Cryptococcus neoformans Biofilm Formation and Quantification

Cryptococcus neoformans is an opportunistic fungal pathogen that heads the Fungal Priority Pathogen List published by the World Health Organization (WHO) in 2022. This pathogen is a primary cause of death for immunocompromised individuals (e.g., those with HIV/AIDS, the elderly, immunotherapy recipients), causing approximately 118,000 deaths yearly worldwide. C. neoformans relies on virulence factors that include a polysaccharide capsule, melanin, extracellular enzymes, and thermotolerance to initiate and sustain host infection. Additionally, similar to other fungal pathogens (e.g., Candida albicans), C. neoformans may develop a biofilm organization linked to more persistent cryptococcal infections. Cryptococcal biofilms are highlighted in cases of cryptococcal meningitis, in which biofilm-like structures form that are highly resistant to host immune response and to antifungal therapies. In this regard, fungal biofilm formation has become an important area of study as a means to improve our understanding of the mechanisms regulating biofilm formation and infection and to advance the discovery of antibiofilm therapeutics. To assess biofilm properties and compare across treatments, quantification and evaluation of cell viability are important. Herein, we describe a standardized method to establish a cryptococcal biofilm and quantify total biomass and cell viability. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Culturing and biofilm formation Basic Protocol 2: Biofilm quantification Alternate Protocol: Biofilm viability assay.