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BCYE agar plates and legionella culture

About microscopic forms of life, including Bacteria, Archea, protozoans, algae and fungi. Topics relating to viruses, viroids and prions also belong here.

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BCYE agar plates and legionella culture

Postby lomha » Tue Dec 13, 2011 2:42 pm

Hi every body,
'm new in this forum, I'm looking for a detailed protocol for legionella pneumophila culture on BCYE agar plates.
Thanks in advance
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Postby JorgeLobo » Thu Dec 29, 2011 1:24 pm

What do you want? It's fairly straightforward with addition of characteristic fluorescense under long wave UV.

Here is one supplier's description
http://www.neogen.com/Acumedia/pdf/ProdInfo/7728_PI.pdf
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Postby LedBulb900Lumen » Thu Jan 05, 2012 2:37 pm

Intended Use
BCYE Agar Base is used for the isolation of Legionella spp.
Product Summary and Explanation
In 1977, McDade et al. identified Legionella pneumophila as the causative agent of Legionnaires’ disease, a multisystem disease manifested primarily by pneumonia.1,2 In 1978 a new medium, F-G Agar, resulted in improved growth of L. pneumophila, a very fastidious organism.3 Freely et al. modified F-C Agar by substituting yeast extract as a vitamin source and replacing starch with activated charcoal, producing Charcoal Yeast Extract (CYE) Agar.4 In 1980, Pasculle et al. reported that CYE Agar could be improved by the addition of ACES (N-2-acetamido-2-aminoethane sulfonic acid) buffer.5 One year later, Edelstein further increased the sensitivity of the medium by adding the potassium salt of alpha-ketoglutaric acid.6
Principles of the Procedure
Yeast Extract provides sources of nitrogen, carbon, and vitamins in BCYE Agar Base. Activated Charcoal decomposes hydrogen peroxide, a metabolic product toxic to Legionella spp., and may also collect carbon dioxide and modify surface tension. ACES Buffer is added to maintain the proper pH for optimal growth. -Ketoglutarate stimulates organism growth. Ferric Pyrophosphate supplies iron. Agar is the solidifying agent. BCYE Agar is supplemented with L-Cysteine, an essential amino acid incorporated to satisfy specific nutritional requirements of Legionella spp. Selective agents can be added if necessary.
Formula / Liter Supplements / 10 mL
Yeast Extract ........................................................................... 10 g L-Cysteine (4%), sterile
ACES Buffer ............................................................................ 10 g
Charcoal, Activated ................................................................ 1.5 g
-Ketoglutarate .......................................................................... 1 g
Ferric Pyrophosphate ........................................................... 0.25 g
Agar ......................................................................................... 15 g
Final pH: 6.9 ± 0.2 at 25C
Formula may be adjusted and/or supplemented as required to meet performance specifications.
Precaution
1. For Laboratory Use.
2. IRRITANT. Irritating to eyes, respiratory system, and skin.
Directions
1. Suspend 38 g of the medium in 900 mL of purified water.
2. Adjust pH to 6.9 with 1N KOH.
3. Add water to bring volume to 1000 mL.
4. Heat to boiling with stirring to dissolve.
5. Autoclave at 121C for 15 minutes. Cool to 45 - 50C.
6. Aseptically add 10 mL of a sterile solution of L-Cysteine (4%).
7. Mix and add inhibitor solutions if required.
8. Dispense with agitation.
Quality Control Specifications
Dehydrated Appearance: Powder is homogeneous, free flowing and grey-black.
Prepared Appearance: Prepared medium is opaque and black.
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Postby JorgeLobo » Wed Jan 25, 2012 2:19 pm

The cut and paste misses the application of long wave UV light and addition of selective agents. Unless you're dealing with a pure culture, you''ll ned to differentiate the target organism from other bugs. With UV you can rule out some other miroorganisms. Do you jnow how to differentiate Legionaella spp?
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