Legionella BCYE Supplement ISO

Description

Legionella GVPC selective supplement, when added to the agar base, gives the antibiotic support in order to obtain a selective final medium.

The selectivity is raised by the addition of vancomycin that acts against Gram-positive bacteria, polymyxin B that inhibits Gram-negative bacteria and cycloheximide or natamycin that are antifungal agents and inhibit the yeast growth.

Technique

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

Reconstitute 1 vial of GVPC Selective Supplement with 10 mL of sterile distilled water, in aseptic conditions, and add it to 500 mL of melted Legionella BCYE Agar Base cooled to 47-50 °C, supplemented before with Legionella BCYE growth supplement. Do not overheat once supplemented.

Pour the complete medium into Petri dishes and, once solidified on a flat surface, spread the plates by streaking methodology or by MF method.

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.

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.

Reconstitute the original freeze-dried vial by adding 1 solvent vial:

Note: each vial is sufficient to supplement 500 mL of Legionella CYE Agar Base. In some cases, crystallization can occur in the vial: this does not affect either the quality or the solubility of the product after adding it to the medium.

Physical/chemical control

Microbiological control

Add 1 vial to 500 mL of medium base. DO NOT HEAT once supplemented.

Add Inhibitor Supplement (GVPC).

Analytical methodology according to ISO 11133:2014/Amd 1:2018; A2:2020.

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

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

Sterility control

Add 5 mL of the sample to 100 mL of TSB and to 100 mL of Thioglycollate.

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.