Influence of environmental and nutritional conditions on Nakaseomyces glabratus biofilm development in vitro.

Biofilm formation is a major virulence trait in Nakaseomyces glabratus, contributing to antifungal tolerance and persistent infections. Environmental and nutritional parameters influence fungal biofilm development, but standardized experimental frameworks for evaluating N. glabratus biofilm modulation remain limited. This study investigated how defined environmental conditions, including incubation time, agitation, and carbon source supplementation modulate biofilm development under controlled in vitro conditions. Biofilms were formed for 24-72 h under static (0 rpm) and agitated (120 rpm) conditions in Sabouraud dextrose broth supplemented with glucose or galactose as carbon sources. Biofilm formation was assessed using complementary analytical methods, including crystal violet staining for total biomass, XTT reduction assay for metabolic activity, viable cell counts, and scanning electron microscopy for structural characterization. Incubation time and culture medium composition significantly affected biofilm development, whereas agitation exerted a comparatively minor effect. High carbon supplementation acted as a controlled metabolic challenge, enabling the investigation of biofilm adaptability under carbon-excess conditions. Overall, this study establishes a reproducible in vitro framework for investigating environmental and nutritional modulation of N. glabratus biofilms and provides a basis for future functional and antifungal studies.