BUFFERED CHARCOAL YEAST EXTRACT (BCYE) AGAR

Cat. no. G07 BCYE Agar, 15x100mm Plate, 18ml 10 plates/bag
Cat. no. G08 BCYE Selective Agar, 15x100mm Plate, 18ml 10 plates/bag
Cat. no. G108 BCYE without Cysteine, 15x100mm Plate, 18ml 10 plates/bag
Cat. no. G209 BCYE Agar with DGVP, 15x100mm Plate, 18ml 10 plates/bag
Cat. no. H08 BCYE Agar, 15x150mm Plate, 68ml 10 plates/bag
Cat. no. P251 BCYE Agar, 15x60mm Contact Plate, 15ml 10 plates/bag
Cat. no. W169 BCYE Selective Agar with GPVC, 15x100mm Plate, 26ml 10 plates/bag
Cat. no. G170 BCYE Selective Agar with CCVC, 15x100mm plate, 24ml 10 plates/bag

INTENDED USE

Hardy Diagnostics Buffered Charcoal Yeast Extract (BCYE) Agar formulations are recommended for use in the cultivation and primary isolation of Legionella spp. in water and other samples suspected of harboring the bacteria.

Cat. no. P251 is not intended to be used for the diagnosis of human disease.

SUMMARY

In 1977, MacDade et al. were the first to isolate the bacterium, Legionella pneumophila, responsible for causing Legionnaire's Disease. Following this discovery, there were numerous reports of Legionella being isolated from fresh water environments such as water distribution systems, evaporative condensers, air conditioning units, cooling towers, fountains, humidifiers, dentistry tools, shower heads, faucets and whirlpool spas or Jaccuzis®. To date, there are approximately 50 known species and 70 serogroups of Legionella that have been identified.

In 1978, Feeley et al. developed a medium to provide consistent isolation of Legionella species.(6) The medium demonstrated differential growth characteristics to aid in the identification of Legionella species. Feeley later modified the medium by substituting yeast extract for casein hydrolysate and beef extract, and replacing starch with activated charcoal. Feeley called this modified formula Charcoal Yeast Extract (CYE) Agar.(7) A further modification was made by Pasculle et al. in 1980.(8) This version employed the addition of ACES (N-2-acetamido-2-aminoethane sulfonic acid) buffer in order to maintain the proper pH for optimal growth. The new medium, designated BCYE for Buffered Charcoal Yeast Extract, could also be incubated aerobically. In 1981, Edelstein et al. increased the sensitivity of the medium by adding the potassium salt of alpha-ketoglutaric acid.(9) That same year, Wadowsky and Yee made the medium more selective by suggesting that glycine, vancomycin and polymyxin be added, resulting in the formation of GVP medium.(10) Another modification in 1984 by Dennis et al. made the medium even more selective for Legionella by the addition of cycloheximide, resulting in GVPC medium.(11)

Hardy Diagnostics BCYE Agar (Cat. no. G07, G08, G108, G209, H08, P251, W169, and G170) is based on Edelstein's formula and is formulated in accordance with ISO 11371:(1998)E.(15) This formulation employs the use of L-Cysteine, soluble ferric pyrophosphate, and alpha-ketoglutarate to enhance the growth of Legionella species. Activated charcoal removes toxic metabolic products. Protein and other growth nutrients are supplied by yeast extract.

BCYE Selective Agar (Cat. no. G08) contains anisomycin, colistin, and vancomycin. Anisomycin inhibits the growth of fungi. Gram-negative organisms are inhibited by colistin. Gram-positive organisms are inhibited by vancomycin.

It has been shown that Legionella spp. require the addition of L-cysteine for growth. BCYE without Cysteine (Cat. no. G108) can be used in conjunction with traditional BCYE medium. Organisms that fail to grow on BCYE without Cysteine but grow on traditional BCYE Agar can be presumptively identified as Legionella spp.

BCYE with DGVP (Cat. no. G209) contains the dyes bromcresol purple and bromothymol blue, along with glycine, vancomycin and polymyxin. Antibiotics in the medium improve the recovery of Legionella species by inhibiting the growth of contaminating microorganisms, while dyes in the medium facilitate the differentiation and identification of Legionella.

BCYE Selective Agar with GPVC (Cat. no. W169) contains glycine, vancomycin, polymyxin and cycloheximide for the greater inhibition of secondary microflora and further selective isolation of Legionella spp.

