Legionella BCYE Agar w/o Cysteine ISO

Collect, dilute and prepare samples and volumes as required according to specifications, directives, official standard regulations and/or expected results.

This medium is also well suited for air environmental sampling (total compatibility with commercially available air samplers).

Spread the plates by streaking methodology or by spiral method.

Incubate the plates right side up at 36 ± 2 °C for up to 5-10 days in a chamber/jar with high humidity.

(Incubation times longer than those mentioned above or different incubation temperatures may be required depending on the sample, on the specifications, etc.)

After incubation, enumerate all the colonies that have appeared on the surface of the agar.

Colonies are usually visible after 3 to 4 days of incubation. They have a mucous, glossy, white-grey appearance and measure 2 to 3 mm in diameter.

Legionella will not grow on media which have no cysteine enrichment.

Microscopic examination shows Legionella to be extremely polymorphic, with short rod-shaped forms and long filaments.

They are Gram-negative and weakly oxidase and catalase positive.

A total identification should be carried out using immunological and chromatographic techniques.

Each laboratory must evaluate the results according to their specifications, comparatively with the results obtained using a medium with addition of cysteine.

Calculate total microbial count per mL of sample by multiplying the average number of colonies per plate by the inverse dilution factor if a diluted sample was streaked. Report results as colony forming units (CFU) per mL or g along with incubation time and temperature.

Physical/chemical control

Microbiological control

Spiral spreading: practical range 100 ± 20 CFU; min. 50 CFU (productivity) / 10⁴-10⁶ CFU (selectivity).

Microbiological control according to ISO 11133:2014/Amd 1:2018.

Aerobiosis. Incubation at 36 ± 2 °C. Reading 3-5 days.

Sterility control

Incubation 48 hours at 30-35 °C and 48 hours at 20-25 °C: NO GROWTH.

Check at 7 days after incubation in same conditions.

• ATLAS, R.M. & L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press. Boca Raton. Fla. USA.
• CLESCERI, L.S., A.E. GREENBERG & A.D. EATON (1998) Standard methods for the examination of water and wastewater. 9-106. 20th edition. APHA-AWWA-WEF. Washington DC, USA.
• EDELSTEIN, P.H. (1981) Improved semiselective medium for the isolation of Legionella pneumoniae from contaminated clinical and environmental specimens. J. Clin. Microbiol. 14(3):298.
• FEELEY, J.C., R.J. GIBSON, G.W. GORMAN, N.C. LANGFORD, J.K. RASHEED, C.D. MACKEL & W.B. BAINE (1979) Charcoal-Yeast Extract Agar: Primary isolation medium for Legionella pneumophila. J. Clin. Microbiol. 10(4) 437.
• ISO 11731 Standard (2017) Water Quality - Enumeration of Legionella.
• ISO 11133:2014/Amd 1:2018. Microbiology of food, animal feed and water. Preparation, production, storage and performance testing of culture media.
• MacFADDIN, J.F. (1985) Media for Isolation-Cultivation-Identification-Maintenance of Medical Bacteria.
• PASCULLE, A.W., J.C. FEELEY, R.J. GIBSON, L.G. CORDES, R.L. MYEROWITZ, C.M. PATTON, G.W. GORMAN, C.L. CARMACK, J.W. EZZELL & J.N. DOWLING (1980) Pittsburgh pneumonia agent: Direct isolation from human lung tissue. J. Infect. Dis., 141:727.
• UNE-EN ISO 11133 (2014). Microbiología de los alimentos para consumo humano, alimentación animal y agua - Preparación, producción, conservación y ensayos de rendimiento de los medios de cultivo.
• WARD, K.W. (1995) Processing and interpretation of specimens for Legionella spp. In "Clinical Microbiology Procedures Handbook" Chap. 12.1 edited by H.D. Isenberg. ASM Press. Washington DC, USA.