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LEGIONELLA CYE AGAR BASE

Code: CM0655

Charcoal Yeast Extract Agar for the isolation of Legionellaceae when used with Legionella BCYE Growth Supplement SR0110 (Edelstein BYCE Agar)

Typical Formula*

gm/litre

Activated charcoal

2.0

Yeast extract

10.0

Agar

13.0

* Adjusted as required to meet performance standards 

LEGIONELLA BCYE GROWTH SUPPLEMENT

Code: SR0110

Vial contents

SR0110A
(1 vial per 100ml medium)

SR0110C
(1 vial per 500ml medium)

Supplement
per litre
Buffer/Potassium hydroxide
1.0g
5.0g
10g

Ferric pyrophosphate

0.025g

0.125g
0.25g

L-cysteine HCl

0.04g

0.2g
0.4g

a -Ketoglutarate

0.1g

0.5g
1.0g

Directions to prepare BCYE Agar
Suspend 2.5g of Legionella CYE Agar Base per 90ml of distilled water and bring gently to the boil to dissolve completely. Sterilise by autoclaving at 121°C for 15 minutes. Reconstitute one vial SR0110A per 100ml medium or one vial SR0110C per 500ml medium, as directed. Allow medium to cool to 50°C and aseptically add the vial contents. Mix gently and pour into sterile Petri dishes. The final pH of the medium should be 6.9 ± 0.2.

Description
The discovery of the causative organism of Legionnaires’ disease has been reviewed by Fallon1. Since that review further progress has been made in culturing the organism from clinical specimens and also in the enumeration of Legionella species from environmental samples. Feeley et al.2 described a modification of F-G Agar3 in which acid hydrolysed casein was replaced by yeast extract as the source of protein and starch was replaced by activated charcoal (Norit A) at a final concentration of 0.2% (w/v). This medium, which they named CYE Agar2 has been further supplemented with ACES Buffer and a-ketoglutarate and is described in the literature as BCYE-a Medium4. BCYE-a Medium has been shown to yield optimal recovery of Legionellaceae in a shorter incubation period from environmental samples and clinical specimens5.

Oxoid BCYE Medium is based on the formulation of Edelstein4 and is prepared from Legionella CYE Agar Base (CM0655) and Legionella BCYE Growth Supplement (SR0110). The sterile lyophilised supplement contains ACES Buffer/potassium hydroxide, a-ketoglutarate, ferric pyrophosphate and L-cysteine HCl. When added to CYE Agar Base it stabilises the pH of the medium at 6.9 ± 0.2 and provides essential growth factors.

This medium can be further supplemented with one of the following:

  • with Legionella BMPA Selective Supplement (SR0111) to give Legionella BMPA Selective Agar
  • with Legionella GVPC Selective Supplement (SR0152) to give Legionella GVPC Selective Agar
  • with Legionella GVPN Selective Supplement (SR0215) to give Legionella GVPN Selective Agar
  • with Legionella MWY Selective Supplement (SR0118) to give Legionella MWY Selective Agar

Additionally, a medium omitting L-cysteine may be prepared from Legionella CYE Agar Base (CM0655) and BYCE Growth Supplement (SR0175).
Legionellaceae have an absolute nutritional requirement for L-cysteine. Presumptive Legionella spp. colonies can be subcultured onto both BCYE Medium with L-cysteine (CM0655) and (SR0110), and BCYE Medium without L-cysteine (CM0655) and (SR0175).

All plates are incubated at 35°C. Colonies which have grown on BCYE Medium with L-cysteine, but not on BCYE Medium without L-cysteine, can be regarded as presumptive Legionella spp.

Storage conditions and Shelf life
Store the dehydrated medium below 30°C and use before the expiry date on the label.
Store the prepared plates at 2-8°C away from light.

Appearance
Dehydrated medium: Black, free-flowing powder
Prepared medium: Black gel

Quality control

Positive controls:

Expected results

Legionella pneumophila ATCC® 33152 *

Good growth; grey/white coloured colonies

Legionella pneumophila NCTC 12821

Good growth; grey/white coloured colonies

Negative control:

 

Uninoculated medium

No change

Selective media 
Positive controls:  
Legionella pneumophila ATCC® 33152 *Good growth; grey/white-blueish coloured colonies
Legionella pneumophila NCTC 12821Good growth; grey/white-blueish coloured colonies
Negative control:  
Staphylococcus epidermidis ATCC® 12228 *Inhibited
* This organism is available as a Culti-Loop®

References
1. Fallon J. Oxoid Limited. Culture September 1979, P. 3-4.
2. Feeley J.C., Gibson R.J., Gorman G.W., Langford N.C., Rasheed J.W., Mackel D.C. and Baine W.B. (1979) J. Clin. Micro. 10. 437-441.
3. Feeley J.C. Gorman G.W., Weaver R.E., Mackel D.C. and Smith H.W. (1978) J. Clin. Micro. 8. 320-325.
4. Edelstein P.H. (1981) J. Clin. Micro. 14. 298-303.
5. PHLS Communicable Diseases Report (1983) CDR 83/49.

 
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