BCYE Selective Agar with CCVC (Cat. no. G170) contains cephalothin, colistin, vancomycin, and cycloheximide. This is based on the formulation of Bopp et al.(14), where they obtained the improved recovery of L. pneumophila with this selective media with an acid wash treatment to reduce the contaminating microbial flora present in environmental water samples.

FORMULA

Ingredients per liter of deionized water:*

BCYE Agar (Cat. no. G07, H08 and P251):
Yeast Extract 10.0gm
ACES Buffer 10.0gm
Activated Charcoal 2.0gm
Potassium Hydroxide 2.8gm
Alpha-Ketoglutarate 1.0gm
L-Cysteine 0.4gm
Ferric Pyrophosphate 0.25gm
Agar 12.0gm

BCYE without Cysteine (Cat. no. G108) is made using the above formulation, without the addition of L-cysteine.

In addition, BCYE Selective Agar (Cat. no. G08) contains:

Anisomycin 80.0mg
Colistin 10.0mg
Vancomycin 0.5mg

In addition, BCYE Agar with DGPV (Cat. no. G209) contains:

Bromcresol Purple 5.0ml
Bromothymol Blue 10.0ml
Glycine 3.0gm
Polymyxin 3.0ml
Vancomycin 1.0mg

In addition, BCYE Selective Agar with GPVC (Cat. no. W169) contains:

Glycine 3.0gm
Polymyxin 80,000iu
Cycloheximide 80.0mg
Vancomycin 1.0mg

In addition, BCYE Selective Agar with CCVC (Cat. no. G170) contains:

Cycloheximide 40.0mg
Colistin Solution 3.0ml
Cephalothin 4.0mg
Vancomycin 1.0mg

Final pH 6.9 +/- 0.2 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8ºC. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

For Cat. nos. G07, G08, G108, G170, G209, H08, and W169.

For Cat. no. P251

PROCEDURE

Specimen Collection: Infectious material should be submitted directly to the laboratory without delay and protected from excessive heat and cold. If there is to be a delay in processing, the specimen should be inoculated onto an appropriate transport media and refrigerated until inoculation. Consult listed references for information on specimen collection.(1-5)

The media should be brought to room temperature prior to use and inoculated to produce isolated colonies. Incubate aerobically in a humidified atmosphere at 35ºC. Incubate for a minimum of four days and examine daily for growth. Media may be reincubated for up to seven days.

Method of Use (Environmental Samples):

1. Obtain a water sample under aseptic conditions and prepare the sample according to the protocol of the standard being applied. Analysis of the sample can be accomplished either by direct seeding or filtration, usually followed by concentration, heat and/or acid treatment, then plating.

2. In direct seeding, spread 0.1 to 0.5ml of the water sample or dilution, using a sterile spreading device, across the agar surface. If using a membrane filter, pour the water sample through a 0.22um polycarbonate membrane filter, place the membrane in a sterile flask containing 5ml of the initial water sample and centrifuge or sonicate the sample to concentrate. Proceed with the appropriate protocol, as required. If plating the filter directly, place the membrane, filtered side up, on the agar surface.

3. Incubate plates at 35 +/- 2ºC. for a minimum of 3 days. Growth is typically visible after 3-4 days, but may take up to 2 weeks.

4. Note the number of each colony type and select at least three distinct colonies of Legionella from each plate.

5. Re-streak each colony onto a separate plate of BCYE without Cysteine (Cat. no. G108) and incubate using the same parameters as above.

6. Consider as positive for Legionella all colonies that develop on BCYE Agar, but present no growth on BCYE without Cysteine.

7. Proceed with immunofluorescence or serological tests for further identification.

INTERPRETATION OF RESULTS

In general, colonies of Legionella spp. present a white to gray coloration. They may also have blue, pink, purple, maroon, greenish-yellow or dark red pigmentation that fades, becoming whiter and more filamentous with age. The colony surface is generally smooth with precise edges, but some strains may give a ground glass or "fried egg" appearance when viewed microscopically. Some species fluoresce under UV light.

On BCYE and BCYE Selective Agars, colonies of Legionella pneumophila appear white-gray to blue-gray and fluoresce yellow-green under long-wave UV light. Colonies of Legionella bozemanii appear white-gray to blue-gray and fluoresce blue-white under long-wave UV light. Colonies of Legionella micdadei do not fluoresce under long-wave UV light.

