Plot No. C40, Road No. 21Y, MIDC, Wagle Industrial Estate,
Thane(W)-400604, MH, India.
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Buffered Charcoal Yeast Extract Agar Base, Modified
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.
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Intended use
Recommended for selective isolation and cultivation of Legionella species from cooling towers, all kinds of water samples and other materials. The composition and performance criteria of this medium are as per the specifications laid down in ISO 11731-1: 2017 & 11731-2: 2017.
Composition**
BCYE Agar As per ISO 11731
| Ingredients | g/L |
|---|---|
| Yeast extract (Bacteriological grade) | 10.000 |
| Activated Charcoal | 2.000 |
| α-ketoglutarate, monopotassium salt | 1.000 |
| ACES Buffer | 10.000 |
| Agar | 12.000 |
| Potassium hydroxide (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 M8131
| Ingredients | Quantity |
|---|---|
| Yeast extract | 10.000 g/L |
| Activated Charcoal | 2.000 g/L |
| α-ketoglutarate, monopotassium salt | 1.000 g/L |
| ACES Buffer | 10.000 g/L |
| Agar | 12.000 g/L |
| Final pH (at 25°C) | 6.8±0.2 |
| Legi Growth Supplement FD041A w/o SS (Twin Pack) | |
| Part A L-Cysteine hydrochloride | 200mg |
| Part B Ferric pyrophosphate, soluble | 125mg |
| Distilled water | 5ml |
Selective culture medium
For Buffered charcoal yeast extract agar with selective supplement (BCYE+AB)
| Medium Components | PCP Supplement FD347 Components | ||
|---|---|---|---|
| Polymyxin B sulfate | 80,000 IU | Polymyxin B sulfate | 80,000 IU |
| Sodium cefazolin | 0.009 | Cefazolin sodium | 0.009 |
| Pimaricin (syn Natamycin) | 0.070 | Pimaricin (Natamycin) | 0.070 |
For Buffered charcoal yeast extract agar with selective supplement (BCYE+GVPC)
| Medium Components | GVPC Selective Supplement FD143 Components | ||
|---|---|---|---|
| Ammonium-free Glycine | 3.000 | Glycine | 1.500g |
| Vancomycin hydrochloride | 0.001 | Vancomycin hydrochloride | 0.500mg |
| Polymyxin B sulphate | 80,000 IU | Polymyxin B sulphate | 40000IU |
| Cycloheximide | 0.080 | Cycloheximide | 40mg |
For Modified Widomski Yee (MWY) Agar
| Medium Components | MWY Selective Supplement FD040 Components | ||
|---|---|---|---|
| Polymyxin B sulphate | 50,000 IU | Polymyxin B sulphate | 25000Unit |
| Ammonium-free glycine | 3.000g | Glycine | 1.500g |
| Anisomycin | 0.080 | Anisomycin | 40mg |
| Vancomycin hydrochloride | 0.001 | Vancomycin | 0.500mg |
| Bromo thymol blue | 0.010 | Bromo thymol blue | 5mg |
| Bromo cresol purple | 0.010 | Bromo cresol purple | 5mg |
Directions
Suspend 35.0 grams in 1000 ml purified/distilled water. Add 2.4 grams KOH pellets and mix to dissolve. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at (121 ± 3) °C for (15 ± 1) minutes. Cool to 45-51°C. Aseptically add sterile rehydrated contents of 2 vials each of Legi Growth Supplement w/o SS (Twin Pack) (FD041A, Part A and Part B). Mix well and pour into sterile Petri plates with constant stirring to ensure that charcoal particles get evenly distributed.
Note: As per standard it is recommended to use 2.8 grams of Potassium hydroxide pellets.
Preparation of Specific Media Variants
Buffered charcoal yeast extract agar without L-cysteine (BCYE-cys)
Prepare same as BCYE Agar composition and aseptically add contents of two vials of Legi Growth Supplement w/o SS (Twin Pack) (FD041A, Part B only).
For Buffered charcoal yeast extract agar with selective supplement (BCYE+AB)
Aseptically add rehydrated contents of one vial of PCP Supplement (FD347).
For Buffered charcoal yeast extract agar with selective supplement (BCYE+GVPC)
Aseptically add rehydrated contents of two vials of GVPC Selective Supplement (FD143).
For Modified Widomski Yee (MWY) Agar
Aseptically add the rehydrated contents of one vial of MWY Selective Supplement (FD040- per 100 ml).
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 α-ketoglutarate stimulate growth of Legionella species. For selective isolation, antibiotic supplements can be used to suppress contaminating microorganisms. PCP Supplement (FD347) containing Polymyxin B, Sodium cefazolin and Pimaricin or Legionella (GVPC ) Selective Supplement (FD143) containing glycine, Polymyxin B sulphate, vancomycin and cycloheximide 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
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 specimens. Safety guidelines may be referred in individual safety data sheets.
Limitations
- 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.
- 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 1.2% Agar gel.
Colour and Clarity of prepared medium
Grey-black coloured opalescent gel forms in Petri plates.
Reaction
Reaction of 3.5% w/v aqueous solution at 25°C. pH: 6.8±0.2
pH
6.60-7.00
Cultural Response
Productivity: Cultural response was observed after an incubation (90% humid atmosphere) at 36 ± 2°C for 2-5 days, with added sterile Legi Growth Supplement w/o SS (Twin Pack) (FD041A, Part A and Part B). Recovery rate is considered as >=50% on BCYE
Selectivity: Cultural response was observed after an incubation (90% humid atmosphere) at 36 ± 2°C for 2-5 days, with added sterile Legi Growth Supplement w/o SS (Twin Pack) (FD041A, Part A and Part B).
| Organism | Inoculum (CFU) | Growth | Recovery | Colour of colony |
|---|---|---|---|---|
| Productivity | ||||
| Legionella pneumophila ATCC 33152 (00107*) | 50-100 | luxuriant | >=50% | white-grey-blue purple colonies with an entire edge exhibiting a characteristic ground glass appearance |
| Legionella pneumophila ATCC 33156 (00180*) | 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) |
| Selectivity | ||||
| Enterococcus faecalis ATCC 29212 (00087*) | >=104 | inhibited | 0% | |
| Enterococcus faecalis ATCC 19433 (00009*) | >=104 | inhibited | 0% | |
| Escherichia coli ATCC 25922 (00013*) | 50-100 | none-poor | <=10% | |
| Escherichia coli ATCC 8739 (00012*) | 50-100 | none-poor | <=10% | |
| ^Pseudomonas paraeruginosa ATCC 9027 (00026*) | 50-100 | none-poor | <=10% | |
| Pseudomonas aeruginosa ATCC 27853 (00025*) | 50-100 | none-poor | <=10% | |
Key: (*) - Corresponding WDCM numbers
^ Formerly known as Pseudomonas aeruginosa
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 sample must be decontaminated and disposed of in accordance with current laboratory techniques (10,11).
| Product Name | Buffered Charcoal Yeast Extract Agar Base, Modified |
|---|---|
| SKU | M813I |
| 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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