pH remains within a range that is conducive to the growth and survival of these bacteria.

Furthermore, the BCYE Agar formulation includes L-cystine hydrochloride, ferric pyrophosphate, and α-ketoglutarate. These components stimulate the growth of Legionella species by promoting the activity of oxygen-scavenging enzymes.

Agar, a solidifying agent, is used in BCYE Agar to provide a solid surface for bacterial growth and facilitate the isolation of individual colonies.

In certain cases, selective agents can be added to BCYE Agar to inhibit the growth of unwanted microorganisms and selectively promote the growth of Legionella, if necessary.

Overall, the principle of BCYE Agar revolves around creating an enriched and specialized medium that fulfills the specific growth requirements of Legionella bacteria. By incorporating essential amino acids, vitamins, buffers, and detoxicants, BCYE Agar provides an optimal environment for the isolation and cultivation of Legionella species from various samples suspected of harboring these bacteria.

Preparation of BCYE Agar

To prepare BCYE Agar, the following steps can be followed:

1. Suspend 20 grams of BCYE Agar in 500 ml of distilled water. Ensure that the medium is evenly dispersed in the water.
2. Heat the suspension to boiling while stirring to aid in the dissolution of the medium. Continue heating until the BCYE Agar is completely dissolved.
3. Sterilize the prepared medium by autoclaving at 15 pounds of pressure (121°C) for 15 minutes. Autoclaving helps to eliminate any potential contaminants and ensure the sterility of the medium.
4. Allow the sterilized BCYE Agar to cool down to approximately 50°C. Cooling is necessary to prevent heat damage to the medium and to allow for the subsequent addition of supplements.
5. Aseptically add the sterile rehydrated contents of 1 vial each of Legionella Supplement to the cooled BCYE Agar. The Legionella Supplement provides additional nutrients and selective agents to support the growth of Legionella bacteria.
6. Thoroughly mix the BCYE Agar with the added Legionella Supplement to ensure proper distribution. Stir the mixture continuously to evenly disperse the charcoal particles present in the medium.
7. Pour the BCYE Agar into sterile Petri dishes or other appropriate containers. It is important to maintain aseptic conditions throughout this process to prevent contamination.
8. While pouring, continue to stir the mixture to ensure that the charcoal particles are evenly distributed in the agar.
9. Allow the BCYE Agar to solidify and cool completely before using it for inoculation or storage. Solidification typically takes place at room temperature or can be expedited by refrigeration.

Inoculation Procedure on BCYE Agar

When performing the inoculation procedure on BCYE Agar, the following steps can be followed:

1. Inoculation Timing: Inoculate and streak the specimen as soon as possible after it is received in the laboratory. This helps ensure the viability and recovery of microorganisms present in the specimen. It is recommended to inoculate both selective and nonselective media to capture a wide range of microorganisms, as some may be inhibited on selective agar.
2. Swab Specimen: If a swab specimen is received, roll the swab over a small area of the BCYE Agar surface. This ensures transfer of the microorganisms onto the agar. Afterward, streak the agar surface using a sterile inoculating loop or swab to promote isolation of individual colonies.
3. Fluid Specimen: If a fluid specimen is received, inoculate the BCYE Agar by placing a portion of the specimen onto the plate. This can be achieved by using a sterile pipette or loop to transfer a small volume of the fluid onto the agar surface. Streak the plate to facilitate the isolation of colonies.
4. Incubation: Incubate the BCYE Agar plates aerobically at a temperature between 33-37°C (91-99°F). Incubation should continue for a minimum of 4 days. Legionella colonies typically require several days to develop, and growth is usually visible within 3 to 4 days. However, it is important to note that some strains may take up to two weeks to appear. Longer incubation periods may be necessary to ensure optimal recovery.
5. Colony Examination: After the appropriate incubation period, examine the BCYE Agar plates for typical colony morphology and color. Legionella pneumophila colonies are typically white with a barely discernible green color. Colonies of Tatlockia micdadei, another species of Legionella, appear shiny and purple. Note any distinct characteristics or variations in colony appearance for further analysis and identification.

By following these steps during the inoculation procedure on BCYE Agar, it is possible to promote the growth and isolation of Legionella species and other microorganisms present in the specimen. The subsequent examination of colony morphology and color aids in the preliminary identification and differentiation of different Legionella species.

Result Interpretation on BCYE Agar

When interpreting results on BCYE Agar, the following characteristics can help identify Legionella pneumophila:

1. Colony Appearance: Legionella pneumophila typically forms colonies that vary in size from small to large. The colonies may initially appear colorless or pale, but as they mature, they can develop a blue-grey hue. These colonies can also exhibit a slightly mucoid texture.
2. Color Change and Fluorescence: As the colonies age, they tend to become whiter and more filamentous. When exposed to longwave ultraviolet (UV) light, Legionella pneumophila colonies on BCYE Agar exhibit a distinct yellow-green fluorescence. This fluorescence is a key characteristic used in the identification of the bacteria.
3. Surface and Edges: The colony surface of Legionella pneumophila on BCYE Agar is generally smooth, and the edges are well-defined and precise. However, it is important to note that certain strains of Legionella pneumophila may show a unique appearance when viewed microscopically. This can include a “ground glass” appearance or a “fried egg” appearance.

