cryptococcus

Introduction

Staib agar medium has been widely used for isolation and identification of yeasts from the genus Cryptococcus. It was developed in 1962, by the German mycologist Friedrich Staib, who thought of a culture medium made of Guizotia abyssinica, an oval black seed used for bird feeding. It is also used in India and Ethiopia for oil production (1, 2).

In his first studies, Staib employed powdered canary droppings, which is used by Cryptococcus neoformans as a nitrogen source. Thus, Staib observed a color change in the colonies, which grew in this medium, turning into a brown color after 4 days of incubation (3).

Later, Staib found that the color variation was not only dependent on the canary droppings, but on the food these birds consumed, principally made of Guizotia abyssinica seeds (3).

These seeds contain cafeinic acid, among other phenolic compounds, in which O-diphenol undergoes an oxidation process by action of the phenoloxidase enzyme, produced by C. neoformans, originating a melanin pigment which covers the yeast wall, thus turning the colonies growing in culture to a dark brown color. This feature differentiates C. neoformans from other yeasts. This reaction was named “Brown color effect” (BCE) by this author (2, 4). Then, BCE depends on the enzyme diphenoloxidase, an antioxidant, which helps the yeast to survive inside the host by the production of melanin (5).

This medium is prepared with glucose, creatinine and potassium phosphate (6). Creatinine is assimilated by C. neoformans; this is possible only in the presence of thiamine or other compounds like thiazol and pirimidine, which are in the seeds. Creatinine is used by the yeast as nitrogen source, its assimilation is especially important in serotypes B and C, but not A and D. This assimilation has been demonstrated by the presence of a green diffusible pigment (1, 2, 3, 7).

Staib medium is used routinely since the beginning of the AIDS epidemic, which increased the frequency of opportunistic infections, including fungal. Culture of samples (especially sputum) from AIDS patients directly onto this medium shows brown colonies of C. neoformans (5, 7-13).

For these reasons, we started to use this medium in our medical mycology laboratory, for the isolation and identification of C. neoformans in clinical samples from AIDS patients.

Initially, Staib agar was being prepared with the traditional method, but further on in the study there was a failure in the supply of some of the compounds, especially creatinine. For this reason the medium was modified, and prepared without creatinine. Surprisingly, C. neoformans colonies showed a deeper brown color than in the original Staib agar. We decided to continue using this medium with excellent results.

The aim of this study was to assess the usefulness of Staib agar without creatinine for the identification of C. neoformans, by the observation of the presence of pigment, macro and microscopic morphological development and time of growth, using strains from the Medical Mycology Department Culture Collection.

Materials and Methods

After certifying their pureness and viability, forty-six C. neoformans strains from the Medical Mycology Department Culture Collection were identified by morphological and biochemical criteria, using urease test (Christensen urea agar) (14) and culture in Sablac medium, which allows better capsule development. They were also identified with the Microscan™ Dade Behring Automated System.

C. neoformans (WC 1400 IMT) and Candida albicans (ATCC 90028) were used as urease positive and negative control isolates, respectively.

Additionally 5 isolates of Candida dubliniensis (S2-5, S2-6, S2-14, S636 and S645) from the Health Science Center, University of Texas (supplied by Dr. Anette Fothergill) were used to assess their development in Staib agar with and without creatinine.

All strains were then transferred into standard Staib and Staib without creatinine agar plates (two isolates per plate), they were incubated at room temperature (26-28°C) for one week, and then the morphological features were evaluated.

Canavanine-glycine-bromotimol blue (CGB) medium (15) was used to identify Cryptococcus isolates. All isolates were inoculated in this medium and incubated at room temperature for 5 days. Interpretation of results: yellow color: C. neoformans var neoformans, dark blue color: C. neoformans var gattii.

Descriptive statistics were used to analyze data obtained, using frequency tables, and percentage and sensitivity determination.

Results

During the course of this study we were forced to use Staib medium without one of its main components, creatinine. The experiments were continued using the modified medium. Later in the study, we were able to find the creatinine and nevertheless decided to use both kinds of Staib medium, the original and the modified.

Forty-seven C. neoformans strains used for the study were tested for urease production, being all positive. The same strains were tested for phenoloxidase production, culturing in standard and modified Staib agar. They yielded brown color colonies in both media in 43 (91.5%) cases (Figura 1, 2). In 4 (8.5%) of the studied strains, the colonies remained cream color, in spite of being urease positive and identified by automated Microscan™ method (Table 1).

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