A Novel and Robust Method for Investigating Fungal Biofilm

<i>Candida auris</i>, 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 <i>C. auris</i> biofilms on gelatin-coated coverslips, reliably producing multilayer biofilms with extracellular polymeric substances (EPS). This method, applicable to other <i>Candida</i> species like <i>C. glabrata</i> and <i>C. albicans</i>, 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 <i>Candida</i> 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 <i>Candida</i> 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 <i>C. auris</i> 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 <i>C. auris</i> and other <i>Candida</i> species. • The simple, cost-effective technique is ideal for screening novel inhibitors and repurposed drug libraries, facilitating the design/identification of new therapeutics against <i>Candida</i> species.