Visualization and Documentation of Capsule and Melanin Production in Cryptococcus neoformans

Abstract

Cryptococcus neoformans is an opportunistic fungal pathogen primarily targeting immunosuppressed populations in both resource-rich and resource-limited nations. Successful treatment is limited to a few antifungals that have become compromised by cryptococcal resistance, leading to intensive research seeking new drug candidates. Two distinguishing hallmarks of this species are the ability to develop a polysaccharide capsule and melanization of the fungal cells. These also act as virulence factors, protecting this pathogen in the host as well as in the environment. Here we describe two classic methods to document capsule and melanin. Although initially described and documented several decades ago, these methods remain relevant in spite of the advent of more sophisticated methodology, due in part to their simplicity and cost efficiency. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Capsule visualization by India ink counterstaining Basic Protocol 2: Assessment of melanin on solid media Alternative Protocol: Quantification of melanin production in liquid medium.

Keywords: Cryptococcus neoformans; fungal pathogen; melanin; polysaccharide capsule; virulence.

Figure 1.. India ink counterstaining of a C. neoformans wild type (H99) and a capsule-defective mutant strain.

Strains were grown for 48 h in CO₂-independent medium then prepared for microscopy by counterstaining with India ink. Capsule is shown as the clear area between the cell body and the surrounding India ink. Note that the cells from the capsule defective strain have much less capsule when compared to the wild-type cells. Also note the heterogeneity of capsule between individual cells from the same strain. The images were captured using a Zeiss Axio Imager A.1 at 1000x magnification using DIC optics and processed with Zeiss Zen software. Scale bar = 10 microns.

Figure 2.. C. neoformans wild-type and mutant strains screened for melanin on Nyjer seed agar.

Wild type (H99) and putative melanin-defective strains were grown 24 h in YPD media. Strains were washed with PBS and then spotted (5 μl) onto a Nyjer seed agar plate. The plate was incubated for 3 days at 30°C prior to imaging with camera equipped smart phone. The upper left spot is H99 showing well-developed brown pigmentation (melanin). The remaining spots are various mutant strains ranging from no pigmentation (white) to wild type levels of pigmentation.