Journal of Clinical and Diagnostic Research, ISSN - 0973 - 709X

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Thanking you
With sincere regards
Dr. Rajendra Kumar Ghritlaharey, M.S., M. Ch., FAIS
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On May 11,2011

Dr. Shankar P.R.

"On looking back through my Gmail archives after being requested by the journal to write a short editorial about my experiences of publishing with the Journal of Clinical and Diagnostic Research (JCDR), I came across an e-mail from Dr. Hemant Jain, Editor, in March 2007, which introduced the new electronic journal. The main features of the journal which were outlined in the e-mail were extensive author support, cash rewards, the peer review process, and other salient features of the journal.
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On April 2011

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On Jan 2020

Important Notice

Original article / research
Year : 2011 | Month : November | Volume : 5 | Issue : 6 | Page : 1187 - 1189

Comparison of Different Media for the Pigment Production of Cryptococcus neoformans

Ruchi Katiyar, Sachin C Deorukhkar, Santosh Saini

MD (Microbiology), Assistant Professor, Department of Microbiology, Rural Medical College, Pravara Institute of Medical Sciences, Loni, Taluka-Rahata,Ahmednagar, Maharashtra, India-413736. MSc (Medical Microbiology), Assistant Professor, Department of Microbiology, Rural Medical College, Pravara Institute of Medical Sciences, Loni, Taluka- Rahata,Ahmednagar, Maharashtra, India-413736. MD(Microbiology), Professor and Head, Department of Microbiology, Rural Medical College, Pravara Institute of Medical Sciences,Loni,Taluka-Rahata, Ahmednagar, Maharashtra, India-413736.

Correspondence Address :
Ruchi Katiyar,
Assistant Professor, Department of Microbiology,
Rural Medical College, Pravara Institute of Medical Sciences,
Loni, Taluka-Rahata, Ahmednagar,
Maharashtra-413736, India
Phone: +91-9890250597


Background: Melanin production by their phenoloxidase activity is a distinctive property of the Cryptococcus neoformans isolates. An agar medium which contains a precursor of melanin is used to test the pigment production by C.neoformans. Purpose: This study aimed to compare the pigment production of C.neoformans on various media.

Materials and Methods:Twenty strains of C.neoformans which were obtained from various clinical samples were inoculated on different media and observed for the rate of growth and pigment production.

Results: Sunflower seed agar was the best medium, with the mean day of growth and the pigment production being 1.25 and 2.8 respectively. The least suitable medium for the observation of pigments in our study was mustard seed agar.

Conclusion: Sunflower seed agar is a simple and inexpensive tool for the presumptive identification of C.neoformans in clinical microbiology laboratories.


Cryptococcus neoformans, melanin production, phenoloxidase, sunflower seed agar, mustard seed agar

Cryptococcosis is an acute, subacute or chronic fungal disease which is caused by encapsulated basidiomycetous yeasts which belong to the genus, Cryptococcus (1). This yeast is a particularly fascinating fungal pathogen, because it crosses the entire spectrum of the host immunity. For instance, it produces infections in apparently immunocompetent patients without any known underlying disease, but on the other hand, commonly the yeast invades a severely immunosuppressed host as a result of HIV infection, organ transplantation or malignancy or due to treatment with high doses of corticosteroids (2).

Cryptococcosis is one of the Acquired Immuno deficiency Syndrome (AIDS) defining illnesses (3).

The incidence of cryptococcosis has risen dramatically over the past 20 years. The Human Immunodeficiency Virus (HIV) epidemic and other forms of immunosuppression are the common factors which explain this increase. Cryptococcus neoformans var. neoformans (C.neoformans) is the species which has been predominantly reported from immuno-compromised patients (4).