When observing colonies on BCYE Agar, it is crucial to consider the combination of these characteristics for accurate interpretation. The presence of small to large, smooth, colorless to pale, blue-grey colonies that become whiter and more filamentous with age and exhibit yellow-green fluorescence under longwave UV light is indicative of Legionella pneumophila. Additionally, microscopic examination can reveal specific morphological patterns that aid in the identification process.

It is important to note that while BCYE Agar is a useful medium for the cultivation and isolation of Legionella species, further confirmatory tests are required to definitively identify Legionella pneumophila and differentiate it from other related bacteria. These tests may include serological assays, nucleic acid amplification techniques, or other specific laboratory methods.

Uses of Buffered Charcoal Yeast Extract (BCYE) Agar

Buffered Charcoal Yeast Extract (BCYE) Agar is widely used for various purposes related to the isolation and identification of Legionella bacteria. Some key uses of BCYE Agar include:

1. Isolation of Legionella: BCYE Agar is primarily employed for the isolation of Legionella species from clinical and environmental samples. Legionella is known to cause respiratory infections, including Legionnaires’ disease, and BCYE Agar provides a selective and enriched medium to support the growth of these bacteria.
2. Presumptive Identification: BCYE Agar serves as a presumptive identification tool for Legionella. The characteristic colony morphology and fluorescence exhibited by Legionella pneumophila and other Legionella species on BCYE Agar can help in the preliminary identification of these bacteria.
3. Clinical Diagnostics: BCYE Agar is utilized in clinical laboratories to culture and isolate Legionella from patient samples. It aids in the diagnosis of Legionnaires’ disease by providing a suitable medium for the growth of Legionella bacteria, which can then be further confirmed and identified through additional tests and procedures.
4. Environmental Testing: BCYE Agar is also employed in environmental testing, particularly in assessing water sources such as cooling towers, hot tubs, and plumbing systems. It helps identify and monitor the presence of Legionella in these environments, contributing to the prevention and control of Legionnaires’ disease outbreaks.
5. Research and Surveillance: BCYE Agar is used in research and surveillance activities related to Legionella. It allows for the study of Legionella species, their growth characteristics, antimicrobial susceptibility, and epidemiological investigations. BCYE Agar plays a crucial role in understanding the distribution, prevalence, and behavior of Legionella bacteria in various settings.

Overall, BCYE Agar is a valuable tool in the laboratory for the isolation and presumptive identification of Legionella. Its uses extend to clinical diagnostics, environmental testing, research, and surveillance, enabling a better understanding of Legionella-associated infections and aiding in the implementation of appropriate control measures.

Limitations of Buffered Charcoal Yeast Extract (BCYE) Agar

Buffered Charcoal Yeast Extract (BCYE) Agar, while a useful medium for the isolation and presumptive identification of Legionella, has certain limitations that should be considered. These limitations include:

1. Complete Identification: While BCYE Agar provides a preliminary identification of Legionella, complete identification requires further testing using biochemical, immunological, molecular, or mass spectrometry methods. Additional confirmatory tests are necessary to accurately identify the specific species of Legionella present in the colonies obtained from BCYE Agar.
2. Multiple Mediums: It is recommended to use more than one type of medium for isolating Legionella species. This ensures better coverage and increases the likelihood of detecting Legionella, as different species may have varied growth requirements. The use of both non-selective and selective BCYE Agar plates in parallel can enhance the recovery of Legionella.
3. Nutritional Variations: Some strains of Legionella may exhibit poor growth or fail to grow on BCYE Agar due to nutritional variations. Different strains may have specific nutrient requirements that may not be fully met by BCYE Agar alone. Therefore, the absence of growth on BCYE Agar does not completely rule out the presence of Legionella.
4. Incubation Conditions: The Centers for Disease Control and Prevention (CDC) recommends incubating environmental samples on BCYE Agar with 2.5% carbon dioxide (CO2). However, it should be noted that only L. gormanii is known to exhibit enhanced growth under this specific condition. Other species may not necessarily show improved growth under elevated CO2 levels.
5. Filter Appearance: The appearance of Legionella colonies on BCYE Agar may differ depending on whether they develop on white membrane filters or against a black or dark background filter. It is important to consider this variation when interpreting and comparing colony appearances.
6. Avoiding Aerosol Formation: Handling Legionella species should be done with caution to prevent aerosol formation. Legionella can be transmitted through inhalation of contaminated aerosols, which can pose a risk to laboratory personnel. Appropriate safety measures, including the use of personal protective equipment and proper handling techniques, should be implemented to minimize the risk of exposure.

Considering these limitations and taking appropriate measures can help overcome potential challenges and ensure accurate detection and identification of Legionella species. It is important to follow recommended protocols, use multiple methods and mediums, and prioritize safety when working with Legionella in the laboratory setting.

FAQ

References

• https://exodocientifica.com.br/_technical-data/FD142.pdf
• https://www.neogen.com/globalassets/pim/assets/original/10022/official_ncm0037_bcye-agar-legionella-isolation-medium_technical-specifications.pdf
• https://assets.thermofisher.com/TFS-Assets/LSG/manuals/IFU1343.pdf
• https://legacy.bd.com/europe/regulatory/Assets/IFU/US/L007349(07)(0207).pdf
• https://microbeonline.com/buffered-charcoal-yeast-extract-bcye-agar-composition-uses-colony-characteristics/
• https://hardydiagnostics.com/w169
• https://goldbio.com/documents/3542/BCYE%20Agar%20Protocol.pdf