On BCYE with DGVP, all strains of L. pneumophila produce round, shiny and white colored colonies with a barely discernable green hue at 3 days incubation. Colonies will be larger by 5 to 7 days incubation, becoming more distinctly green, flat, dull, and opaque in appearance. Tatlockia micdadei (syn. Legionella micdadei) yields shiny, round, blue-gray colonies. Colonies become larger and more intensely blue in color during prolonged incubation. Colonies of Fluoribacter spp. appear round, shiny and distinctly green in appearance after 3 days incubation; the green coloration will deepen with continued incubation and colonies will remain shiny. Colonies of L. bozemanii will exhibit a bright pastel green coloration that will intensify over prolonged incubation; colonies will fluoresce blue-white under long-wave UV light.

Growth of most other flora should be inhibited or greatly reduced on BCYE Selective Agar, BCYE Agar with DGVP, BCYE Selective Agar with GPVC, and BCYE Selective Agar with CCVC.

LIMITATIONS

It is also recommended that more than one type of medium be used for isolating Legionella spp. and that non-selective and selective BCYE Agar plates be inoculated in parallel.

The Centers for Disease Control and Prevention (CDC) recommend incubation of environmental samples with 2.5% CO2; however, L. gormanii is the only known species with enhanced growth under this condition.(12,13)

This medium is to be used for the isolation and presumptive identification of Legionella.

Colonies of Legionella that develop on white membrane filters may have a different appearance to those that develop against a black or dark background filter.

When handling Legionella spp., it is important to avoid aerosol formation. Thoroughly clean and disinfect all work areas.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, other culture media, swabs, UV lamps, applicator sticks, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
BCYE Agar (Cat. no. G07, H08 and P251):
Legionella pneumophila
ATCC® 33152**
A 48-96hr 35°C Aerobic Growth; white-gray to blue-gray colonies, fluoresces yellow-green under long-wave UV light
Legionella pneumophila
clinical strain
A 48-96hr 35°C Aerobic Growth; white-gray to blue-gray colonies, fluoresces yellow-green under long-wave UV light
Legionella bozemanii
ATCC® 33217
A 48-96hr 35°C Aerobic Growth; white-gray to blue-gray colonies; fluoresces blue-white under long-wave UV light
Legionella micdadei
ATCC® 33204
A 48-96hr 35°C Aerobic Growth; white-gray to blue-gray, no fluorescence under long-wave UV light
BCYE Selective Agar (Cat. no. G08):
Legionella pneumophila
ATCC® 33152**
A 48-96hr 35°C Aerobic Growth; white-gray to blue-gray colonies, fluoresces yellow-green under long-wave UV light
Legionella pneumophila
clinical strain
A 48-96hr 35°C Aerobic Growth; white-gray to blue-gray colonies, fluoresces yellow-green under long-wave UV light
Legionella bozemanii
ATCC® 33217
A 48-96hr 35°C Aerobic Growth; white-gray to blue-gray colonies; fluoresces blue-white under long-wave UV light
Legionella micdadei
ATCC® 33204
A 48-96hr 35°C Aerobic Growth; white-gray to blue-gray colonies, no fluorescence under long-wave UV light
Escherichia coli
ATCC® 25922**
B 24hr 35°C Aerobic Inhibited
Staphylococcus aureus
ATCC® 25923
B 24hr 35°C Aerobic Inhibited
Candida albicans
ATCC® 10231
B 72hr 35°C Aerobic Inhibited
BCYE without Cysteine (Cat. no. G108)
Campylobacter jejuni
ATCC® 33291**
A 24-72hr 35°C Microaerophilic Growth
Legionella pneumophila
ATCC® 33152**
A 48-72hr 35°C Aerobic Inhibited
BCYE Agar with DGVP (Cat. no. G209)
Legionella pneumophila
ATCC® 33152**
A 48-120hr 35°C Aerobic Growth; pale green colonies, fluoresces yellow-green under long-wave UV light
Legionella pneumophila
clinical strain
A 48-120hr 35°C Aerobic Growth; pale green colonies, fluoresces yellow-green under long-wave UV light
Legionella bozemanii
ATCC® 33217
A 48-120hr 35°C Aerobic Growth; bright green colonies; fluoresces blue-white under long-wave UV light
Legionella micdadei
ATCC® 33204
A 48-120hr 35°C Aerobic Growth; blue-gray colonies, no fluorescence under long-wave UV light
Escherichia coli
ATCC® 25922**
B 24hr 35°C Aerobic Partial to complete inhibition
Staphylococcus aureus
ATCC® 25923
B 24hr 35°C Aerobic Partial to complete inhibition
BCYE Agar with GPVC (Cat. no. W169)
Legionella pneumophila
ATCC® 33152**
A 48-96hr 35°C Aerobic Growth; white-gray to blue-gray colonies, fluoresces pale green under long-wave UV light
Legionella pneumophila
clinical strain
A 48-96hr 35°C Aerobic Growth; white-gray to blue-gray colonies, fluoresces pale green under long-wave UV light
Legionella bozemanii
ATCC® 33217
A 48-96hr 35°C Aerobic Growth; white-gray to blue-gray colonies; fluoresces blue-white under long-wave UV light
Legionella micdadei
ATCC® 33204
A 48-96hr 35°C Aerobic Growth; white-gray to blue-gray colonies, no fluorescence under long-wave UV light
Escherichia coli
ATCC® 25922**
B 24hr 35°C Aerobic Partial to complete inhibition
Staphylococcus aureus
ATCC® 25923
B 24hr 35°C Aerobic Partial to complete inhibition
Aspergillus brasiliensis
ATCC® 16404
G 24-48hr 35°C Aerobic Partial to complete inhibition
BCYE Agar with CCVC (Cat. no. G170)
Legionella pneumophila
ATCC® 33152**
A 48-96hr 35°C Aerobic Growth; white-gray to blue-gray colonies, fluoresces pale green under long-wave UV light
Legionella pneumophila
clinical strain
A 48-96hr 35°C Aerobic Growth; white-gray to blue-gray colonies, fluoresces pale green under long-wave UV light
Escherichia coli
ATCC® 25922**
B 24hr 35°C Aerobic Partial to complete inhibition
Staphylococcus aureus
ATCC® 25923
B 24hr 35°C Aerobic Partial to complete inhibition
Aspergillus brasiliensis
ATCC® 16404
G 24-48hr 35°C Aerobic Partial to complete inhibition

