In 1978, Feeley et al developed a medium for isolating Legionella species. They later modified it by replacing casein hydrolysate with beef extract and starch with activated carbon and naming it Charcoal Yeast extract (CYE) Agar. A further modification was made by Pasculle et al in 1980 by the addition of ACES (N-2-acetamido-2-aminoethane sulfonic acid) buffer in order to maintain the proper pH for optimal growth designated as BCYE for Buffered Charcoal Yeast Extract. Edelstein et al modified the medium in 1981 by adding potassium salt to alpha-ketoglutaric acids, which increased the medium’s sensitivity. It is used in primary isolation and cultivation of Legionella species. It is recommended to be used in the cultivation and primary isolation of Legionella spp.
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
Legionella species is a non-spore-forming, narrow-gram-negative rod that causes pneumonia (Legionnaires Disease) or Pontiac fever. The bacteria does not oxidize, ferment, or metabolize carbohydrates in traditional media. They also do well on sheep blood agar. Buffered Charcoal yeast extract agar is better for faster growth. The BCYE medium contains the various components that help in the isolation of and growth of the bacteria. The main sources of energy for Legionella are amino acids. L-cystine is an absolute necessity as it plays a key role in Legionella’s growth metabolism. This amino acid, along with ferric Pyrophosphate, is essential for Legionella growth. Charcoal, which acts as a detoxicant, activated charcoal decomposes hydrocarbonate, which is toxic to Legionella species. It may also collect carbon dioxide, modify surface tension, and act as a detoxicant. Yeast extract is a rich source for vitamins, nitrogen and carbon that are necessary for growth. ACES buffer maintains an optimal pH for growth, while L-cystine Hydrochloride, ferric Pyrophosphate and A-ketoglutarate stimulate Legionella species growth by stimulating oxygen-scavenging enzymes. Agar acts as the solidifying agent. If necessary, you can add select agents.
Preparation of BCYE Agar
- Take 20 grams in 500 ml of distilled water.
- To dissolve the medium completely, heat to boiling
- For 15 minutes, sterilize by heating at 15 lbs pressure (121degC), and then cooling to 50degC.
- Aseptically, add 1 vial of Legionella Supplement to sterile rehydrated.
- Mix thoroughly and pour, stirring constantly to ensure evenly distributed charcoal particles
Result Interpretation on BCYE Agar
- Legionella pneumophila is a small-to-large, smooth, colorless, pale to pale, bluegrey, slightly mucoid colony that becomes whiter with age. It fluoresces yellow green under longwave ultraviolet light.
- Although the colony surface is smooth and has precise edges, some strains can give off a “fried egg” or ground glass appearance when microscopically viewed.
Uses of Buffered Charcoal Yeast Extract (BCYE) Agar
- This medium is used to isolate and presumptive identify Legionella.
- It is used to isolate Legionella from samples taken from clinical or environmental samples.
Limitations of Buffered Charcoal Yeast Extract (BCYE) Agar
- For complete identification, it is important to perform biochemical, immunological and molecular testing on colonies grown in pure culture.
- It is recommended that you use more than one medium for the isolation of Legionella species. Also, that both non-selective BCYE Agar plates and selective BCYE Agar plates are used.
- Some strains can fail to grow or grow well on this medium because of nutritional variation.
- The Centers for Disease Control and Prevention recommend that environmental samples be incubated with 2.5% CO2 for at least two weeks. However, L. gormanii is known to have an enhanced growth rate under this condition.
- Legionella colonies that grow on white membrane filters might have a different appearance than those that grow against a dark background filter.
- It is important to avoid the formation of aerosols when handling Legionella species.