C.neoformans has several well-characterized virulence phenotypes. The three classical phenotypes which were under genetic control have been: (1) capsule production (2) melanin formation; and (3) the ability of a yeast strain to grow at 37°C (5). Melanin production by their phenoloxidase activity is a distinctive property of the C.neoformans isolates. The ability to produce these melanin pigments is one of the most used criteriae for the identification of C.neoformans from clinical and environmental isolates and for the evaluation of Cryptyococcus virulence (6),(7). It has been suggested that a primary mechanism for melanin’s importance is its capacity to act as an antioxidant, but there are other possible mechanisms by which the yeast might use melanin for protection from the host, including cell wall integrity and charge, interference with antifungal susceptibility, abrogating antibody-mediated phagocytosis, and protection from extreme temperatures (8).

Melanin production is usually tested in a proper agar medium which contains a precursor of melanin. For this purpose, agar media which contain L-dopa (9), caffeic acid (10), bird seed (11),(12), sunflower extracts (13), tobacco (14),(15), henna (16) and mustard seed (17) have been reported to be used so far. This study was undertaken to compare the pigment production of C.neoformans on various media.

Material and Methods

This study was carried out in the Department of Microbiology of this medical college. A total of 20 C. neoformans isolates which were obtained from various clinical samples, which were received in the Mycology section were included in the study. The definitive identification of C. neoformans was done on the basis of: (18) • Growth at 370 C. • Hydrolysis of Christensen’s urea agar. • Inositol and nitrate assimilation. • Production of brown pigment.

The pigment production of C.neoformams was observed on the following media: 1. Niger seed agar. 2. Sunflower seed agar. 3. Tobacco agar. 4. Mustard seed agar. 5. Henna agar.

All the media were prepared in the media section of the laboratory as per the techniques which are described by other workers (11), (13), (14), (16), (17).

The media were inoculated with C .neoformans and incubated at 37°C for a period of 2 weeks. The plates were observed daily for growth and pigment production. Candida albicans was used as a negative control.


15 isolates of C.neoformans showed growth on the first day on sunflower seed agar, whereas 11 isolates showed pigment production on the second day(Table/Fig 1)(Table/Fig 2)(Table/Fig 3)(Table/Fig 4).


The need for the rapid and accurate identification of C.neoformans in the clinical laboratories has in recent years, led to the development of many identification tests which are based on the phenoloxidase enzyme activity of the organisms. The concept of using different media for the identification of C.neoformans is not new, but problems like elevated costs, complex media preparation, the time which is required for the growth and pigment production and ill-defined interpretations are frequently encountered.

Sunflower seed agar, which is prepared from pulverised sunflower seed, is the best medium for the early growth and pigment production of C.neoformans. In our study, most of the isolates produced a brown pigment within 48 hours. Khan et al have also reported the production of brown colonies of C.neoformans on this medium (19). Since sunflower seed agar is known to impart a brown pigmentation to the C.neoformans colonies, it has been utilized for the isolation and presumptive identification of C.neoformans from saprophytic and clinical sources (20). It is easy to prepare and inexpensive and the contents are readily available in the market. Sunflower is one of the major crops which are cultivated in Maharashtra.

Tobacco agar is the next suitable medium for the appreciation of the brown pigment of C.neoformans. Tobacco, which is used in this medium, is also widely available over the counter in India. A maximum number of isolates showed pigments on this media within 72 hours. Tendolkar et al also reported the same finding, where all the isolates produced a brown pigment within 48-72 hours of their incubation (14). Khan et al reported the appearance of dark brown pigmented colonies of C.neoformans on this medium after 72 hours of incubation (15). In our study, most of the strains of C.neoformans produced a brown pigment on henna agar within 72 hours of incubation. Nandhakumar et al have reported that all isolates of C.neoformans produced a brown pigment on this medium at 24 hours post inoculation (16). In this study, the rate of growth and pigment production of C.neoformans was late or poor on mustard seed agar, which was in contrast to the observations of Nandhakumar et al, who reported the brown colour effect of C. neoformans at 48 hours post inoculation (17). Since the concept of using mustard seed agar for the appreciation of the pigments of this yeast is new, further evaluation of this medium by using more strains of C.neoformans is required to confirm its utility for the diagnosis of cryptococcosis.

