A Novel and Robust Method for Investigating Fungal Biofilm.

, labeled an urgent threat by the CDC, shows significant resilience to treatments and disinfectants via biofilm formation, complicating treatment/disease management. The inconsistencies in biofilm architecture observed across studies hinder the understanding of its role in pathogenesis. Our novel in vitro technique cultivates biofilms on gelatin-coated coverslips, reliably producing multilayer biofilms with extracellular polymeric substances (EPS). This method, applicable to other species like and , is cost-effective and mimics the niche of biofilm formation. It is suitable for high-throughput drug screening and repurposing efforts, aiding in the development of new therapeutics. Our technique represents a significant advancement in biofilm research, addressing the need for consistent, reproducible biofilm models. We detail a step-by-step procedure for creating a substratum for biofilm growth and measuring biofilm thickness using confocal laser scanning microscopy (CLSM) and ultrastructure by scanning electron microscopy (SEM). This method provides consistent outcomes across various species. Key features • The biofilm formed on gelatin surfaces mimics host conditions, replicating the multilayered structure and EPS, offering a more accurate model for studying biofilms. • This method is highly reproducible and suitable for drug screening and biofilm analysis through three-dimensional (3D) reconstruction. • This in vitro technique aids in studying biofilm formation, related virulence properties, and drug tolerance of and other species. • The simple, cost-effective technique is ideal for screening novel inhibitors and repurposed drug libraries, facilitating the design/identification of new therapeutics against species.