CAFFEIC ACID AGAR - "Bird Seed Agar" for the identification of Cryptococcus neoformans

Cat. no. G213 Caffeic Acid Agar, 15x100mm Plate, 18ml 10 plates/bag
Cat. no. L13 Caffeic Acid Agar, 16x100mm Tube, 5.5ml Slant 20 tubes/box

INTENDED USE

Hardy Diagnostics Caffeic Acid Agar Slant is recommended for the selective isolation and differentiation of Cryptococcus neoformans and Cryptococcus gattii .

SUMMARY

Cryptococcus neoformans is an encapsulated yeast that produces the enzyme phenoloxidase, an enzyme necessary in melanin synthesis. When in the presence of caffeic acid, the enzyme attacks the acid resulting in the production of melanin. Subsequently, melanin is absorbed by the cell wall of the yeast producing tan to brown pigmented colonies.

The brown pigmented colonies of Cryptococcus neoformans were observed by Staib in 1962 when he grew cultures of the yeast on media containing Guizotia abyssinica seeds. (6) It was later determined that the seeds contain caffeic acid, which served as the melanin-producing substrate.

In 1966, Shields and Ajello modified Staib's Birdseed Agar by making the medium selective with an antimicrobic additive. (7) Hardy Diagnostics Caffeic Acid Agar is a modification of the latter formula.

FORMULA

Ingredients per liter of deionized water:*

Ammonium Sulfate 5.0gm
Glucose 5.0gm
Yeast Extract 2.0gm
Dipotassium Phosphate 0.8gm
Magnesium Sulfate 0.7gm
Caffeic Acid 0.18gm
Chloramphenicol 0.05gm
Ferric Citrate Solution 4.0ml
Agar 15.0gm

Final pH 6.5 +/- 0.3 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 and freezing.

PRECAUTIONS

PROCEDURE

Specimen Collection: Infectious material should be submitted directly to the laboratory without delay and protected from excessive heat and cold. Consult listed references for information on specimen collection. (2-5)

Method of Use:

1. Prior to use, allow the medium to equilibrate to room temperature.

2. Using aseptic technique, inoculate sample to medium.

Note: Sputa specimens can be inoculated directly to the agar surface with a sterile loop or rayon-tipped swab. Urine samples should be centrifuged for 15 minutes at 1,500Xg and the sediment recovered with a sterile transfer pipette. Evenly distribute sediment over agar surface.

3. Incubate at room temperature (15-30ºC.) for 3-5 days.

4. Observe daily for the production of brown to black pigmented colonies.

INTERPRETATION OF RESULTS

Development of brown to black pigmented colonies is a positive presumptive identification for C. neoformans .

LIMITATIONS

Yeasts other than C. neoformans may rarely produce brown pigmentation on media containing caffeic acid.

A Sabouraud Dextrose Agar (Cat. no. W70) control should be inoculated in parallel to the Caffeic Acid Agar slant to ensure that a dark pigment is not naturally produced by the colonies. Aureobasidium , Sporothrix , Wangiella , and Phialophora may produce dark brown colonies, but the pigment will not be a result of enzymatic activity which is made evident by pigmentation developing in colonies on all media.

Rare strains of C. neoformans may not produce pigmented colonies.

Specimens heavily contaminated with bacteria may obscure growth and/or pigmentation of C. neoformans .

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, other culture media, swabs, 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
Cryptococcus neoformans
ATCC ® 32045*
A 72-96hr 15-30°C Aerobic Growth; brown to black pigmented colonies
Escherichia coli
ATCC ® 25922*
B 24hr 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

Caffeic Acid Agar should appear slightly opaque, with precipitate, and light gray in color.

C. neoformans growing on Caffeic Acid Agar

Cryptococcus neoformans (ATCC ® 32045) colonies growing on Caffeic Acid Agar (Cat no. L13). Incubated aerobically for 72 hours at 30ºC.

E. coli inhibited on Caffeic Acid Agar

Escherichia coli (ATCC ® 25922) growth inhibited on Caffeic Acid Agar (Cat no. L13). Incubated aerobically for 24 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. Staib, F. 1962. Hyg. Infektionskr. Med. Mikeobiol. Immunol. Virol. ; 148:466-475.

7. Shields, A.B. and Ajello, L. 1966. Medium for Selective Isolation of Cryptococcus neoformans, Service; 151:208-209.

8. Denning, D.W., et al. 1990. Journal of Clinical Microbiology; Vol. 28, No. 11, p. 2565-2567.

9. La Rocco, Mark, Ph.D. 1992. Clinical Microbiology Newsletter; Vol. 14, No. 23, p. 177-181.


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121415hh