It can be concluded from this study, that of the various media which were used for the pigment production of C.neoformans, sunflower seed agar was the most valuable tool for the selective isolation and presumptive identification of C.neoformans.


Cox GM, Perfect JR. Cryptococcus neoformans var. neoformans and gattii and Trichosporon species. In: Topley and Wilson’s Microbiology and Microbial Infections. Ajello, Land – May RJ, editors Arnold: London; 1999; 461-84.
Perfect J, Casadevall A. Cryptococcosis. Infect. Dis. Clin. North. Am 2002; 16:837-74.
Iyer R, Banker D. Cryptococcal meningitis in AIDS. Indian J Med Sci 2002; 56:593-7.
Capoor M R, Nair D, Deb M ,Gupta B, Aggarwal P. Clinical and mycological profile of cryptococcosis in a tertiary care hospital. Indian J Med Microbiol 2007; 25:401-04.
Casadevall A, Perfect J. Cryptococcus neoformans. ASM press, Washington, DC.
Chaskes S, Tyndall RL. Pigment production by Cryptococcus neoformans and other Cryptococcus species from aminophenols and diaminobenzenes. J Clin Microbiol 1978; 7:146-52.
Douchet C,Chandenier J, Barrabes A, Therizol- Ferly M, Lenoble R. Reconatssance rapide de colonies de Cryptococcus neoformans. J Mycol Med 1995; 5:122-3.
Perfect JR. Cryptococcus neoformans: A sugar- coated killer with designer genes. FEMS Immunology and Medical Microbiology 2005; 45:395-404.
Chaskes S, Tyndall RL. Pigment production by Cryptococcus neoformans from para and ortho-diphenols: effect of the nitrogen source. J Clin Microbiol 1975;1:509-14.
Pulverer C, Korth H. Cryptococcus neoformans: Pigment bildung aus verschiedenes polyphenolen. Med Microbiol Immunol 1971; 175:46-51.
Paliwal DK, Randhawa HS. Evaluation of a simplified Guizotia abyssinica seed medium for the differentiation of Cryptococcus neoformans . J. Clin. Microbiol 1978; 7:346-8.
Denning DW, Stevens DA, Hamilton JR. Comparison of the Guizotia abyssinica seed extract (Birdseed) agar with conventional media for the selective identification of Cryptococcus neoformans in patients with Acquired Immunodeficiency Syndrome. J Clin Microbiol 1990; 28:2565-7.
Pal M. Use of Pal’s Sunflower medium for an early diagnosis of Cryptococcus neoformans. Antiseptic 1997; 95:175.
Tendolkar U, Taniwala S , Jog S, Mathur M.. Use of a new medium – tobacco agar, for the pigment production of Cryptococcus neoformans. Indian J .Med. Microbiol 2003;21:277-9.
Khan ZU, Ahmad S, Mokaddas E, Chandy R.Tobacco agar, a new medium for differentiating Candida dubliniensis from Candida albicans. J Clin Microbiol 2004; 42:4796-8.
Nandhakumar B, Menon T, Kumar G. A new henna -based medium for the differentiation of Cryptococcus neoformans. J. Med. Microbiol 2007;568.
Nandhakumar B, Kumar G, Prabhu CP, Menon T. Mustard seed agar, a new medium for the differentiation of Cryptococccus neoformans. J Clin Microbiol 2006;44:674.
Koneman EW, Roberts GD. Practical Laboratory Mycology. 3rd ed. (Williams and Wilkins, Baltimore)1985;143-59.
Khan ZU, Ahmad S, Mokaddas E, Chandy R. Simplified sunflower (Helianthus annuus) seed agar for the differentiation of Candida dubliniensis from Candida albicans. Clin Microbiol Infect 2004; 10: 590-2.
Pal M, Mehrotra. Studies on the efficacy of sunflower seed agar for the isolation and identification of Cryptococcus neoformans. Arogya. J. Health Sci 1982; 8:74-9.

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