** Recommended QC strains for User Quality Control according to the CLSI document M22 when applicable.

User Quality Control

PHYSICAL APPEARANCE

All formulations of BCYE Agar listed should appear opaque, and black in color.

L. pneumophila growing on BCYE Selective Agar

Legionella pneumophila (ATCC® 33152) colonies growing on BCYE Selective Agar (Cat no. G08). Incubated aerobically for 48 hours at 35ºC.

L. pneumophila growing on BCYE Selective Agar

Legionella pneumophila (ATCC® 33152) colonies growing on BCYE Selective Agar (Cat. no. G08) under UV light. Incubated aerobically for 72 hours at 35ºC.

L. bozemanii growing on BCYE Selective Agar

Legionella bozemanii (ATCC® 33217) colonies growing on BCYE Selective Agar (Cat. no. G08) under UV light. Incubated aerobically for 48 hours at 35ºC.

REFERENCES

1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

2. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

3. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

4. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

5. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology, J.B. Lippincott Company, Philadelphia, PA.

6. Bueshing, W.J., et al. 1985. J. Clin. Microbiology; 17:1153-1155.

7. Edelstein, P.H. 1981. J. Clin. Microbiology; 14:298-303.

8. Pasculle, et al. 1980. Pittsburgh Pneumonia Agent: Direct Isolation from Human Lung Tissue. J. Infect. Dis.; 141:727.

9. Edelstein. 1981. Improved Semiselective Medium for Isolation of Legionella pneumophila from Contaminated Clinical and Environmental Specimens. J. Clin. Microbiology; 14:298.

10. Wadowsky, R.M. and R.B. Yee. 1981. Glycine-Containing Selective Medium for Isolation of Legionellaceae from Environmental Specimens. Appl. and Environ. Micro.; 42:768-772.

11. Dennis, P.J.L., C.L.R. Bartlett, and A.E. Wright. 1984. Comparison of Isolation Methods for Legionella spp. In Thronsbury, C. et al. (ed.) Legionella: Proceedings of the 2nd International Symposium. Washington, D.C. ASM.; 294-296.

12. Garrity, G.M. (2005). Bergey's Manual of Systematic Bacteriology. Springer. New York, NY.

13. Centers for Disease Control and Prevention (CDC). 2005. Procedures for the Recovery of Legionella from the Environment. www.cdc.gov/legionella/files/LegionellaProcedures-508.pdf.

14. Bopp, E.A., J.W. Sumner, G.K. Morris, and J.G. Wells. 1981. Isolation of Legionella spp. from environmental water samples by low-pH treatment and use of a selective medium. J. Clin. Microbiology; 13:714-719.

15. International Organization for Standardization: ISO 11731:1998. Water Quality -- Detection and Enumeration of Legionella. 1998.


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