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Year : 2003  |  Volume : 21  |  Issue : 4  |  Page : 277--279

Use of a new medium - Tobacco agar, for pigment production of cryptococcus neoformans

U Tendolkar, S Tainwala, S Jog, M Mathur 
 Department of Microbiology, Lokmanya Tilak Municipal Medical College and General Hospital Sion, Mumbai - 400 022, India

Correspondence Address:
U Tendolkar
Department of Microbiology, Lokmanya Tilak Municipal Medical College and General Hospital Sion, Mumbai - 400 022


Cryptococcus neoformans produces brown colonies on Niger seed (Guizotia abyssinica) agar. Media containing caffeic acid, L-dopa and other diphenolic compounds, have been used for the same purpose. The present report describes a new medium containing tobacco which supports growth of C.neoformans and allows its easy differentiation by formation of brown coloured colonies.

How to cite this article:
Tendolkar U, Tainwala S, Jog S, Mathur M. Use of a new medium - Tobacco agar, for pigment production of cryptococcus neoformans.Indian J Med Microbiol 2003;21:277-279

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Tendolkar U, Tainwala S, Jog S, Mathur M. Use of a new medium - Tobacco agar, for pigment production of cryptococcus neoformans. Indian J Med Microbiol [serial online] 2003 [cited 2020 Jul 3 ];21:277-279
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Cryptococcus neoformans can be easily identified by the dark brown colonies it forms on Niger seed agar.[1] In addition, it produces brown black colonies in 4-5 days on other special media containing caffeic acid,[2] L-dopa,[3] chlorogenic acid,[4] dopamine, epinephrine or norepinephrine.[5] Another useful medium is Pal's medium based on similar principle.[6] These media are useful in selecting colonies of C.neoformans from mixed cultures expected from environmental samples and clinical samples such as respiratory specimens and urine. The property of producing brown colonies on these media provides a definitive identification of C.neoformans and is due to production of a black pigment from various substrates which accumulates in the fungal cell due to an enzyme laccase.[7]

In India, a substantial population of people is habituated to chewing on tobacco leaves in its dry form as such or rolled in fresh betel leaves. This habit if chronic, results in deposition of brown pigment on the teeth. Assuming the nature of these pigments to be melanoid, a new medium was devised using tobacco leaves to see if C.neoformans develops brown colonies on it. To the best of our knowledge this is the first instance of the use of tobacco in the preparation of a differential medium for C.neoformans.

 Materials and methods

Preparation of tobacco agar

The tobacco agar was prepared by a similar technique which is used to prepare niger seed agar.[8] To 50g of tobacco leaves, one litre of distilled water was added. The mixture was boiled for 30 minutes. It was filtered through gauze and the volume was adjusted to one liter with distilled water. To this was added 20g agar. It was boiled till the agar dissolved and autoclaved at 121C for 15 minutes. The agar was poured in plates. Since only pure cultures were tested on this medium, antibiotics were not added. The agar after setting was transparent and brownish yellow in colour.


Four serotypes of C.neoformans, A, B, C and D (CDC Y-589-92, CDC Y-586-92, CDC Y-603-92, CDC Y-600-92 respectively) were inoculated, with Candida albicans as the negative control. Within 48 hours at 30C incubation, all serotypes of C.neoformans showed dark brown colonies which were easily appreciated despite the background brownish yellow colour of the medium, while those of Candida albicans remained white upto a maximum period of incubation upto two weeks [Figure:1]. Fifteen local clinical isolates of C.neoformans also gave the same results.


A number of selective media[2],[3],[4],[5] were devised for C.neoformans following Staib's observation that C.neoformans selectively absorbs a brown pigment from the seeds of a common weed Guizotia abyssinica.[1] The Niger seed agar found a key position in the identification of C.neoformans in the yeast identification schema.[9] The length of duration for incubation of media based on similar principle, after which the medium may be discarded if found to be sterile, is usually taken as two weeks, when the medium is used for primary isolation directly from clinical specimens. Pigment production in C.neoformans is dependent upon presence of exogenous substrates unlike some other fungi which can make pigments endogenously.[3] The pigment of C.neoformans is cell-associated and is a melanin like pigment,[10] formation of which is catalysed by a laccase enzyme.[11]

A persistant habit of chewing tobacco leaves[12] and even smoking often results in staining of teeth. Most dentists associate tobacco use with staining of teeth in smokers.[13] Thus it was hypothesized that tobacco has substrates responsible for melanin like pigment deposition on teeth. Taking this as a basis, the tobacco medium was prepared.

The use of tobacco in this medium is economical. It is widely available over the counter in India and offers a transparent medium which would theoretically allow an early detection of colonies. The only drawback is the high colour of the uninoculated medium. However, despite this background, the dark pigmented colonies of C.neoformans are easily appreciable. A standardization of the quantity of tobacco may solve this problem. Antibiotics may be added to prevent bacterial growth.[8] The nature of the substrate in tobacco which is acted upon by C.neoformans or the basis of formation of pigmented colonies of C.neoformans on this medium are obscure at this stage. It is presumed to be due to laccase.

In a study of 46 cases of cryptococcal meningitis in India,[14] 41.3% of the patients with cryptococcal meningitis were found to be smokers. This rate was significantly higher (pC.neoformans generally occurs through the respiratory route, a structural damage to the lung due to smoking habits may add to the pathogenicity of C.neoformans. There is a possibility of C.neoformans utilizing the substrates of tobacco to its survival benefit.

The laccase of C.neoformans is a virulence factor for the organism.[7] Melanization of the fungal cells has been found to protect the organism against environmental stresses[16],[17] and host defenses.[10] A study on the utilization of substrates (in tobacco smoke / juice) by C.neoformans and subsequent melanization would be interesting. Since this is a preliminary report on the medium, evaluation of the medium with an extensive range of yeasts is warranted.


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