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LEGIONELLA SELECTIVE MEDIA
For the isolation of Legionellaceae from clinical and environmental samples.

LEGIONELLA CYE AGAR BASE

Code: CM0655
Charcoal Yeast Extract Agar for the isolation of Legionellaceae when used with Legionella BCYE Growth Supplement SR110 (Edelstein BYCE Agar).

LEGIONELLA BCYE GROWTH SUPPLEMENT

Code: SR0110

LEGIONELLA BCYE GROWTH SUPPLEMENT
Without L-Cysteine

Code: SR0175

LEGIONELLA BMPAa SELECTIVE SUPPLEMENT

Code: SR0111

LEGIONELLA MWY SELECTIVE SUPPLEMENT

Code: SR0118
Vial contents

Directions to prepare BCYE Agar
Suspend 2.5 g of Legionella CYE Agar Base (CM0655) in 90 ml of distilled water and bring gently to the boil to dissolve completely. Sterilise by autoclaving at 121° C for 15 minutes. Allow to cool to 50°C and aseptically add the contents of one vial of Legionella BCYE Growth Supplement SR0110 reconstituted as directed.. Mix gently and pour into sterile Petri dishes. The final pH of the medium should be 6.9 ± 0.2.

Directions to prepare BCYE without L-cysteine Agar
Suspend 2.5 g of Legionella CYE Agar Base (CM0655) in 90 ml of distilled water and bring gently to the boil to dissolve completely. Sterilise by autoclaving at 121° C for 15 minutes. Allow to cool to 50°C and aseptically add the contents of one vial of Legionella BCYE Growth Supplement without L-cysteine SR0175 reconstituted as directed.. Mix gently and pour into sterile Petri dishes. The final pH of the medium should be 6.9 ± 0.2.

Directions to prepare BMPAa and MWY Legionella Selective Media
Suspend 2.5 g of Legionella CYE Agar Base (CM0655) in 90 ml of distilled water and bring gently to the boil to dissolve completely. Sterilise by autoclaving at 121° C for 15 minutes. Allow to cool to 50°C and aseptically add the contents of one vial of Legionella BYCE Growth Supplement SR0110 and one vial of either Legionella BMPAa Selective Supplement SR0111, or Legionella MWY Selective Supplement SR0118 reconstituted as directed. Mix gently and pour into sterile Petri dishes. The final pH of both media 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.
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.

Quality Control

* This organism is available as a Culti-Loop®
Edelstein and Edelstein later compared agars used in the manufacture of buffered charcoal yeast extract medium and found significant differences in growth occurred. Oxoid Agar L11 gave the best results in the series tested⁶.

Characteristics of Legionella species ( Adapted from Renner & Tseng¹⁰ )

1. BMPAa Medium containing polymyxin 80 IU/ml, anisomycin 80 µg/ml and cefamandole 4 µg/ml. This semi-selective medium was recommended by Edelstein⁴ for the isolation of Legionella pneumophila from contaminated clinical and environmental specimens and can be prepared by supplementing BCYE Agar with Legionella BMPAa Selective Supplement SR111.
2. Wadowsky and Yee Medium⁷ modified by Edelstein (MWY Medium)⁸ containing polymyxin 50 IU/ml, anisomycin 80 µg/ml, vancomycin 1 µg/ml and glycine 0.3% w/v. Bromocresol purple 10 µg/ml and Bromothymol blue 10 µg/ml colour the colonies and aid in the identification of the organisms^9,10. Edelstein⁸ considered the medium to be the best for isolating Legionella pneumophila from potable water samples. This medium can be prepared by supplementing BCYE Agar with Legionella MWY Selective Supplement SR0118.
Environmental samples should be pre-treated with an acid buffer (pH 2.2)¹¹ or by heat treatment¹². They should be plated both before and after treatment to maximise recovery (see Technique).
MWY medium has been used successfully for examination of clinical specimens⁵.
3. GVPC Medium based on the formula by Dennis et al. This medium contains glycine, vancomycin, polymyxin B and cycloheximide. A complete description follows.

Technique: Clinical Samples
For the isolation of Legionella spp. from patients with clinical evidence of Legionnaires' disease, greatest success has been achieved by the examination of lung tissue and bronchial aspirate.
1. Homogenise the patient's specimen in sterile distilled water.
2. Examine microscopically for Legionella by the Fluorescent Antibody Method (FA) and for other bacteria by Gram's stain.
3. Inoculate specimens that are FA-positive but with no other organisms present on to plates of BCYE Medium. Both FA-positive and FA-negative specimens in which other organisms have been detected by the Gram stain, are inoculated on to the selective medium BMPAa .
4. Incubate the plates at 35° C in a 90% relative humidity atmosphere.
5. Growth usually appears in 2-3 days but continue to examine daily for 14 days before discarding the plates.

