Ready-to-use media
Legionella BCYE Agar ISO
Solid medium base used for the detection, isolation and enumeration of Legionella spp. from water according to the ISO Standards.
- Cat. No.
- LC-944
- Category
- Ready-to-use media
- Formats
- 2 formats
- ISO 17025 quality control
- Yes
- Standards
- ISO 11133ISO 11731
The current formulation of this medium is according to the ISO Standards 11731, but BCYE Agar is based on a modification of a previously described medium. In 1979 Feeley and collaborators described Charcoal Yeast Extract (CYE) Agar as a modification of the F-G Agar. They replaced the starch in the F-G Agar with activated charcoal and substituted yeast extract for casein hydrolysate, resulting in a better recovery of Legionella pneumophila. Pasculle, in 1980, reported that CYE Agar could be improved by buffering the medium with ACES buffer and a year later Edelstein increased the sensitivity of the medium by adding alpha-ketoglutarate, which is the present formulation (BCYE Agar).
Key features
Applications
Selective enumeration of Legionella.
Industry
Clinical, pharmaceutical/veterinary.
Standards
ISO 11133, ISO 11731.
Testing
Microbiological control according to ISO 11133:2014/Amd 1:2018.
Description and technique
Description
The medium consists of a medium base supplemented with growth factors (BCYE Agar) and the selective medium supplemented with inhibitors of undesirable accompanying flora. The yeast extract supplies the basic nutrients as the medium contains no fermentable carbohydrates. L-cysteine, ferric pyrophosphate and alpha-ketoglutarate are incorporated to satisfy the specific nutritional requirements of Legionella species.
The activated charcoal decomposes hydrogen peroxide, a toxic metabolic product, and may also collect CO₂ and modify surface tension. The addition of the buffer helps maintain the proper pH for optimal growth. The selectivity is increased by the addition of vancomycin and polymyxin B, which inhibit Gram-positive bacteria, and cycloheximide or natamycin, which are antifungal agents and inhibit the yeast growth.
Technique
Refer to the ISO Standards 11731 or other standard procedures to obtain isolated colonies from specimens and samples. Allow the inoculated plates to stand until the inocula have been absorbed. Invert the plates and incubate at 36 ± 2 °C for up to 5 days. To ensure the atmosphere in the incubator is humid, place a tray of water in the bottom of the incubator. Top up this tray with fresh water (if necessary) each time the plates are examined. Incubation in an atmosphere of air with 2.5% (volume fraction) CO₂ may be beneficial for the growth of some Legionella, but it is not essential.
Examine the plates with a plate microscope on at least three occasions at intervals of 2 to 4 days during the 10-day incubation period, as Legionella grow slowly and can be masked by the growth of other organisms. Record the number of each type of colony present.
Colonies of Legionella are often white-grey-blue-purple in colour, but may be brown, pink, lime-green or deep-red. They are smooth with smooth edges and exhibit a characteristic ground-glass appearance. Under ultraviolet light colonies of several species autofluoresce brilliant white, but others are red and L. pneumophila appears dull green often tinged with yellow. All presumptive colonies must be confirmed by cultural, biochemical, serological or genetic methods.
Presentation
| Code | Presentation | Filling | Packaging |
|---|---|---|---|
| LC-944 | 20 prepared plates, 90 mm | 22 ± 2 mL | 1 box with 2 packs of 10 plates/pack. Single cellophane. |
| LC-4737 | 30 membrane filtration plates, 55 mm, for filtration purposes | 12 ± 1 mL | 1 box containing 6 plastic bags with 5 plates of 55 mm/bag. |
Composition
| Ingredient | g/L |
|---|---|
| Activated charcoal | 2.0 |
| Yeast extract | 10.0 |
| ACES buffer | 10.0 |
| Potassium hydroxide | 2.8 |
| Alpha-ketoglutarate | 1.0 |
| Cysteine | 0.4 |
| Ferric pyrophosphate | 0.25 |
| Agar | 15.0 |
Quality control
Physical/chemical control
| Color | Black |
|---|---|
| pH | 6.8 ± 0.2 at 25 °C |
Microbiological control
Inoculate: practical range 100 ± 20 CFU; min. 50 CFU (productivity).
Microbiological control according to ISO 11133:2014/Amd 1:2018.
Aerobiosis. Incubation at 36 ± 2 °C. Reading 3-5 days.
| Microorganism | Growth |
|---|---|
| Legionella pneumophilaATCC 33152, WDCM 00107 | Good (≥ 70%) |
LC-4737: microbiological control
Membrane filtration; practical range 100 ± 20 CFU; min. 50 CFU (productivity).
Microbiological control according to ISO 11133:2014/Amd 1:2018.
Analytical methodology according to ISO 11133:2014/Amd 1:2018; A2:2020.
Aerobiosis. Incubation at 36 ± 2 °C. Reading 2-5 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.
Bibliography
- 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.
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