Evaluation of tobacco agar for differentiation of Cryptococcus neoformans

Z.U. Khan, R. Chandy

Author address: 

Kuwait, KW


Background and objective: Cryptococcus neoformans is the etiologic agent of cryptococcosis, a systemic mycosis of humans and animals with a world-wide distribution. The aim of the study was to evaluate the efficacy of tobacco agar as a differential medium for C. neoformans and its serotypes. Methods: One hundred and sixty-eight isolates of C. neoformans originating from clinical specimens (n = 8), and environmental sources (n = 160) were evaluated for their ability to produce brown melanin-like pigment on tobacco agar. In addition, reference strains or clinical isolates of C. neoformans (n = 10), C. laurentii (n = 1), C humicola (n = 1), Candida albicans (n = 55), C. parapsilosis (n = 9), C. tropicalis (n = 8), C. krusei (n = 5) and C. glabrata (n = 7) were also tested. Twenty-five gram of tobacco obtained from commercially available cigarette brand (Marlboro; tar 8 mg, nicotine 0.6 mg; Philip Morris Products SA, Richmond, VA, USA) was mixed with 1 l of distilled water. The mixture was boiled for 30 min and then filtered through several layers of gauze. To this filtrate 20 g of agar was added and the volume was made up to 1 l. The pH of the medium at this point was 5.4. It was autoclaved at 121 °C for 15 min. Twenty millilitres medium was poured into each plate. The test cultures were streaked on the medium and plates were incubated at 280 °C and examined for development of brown-coloured colonies up to 96 h. Results: All the isolates of C. neoformans developed brown-coloured colonies within 48 h. Induction of brown pigmentation was discernible as early as12 h. Isolates belonging to serotypes B (n = 62) and C (n = 1, reference strain) produced more intense pigmentation (cherry brown) as compared to serotypes A (n = 106) and D (n = 1, reference strain). Conclusion: The results of the study suggest that tobacco agar can be used as a differential medium for presumptive identification of C. neoformans from other Cryptococcus species and Candida species. While isolates of serotype B produced more intense brown pigmentation as compared to serotype A, this phenomenon needs to be evaluated more extensively with respect to serotypes C and D. Acknowledgements: The authors are thankful to Prof. H. S. Randhawa and Dr. Anuradha Chowdhary for providing environmental isolates and some reference strains of C. neoformans.

abstract No: 


Full conference title: 

16th European Congress of Clinical Microbiology and Infectious Diseases
    • ECCMID 16th (2006)