Environmental Samples
1. Take 10 ml of the concentrated sample and centrifuge at 2,500 rpm for 20 minutes (using sealed buckets).
2. Remove the supernatant to leave approximately 1ml of fluid. Resuspend the deposit. This constitutes the inoculum.
3. Spread 0.1 ml on to plates of BCYE Medium with and without selective agents using a sterile spreader.
4. Add 9 ml of HCl-KCl buffer* (pH 2.2); shake gently and leave for 5 minutes.
*HCl-KCl buffer: 3.9 ml of 0.2 M HCl; 25 ml of 0.2 M KCl; Adjust the pH to 2.2 using 1M KOH.
Alternatively, heat 10 ml of the sample concentrate in a 50° C water bath for 30 minutes.
Important
ACID AND HEAT PRETREATMENT OF SAMPLES MUST NOT BE COMBINED.
5. Spread 0.1 ml on to plates of BCYE Medium using a sterile spreader.
6. Incubate the plates at 35° C and examine daily for up to seven days.
Colonies suspected of being Legionella are subcultured to Tryptone Soya Agar containing 5% sheep blood and BCYE Agar.
Isolates that grow on BCYE Agar but fail to grow on TSA Blood Agar and have characteristic morphology, may be presumed to be Legionella. Confirmation must be made by biochemical and serological tests¹².
As the media described are not completely selective for Legionella species, it is recommended⁸ that the following criteria are used for the examination of plates:
1. The colonies must have characteristic colour, size and appearance when examined under a dissecting microscope.
2. The isolates should not grow on blood agar or Legionella BCYE Agar without L-cysteine.
3. The organisms should show characteristic Gram morphology.
Legionella spp. cannot be identified solely on growth characteristics on various media or by biochemical tests. Further studies with DNA homology, cellular fatty acids and serotyping must be undertaken.

Colony morphology after incubation at 35° C for 2-3 days on CYE/BCYE media
Legionella pneumophila: diameter 1-2 mm (increase in size on further incubation). White, glistening, circular, smooth, raised with an entire edge.
Legionella gormanii: diameter 1-2 mm. Buff-white or cream, slight raised, mucoid.
Other legionellae: (Legionella micdadei, Legionella bozemanii, Legionella dumoffii, Legionella longbeachae and Legionella jordanis) - indistinguishable from Legionella pneumophila.

Characteristic Gram morphology on first isolation
Gram-negative, short, pleomorphic rods 1 mm long.
Carbol-fuchsin should be used as a counterstain in the Gram films of legionellae because they resist staining with safranin and basic fuchsin.

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

* This organism is available as a Culti-Loop®

Precautions
Legionella spp. are highly pathogenic organisms if inhaled. Avoid creating aerosols and handle liquid cultures or suspensions of organisms in a protective cabinet. Decontaminate working surfaces with 5% hypochlorite. Autoclave all materials before discarding.
Incubate cultures at 35°C in 65% humidity for up to 10 days.
No CO₂ is required for BCYE cultures but 2.5% CO₂ is recommended for other media and water samples^7,13.
Organisms that grow on blood agar as well as Legionella media are not legionellae. Some thermophilic spore-bearing organisms mimic Legionella colonies after 35°C incubation. These organisms can be detected by incubating parallel plates at 35°C and 55°C when the thermophilic organisms will grow at the higher temperature. Legionella species will not grow above 45°C.

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.
6. Edelstein P.H. and Edelstein M.A.C. (1991) J. Clin. Microbiol. 29. 190-191.
7. Wadowsky R.M. and Yee R.B. (1981) Clin. Microbiol. Newsletter 4. 768-772.
8. Edelstein P.H. (1982) J. Clin. Micro. 16. 697-699.
9. Vickers R.M., Brown A. and Garrity G.M. (1981) J. Clin. Micro. 13. 380-283.
10. Renner E.D. and Tseng C.H. (1982) Clin. Microbiol. Newsletter 4. 139, 142.
11. Bopp C.A., Sumner J.W., Morris G.K. and Wells J.G. (1981) J. Clin. Micro. 13. 714-719.
12. Vesey G., Dennis P.J., Lee J.V. and West A.A. (1988) J. Appl. Bact. 65. 339-345.