Legionella Agar GVPC ISO

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 and 11731-2 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 ± 1 °C for up to 5-10 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.

Note: if the medium is used with the membrane filter method, the colour and growth of the colonies may be affected. It is advisable that the technician performs a validation of the membrane filter used.

LC-985 (20 plates, 90 mm) and LC-4730 (membrane filtration plates)

The current formulation of this medium is according to the ISO Standard 11731:2017, but BCYE Agar is based on a modification of a previously described medium.

LC-985: the selectivity is raised by the addition of vancomycin or sodium cefazolin, which act against Gram-positive bacteria, polymyxin B, which inhibits Gram-negative bacteria, anisomycin, which has a broad spectrum of activity, and cycloheximide or natamycin, which as antifungal agents inhibit the yeast growth.

LC-4730: the selectivity is raised by the addition of vancomycin, which acts against Gram-positive bacteria, polymyxin B, which inhibits Gram-negative bacteria, and cycloheximide, which as an antifungal agent inhibits the yeast growth.

Refer to the ISO Standard 11731:2017 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 2, 3, 5-10 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, 3 to 5 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.

LC-985 (20 plates, 90 mm) and LC-4730 (membrane filtration plates)

Physical/chemical control

Microbiological control

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

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 for L. pneumophila, 5-10 days for L. anisa, and 3 days for selectivity.

The reference medium is validated GVPC.

LC-4730, membrane filtration plates: microbiological control

Membrane filtration / practical range 100 ± 20 CFU, min. 50 CFU (productivity) / 10⁴-10⁶ CFU (selectivity) / ≥ 10³ CFU (specificity).

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 for L. pneumophila, 5-10 days for L. anisa, and 3 days for selectivity.

Sterility control

Incubation 48 h at 30-35 °C and 48 h 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.