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, water samples, clinical and other materials. The composition and performance criteria of this medium are as per the specifications laid down in ISO 11731-2017 (Ε).
Composition**
BCYE Agar As per ISO 11731:2017(E)
| Ingredients | g/L |
|---|---|
| Yeast extract (Bacteriological grade) | 10.000 |
| Activated Charcoal | 2.000 |
| a-ketoglutarate, monopotassium salt | 1.000 |
| ACES Buffer | 10.000 |
| Agar | 12.000 |
| KOH pellets | 2.800 |
| L-cystine hydrochloride monohydrate | 0.400 |
| Iron (III) pyrophosphate | 0.250 |
| Final pH (at 25°C) | 6.8±0.2 |
BCYE Agar
| Ingredients | g/L |
|---|---|
| Yeast extract | 10.000 |
| Activated Charcoal | 2.000 |
| alpha ketoglutarate, monopotassium salt | 1.000 |
| ACES Buffer | 10.000 |
| Agar | 12.000 |
| Final pH (at 25°C) | 6.8±0.2 |
Legi Growth Supplement FD041A w/o SS (Twin Pack)
| Ingredients | |
|---|---|
| Part A | " |
| L-Cysteine hydrochloride | 200mg |
| Part B | " |
| Ferric pyrophosphate, soluble | 125mg |
| Distilled water | 5ml |
Directions
Either streak, inoculate or surface spread the test inoculum (50-100 CFU) aseptically on the plate. Membrane filters through which water sample has been filtered can be placed directly on the agar plate.
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 tryptone 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. The formulation of Buffered Charcoal Yeast Extract Agar Base is as per specification laid in ISO 11731-2 (5).
Legionella species are non-spore forming, narrow, gram-negative rods. Legionella causes pneumonia (Legionnaires disease) (6) 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 (2,7).
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 (8). This amino acid as well as ferric pyrophosphate helps for the growth of Legionella.
The media contains charcoal, which acts as 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. Wear gown, mask and gloves while handling Legionella cultures. Work in a safety hood (8).
Type of specimen
Water samples
Specimen Collection and Handling
For water samples, follow appropriate techniques for sample collection, processing as per guidelines and local standards (5). After use, contaminated materials must be sterilized by autoclaving before discarding.
Warning and Precautions :
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.
- Individual organisms differ in their growth requirement and may show variable growth patterns on the medium
- Each lot of the medium has been tested for the organisms specified on the COA. It is recommended to users to validate the medium for any specific microorganism other than mentioned in the COA based on the user's unique requirement.
Performance and Evaluation
Performance of the medium is expected when used as per the direction on the label within theexpiry period when stored at recommended temperature.
Quality Control
Appearance
Sterile Buffered Charcoal Yeast Extract Agar in 90mm disposable plate.
Colour
Grey-black coloured opalescent medium
Quantity of medium
25 ml of medium in 90mm plate
Reaction
6.60 -7.00
Sterility test
Passes release criteria
Cultural Response
Cultural characteristics observed in 90% humid atmosphere after an incubation at 34-38°C for 2-5 days.
| Organism | Inoculum (CFU) | Growth | Recovery | Colour of colony |
|---|---|---|---|---|
| Legionella pneumophila ATCC 13152(00107*) | 50-100 | luxuriant | >=50% | white-grey-blue purple colonies with an entire edge exhibiting a characteristic ground glass appearance |
| Legionella anisa ATCC 35292 (00106*) | 50-100 | luxuriant | >=50% | white-grey-blue purple colonies with an entire edge exhibiting a characteristic ground glass appearance (incubated for 5-10 days) |
| Legionella dumofii ATCC 33343 | 50-100 | luxuriant | >=50% | light blue - grey |
Key: (*) - Corresponding WDCM numbers
Storage and Shelf Life
On receipt store between 2-8°C. Use before expiry date on the label. 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 sample must be decontaminated and disposed of in accordance with current laboratory techniques (9,10).
| Product Name | BCYE Agar Plate |
|---|---|
| SKU | MP813I |
| Customized Product Available | No |
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