CGB Agar

Cat. no. G113 CGB Agar, 15x100mm Plate, 20ml 10 plates/bag

INTENDED USE

Hardy Diagnostics CGB Agar is recommended for the differentiation of Cryptococcus neoformans var. neoformans and Cryptococcus gattii (formerly C. neoformans var. gattii ).

SUMMARY

Cryptococcosis, or torulosis, is an opportunistic and systemic infection that affects humans and mammals. It is a subacute or chronic infection most frequently involving the tissue of the central nervous system but occasionally producing lesions in the skin, bones, lungs, or other internal organs. (1,3) Cryptococcal meningitis is extremely common in AIDS patients. Also, Cryptococcus spp. have been known to parasitize cats in some areas, although strains vary in virulence. (9-11) The other species of this genus are commonly considered nonpathogenic but may occasionally cause disease in severely immunosuppressed patients.

Cryptococcosis is typically caused by Cryptococcus neoformans , an encapsulated yeast found in soil, specifically in soil containing manure from pigeons, chickens, or turkeys. The increased pH and concentration of nitrogen allows for proliferation of this organism. The yeast cells can easily become airborne due to sweeping, cleaning and construction. C. gattii is also commonly associated with certain species of Eucalyptus in India; consequently, this organism has been implicated in cases of chronic cryptococcal meningitis. (8)

Cryptococcal cells are spherical and produce a mucoid polysaccharide capsule, which may vary in width from very thin to several times the radius of the parent cell, including buds. (2) All species of Cryptococcus lack fermentative ability. Four major serotypes (A-D) exist within the capsular polysaccharide antigen, with 80-85% of isolates being either A or B. C. neoformans var. neoformans corresponds to serotypes A and D, while serotypes B and C are associated with C. gattii .

In 1981, Kyung J. Kwon-Chung et al. reported in the Journal of Clinical Microbiology that the most effective way to distinguish between the two species was to culture on a solid agar medium containing canavanine, glycine and bromothymol blue (CGB). (6) This medium is a modification of CDB agar developed in 1978, which used dextrose instead of glycine, and was prone to false-negatives resulting in improper identification. According to Kwon-Chung et al., the CGB formulation effectively eliminates the false reactions and allows for a positive color change from yellow-green to cobalt blue. The blue color is the result of an alkaline pH shift produced when creatine is degraded to ammonia. All of the C. gattii strains tested in this study, serotypes B and C, were positive by day five. C. neoformans , serotypes A and D, all produced no color change at two and five days. (6,7)

This formula contains agar for solidification, glycine as a source of carbon, and potassium phosphate, magnesium sulfate and thiamine hydrochloride as vitamins and nutrients. L-canavanine sulfate allows for the isolation of Cryptococcus as they are the only yeasts known to have a natural resistance. (7) Bromothymol blue is added to indicate the shift in pH to 7.0 when the media appears cobalt blue.

FORMULA

Ingredients per liter of deionized water:*

Glycine 10.0gm
Potassium Phosphate 1.0gm
Magnesium Sulfate 1.0gm
Bromothymol Blue 0.08gm
L-Canavanine Sulfate 0.03gm
Thiamine Hydrochloride 0.001gm
Agar 20.0gm

Final pH 5.8 +/- 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

PROCEDURE

Inoculate the medium with an isolate of Cryptococcus (see note under limitations) and incubate between 25 and 30ºC. for up to five days. (6)

INTERPRETATION OF RESULTS

Growth and a color change to medium blue indicates C. gattii ; no growth or minimal growth that is medium yellow or green may be interpreted as C. neoformans var. neoformans . (2,5,6)

LIMITATIONS

A very heavy inoculum (a heaping loopful) may produce a slight color change from yellow-green to light green with the C. neoformans variety. (6)

Note : Some yeast isolates other than C. gattii may be CGB Agar positive. (12) Therefore, suspected cryptococcal isolates should be initially identified by a positive urease test using Urea Agar (Cat. no. R42 or L65) or Urea Disks (Cat. no. DU7525) and by a positive phenoloxidase test when grown on Caffeic Acid Agar (Cat. no. G213 or L13) or through the use of Caffeic Acid Disks (Cat. no. Z118). Cryptococci are urease positive and most other fungi, except Trichosporon spp. and some Candida spp., are urease negative. (12) C. neoformans and C. gattii may be differentiated from other Cryptococci by observing colonies for melanin production in the presence of caffeic acid. (12)

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, swabs, applicator sticks, other culture media such as Caffeic Acid Agar (Cat. no. G213 or L13) or Urea Agar (Cat. no. R42 or L65), incinerators, and incubators, etc., as well as serological and biochemical reagents such as Urea Disks (Cat. no. DU7525) or Caffeic Acid Disks (Cat. no. Z118), are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Cryptococcus gattii
ATCC ® MYA-4560**
B up to 5 days 15-30°C Aerobic Growth; deep blue color change
Cryptococcus neoformans var. neoformans
ATCC ® 204092**
B up to 5 days 15-30°C Aerobic Growth minimal; if present medium yellow or green

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

USER QUALITY CONTROL

Physical Appearance

CGB Agar should appear clear, and bright yellow-green in color.

C. gattii growing on CGB Agar

Cryptococcus gattii (ATCC ® MYA-4560) colonies growing on CGB Agar (Cat. no. G113). Incubated aerobically for 72 hours at 30ºC.

REFERENCES

1. Hensyl, William R., et al. 1990. Stedman's Medical Dictionary, 25th ed. Williams & Wilkins, Baltimore, MD.

2. St. Germain, Guy, et al. 1996. Identifying Filamentous Fungi. Star Publishing Company, Belmont, CA.

3. Larone, D.H. Medically Important Fungi: A Guide to Identification, American Society for Microbiology. Washington, D.C.

4. The Oxoid Vade-Mecum of Microbiology. 1993. Unipath Ltd., Basingstoke, UK.

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

6. Kwon-Chung, K. J., I. Polacheck, and J.E. Bennett. 1982. Improved Diagnostic Medium for Separation of Cryptococcus neoformans var. neoformans (serotypes A and D) and Cryptococcus neoformans var. gattii (serotypes B and C). Journal of Clinical Microbiology ; 15:525-537.

7. Polacheck, I. and K. J. Kwon-Chung. 1985. Canavanine Resistance in Cryptococcus neoformans. Antimicrobial Agents and Chemotherapy; 29.3:468-473.

8. Chakrabarti, Arunaloke, et al. 1997. Isolation of Cryptococcus neoformans var. gattii from Eucalyptus camaldulensis in India. Journal of Clinical Microbiology; 35.12:3340-3342.

9. Gionfriddo, J. R. 2000. Feline Systemic Fungal Infections. Vet Clin North Am Small Anim Pract. 30(5):1029-50.

10. Gerds-Grogan S. and B. Dayrell-Hart B. 1997. Feline Cryptococcosis: A Retrospective Evaluation. J. Am. Anim. Hosp. Assoc. 33(2):118-22.

11. Malik R., D.I. Wigney, D.B. Muir, D.J. Gregory and D.N. Love. 1992. Cryptococcosis in Cats: Clinical and Mycological Assessment of 29 Cases and Evaluation of Treatment Using Orally Administered Fluconazole. J. Med. Vet. Mycol. 30(2):133-44.

12. Klein, K.R., L. Hall, S.M. Deml, J.M. Rysavy, S.L. Wohlfiel and N.L. Wengenack. 2009. Identification of Cryptococcus gattii by Use of L-Canavanine Glycine Bromothymol Blue Medium and DNA Sequencing. J. Clin. Micro. 47(11):3669-3672.


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