Plot No. C40, Road No. 21Y, MIDC, Wagle Industrial Estate,
Thane(W)-400604, MH, India.
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Intended Use
Recommended for selective isolation and cultivation of Legionella species from cooling towers, clinical and other specimens.
Composition
| Ingredients | g/L |
|---|---|
| Yeast extract | 10.000 |
| Charcoal activated | 2.000 |
| ACES buffer | 10.000 |
| a-Ketoglutarate monopotassium salt | 1.000 |
| Agar | 17.000 |
| Final pH (at 25°C) | 6.9±0.2 |
Formula adjusted, standardized to suit performance parameters
Directions
Suspend 20.0 grams in 500 ml purified/distilled water. Add 2.4 grams of KOH pellets and mix to dissolve. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Cool to 45-50°C. Aseptically add sterile rehydrated contents of 1 vial each of MWY Selective Supplement (FD040) and Legi Growth Supplement w/o SS (Twin Pack) (FD041A). Mix well and pour with constant stirring into sterile Petri plates, taking care that charcoal particles get evenly distributed. For additional selectivity, VCAT Selective Supplement (FD017), C3V Selective Supplement (FD037) and PGV Selective Supplement (FD038) may be added to molten medium as per choice.
Principle And Interpretation
Feeley et al (1) originally formulated Charcoal Yeast Extract (CYE) Agar. This medium was a modification of the existing F-G Agar (2). F-G Agar had starch and casein enzymic hydrolysate as ingredients in the composition. Feely et al (1,2) replaced these two with charcoal and yeast extract respectively, and reported better recovery of Legionella pneumophilla. Later Paseulle (3) reported that supplementation of the Charcoal Yeast Agar with ACES buffer improved the performance of the medium. Edelstein (4) further modified the medium by adding alpha-ketoglutarate. This addition helped in improving the sensitivity of the medium. Buffered Charcoal Yeast Extract Agar Base is based on Edelsteins Modification. Legionella species are non-spore forming, narrow, gram-negative rods. Legionella causes pneumonia (Legionnaires disease) (5) or a milk, febrile disease (Pontiac fever). They do not oxidize or ferment carbohydrates in conventional media or grow on sheep blood agar. Growth is much better and more rapid on Buffered Charcoal Yeast Extract Agar (2,6). Amino acids are the major sources of energy for Legionella. The amino acid L-cystine holds an absolute requirement as it plays major role in growth metabolism of Legionella (7). This amino acid as well as ferric pyrophosphate helps for the growth of Legionella. The media contains charcoal, which acts as a detoxicant. Yeast extract acts as a rich source of vitamins, nitrogen as well as carbon. ACES Buffer maintains optimal pH for growth while L-cystine hydrochloride; ferric pyrophosphate and a-ketoglutarate stimulate growth of Legionella species. For selective isolation, antibiotic supplements can be used to suppress contaminating microorganisms. C3V Selective Supplement (FD037) containing cephalothin, colistin, vancomycin and cycloheximide (8) or MWY Selective Supplement (FD040) containing glycine, polymyxin B, anisomycin, vancomycin, bromothymol blue and bromocresol purple (9) are often used. Wear gown, mask and gloves while handling Legionella cultures. Work in a safety hood.
Type of specimen
Clinical samples - Urine; Water samples
Specimen Collection and Handling
For clinical samples follow appropriate techniques for handling specimens as per established guidelines (10,11).
For water samples, follow appropriate techniques for sample collection, processing as per guidelines and local standards (12). After use, contaminated materials must be sterilized by autoclaving before discarding.
Warning and Precautions
In Vitro diagnostic use. For professional use only. Read the label before opening the container. Wear protective gloves/ protective clothing/eye protection/ face protection. Follow good microbiological lab practices while handling specimens and culture. Standard precautions as per established guidelines should be followed while handling clinical specimens. Safety guidelines may be referred in individual safety data sheets.
Limitations
- Further biochemical confirmation has to be carried out for further confirmation.
Performance and Evaluation
Performance of the medium is expected when used as per the direction on the label within the expiry period when stored at recommended temperature.
Quality Control
Appearance
Grey to black homogeneous free flowing powder
Gelling
Firm, comparable with 1.7% Agar gel.
Colour and Clarity of prepared medium
Grey-black coloured opalescent gel forms in Petri plates.
Reaction
Reaction of 4.0% w/v aqueous solution at 25°C. pH : 6.9±0.2
pH
6.70-7.10
Cultural Response
Cultural characteristics observed in 90% humid atmosphere with added MWY Selective Supplement (FD040) and Legi Growth Supplement w/o SS (Twin Pack) (FD041A), after an incubation at 35-37°C for 3-4 days.
| Organism | Inoculum (CFU) | Growth | Recovery | Colour of colony |
|---|---|---|---|---|
| Escherichia coli ATCC 25922 (00013*) | 50-100 | none-poor | <=10% | |
| Legionella dumoffii ATCC 33343 | 50-100 | luxuriant | >=50% | light blue-grey |
| Legionella pneumophila ATCC 33153 | 50-100 | luxuriant | >=50% | white grey to blue grey |
| Staphylococcus epidermidis ATCC 12228 (00036*) | 50-100 | none-poor | <=10% |
Key: (*) - Corresponding WDCM numbers
Storage and Shelf Life
Store between 10-30°C in a tightly closed container and the prepared medium at 2-8°C. Use before expiry date on the label. On opening, product should be properly stored dry, after tightly capping the bottle in order to prevent lump formation due to the hygroscopic nature of the product. Improper storage of the product may lead to lump formation. Store in dry ventilated area protected from extremes of temperature and sources of ignition Seal the container tightly after use. Product performance is best if used within stated expiry period.
Disposal
User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this product. Follow established laboratory procedures in disposing of infectious materials and material that comes into contact with clinical sample must be decontaminated and disposed of in accordance with current laboratory techniques (10,11).
| Product Name | Buffered Charcoal Yeast Extract Agar Base |
|---|---|
| SKU | M813 |
| Product Type | Regular |
| Physical Form | Powder |
| Origin | Animal Free (Microbial) |
| Packaging type | HDPE |
| References | 1. Broome C. V., Fraser D. W., 1979, Epidemiol. Rev 1:1-16. |
| Customized Product Available | No |
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