Buffered Charcoal Yeast Extract (BCYE) Agar

Buffered charcoal yeast extract agar (BCYE) was developed by Feeley et al and then further modified by Edelstein for selective cultivation of Legionella species. BCYE agar is an enrichment medium that is used for the isolation and cultivation of Legionella species from environmental samples and clinical specimens.

The Legionellaceae are aerobic and nutritionally fastidious organisms. They require L-cysteine and iron salts for growth. Growth on BCYE agar after 3 or more days of incubation with no growth on blood agar and/or chocolate agar is one of the most useful presumptive clues that an isolate could be a species of Legionella.

The clinician should inform the laboratory if infection with Legionella is suspected so that the appropriate media are set up. BCYE agar provides L-cysteine and other nutrients required for the growth of Legionella pneumophila, the causative agent of Legionnaires’ disease.

Composition of BCYE Agar

1. Yeast extract: Yeast extract supplies the protein and other nutrients necessary to support growth
2. L-cysteine HCl: L-cysteine is an essential amino acid incorporated to satisfy specific nutritional requirements of Legionella species
3. Charcoal activated: Activated charcoal decomposes hydrogen peroxide, a metabolic product toxic to Legionella species, and may also collect carbon dioxide and modify the surface tension
4. Ferric pyrophosphate: Used as an iron supplement; iron source
5. ACES (N-(2-Acetamido)-2-aminoethanesulfonic acid) buffer: ACES buffer is added to maintain the proper pH for optimal growth.
6. Ketoglutarate monopotassium salt: Alpha-ketoglutarate is added to stimulate growth
7. Agar

Final pH ( at 25°C) 6.9±0.2

Preparation

1. Suspend 20 grams in 500 ml distilled water.
2. Heat to boiling to dissolve the medium completely.
3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
4. Cool to 50°C.
5. Aseptically add sterile rehydrated contents of 1 vial each of Legionella supplements.
6. Mix well and pour with constant stirring to ensure that charcoal particles get evenly distributed.

Quality Control

 Legionella pneumophila ATCC 33153 grows well on BCYE agar and gives white grey to blue-grey colonies.

Colony Characteristics

After 3-4 days of incubation, small white colonies of Legionella appear in BCYE agar which becomes grey in a few days. The colonies are round and convex with entire edges. The isolate may show a typical ground glass (or iridescent) colony appearance.

Colonies may be examined under long-wave UV light (360 nm ± 20nm) to reveal yellow pigment on BCYE or auto-fluorescence of Legionella colonies.

References

1. Madigan Michael T, Bender, Kelly S, Buckley, Daniel H, Sattley, W. Matthew, & Stahl, David A. (2018). Brock Biology of Microorganisms (15th Edition). Pearson.
2. Color Atlas and Textbook of Diagnostic Microbiology, Koneman, 5th edition
3. Raad, I., Rand, K., & Gaskins, D. (1990). Buffered charcoal-yeast extract medium for the isolation of brucellae. Journal of clinical microbiology, 28(7), 1671–1672. https://doi.org/10.1128/jcm.28.7.1671-1672.1990