Hyphal formation is a key virulence trait in Candida albicans, playing a crucial role in the development and progression of candidiasis. As a result, it remains a significant area of interest for antifungal drug target studies. Conventional hyphal induction medium typically contain multiple components, making them complex to prepare. In this study, we have optimized a simple and cost-effective medium with minimal components that induces hyphal formation. Among different combination, MF8 with Peptone 0.16 %, Dextrose 0.4 % and BSA 0.25 % caused moderate hyphal formation. Further addition of N-acetylglucosamine (GlcNAc) enhanced hyphal formation and caused clumped mycelial growth. To determine the different combinations of GlcNAc and MgSO₄ that cause hyphal formation, we employed a Design of Experiment (DOE) approach using JMP software. GlcNAc was found to be a significant component in hyphal induction (p < 0.05, R2 = 0.26) and MgSO4 was not significant (p > 0.05). Further gene expression and pH was analysed under Run 20 conditions (30 mM GlcNAc, 1 mM MgSO₄) which revealed significant transcriptional activation of hyphal-specific genes hyphal wall protein 1(HWP1) and Hypha-specific G1 cyclin 1(HGC1) in the optimized medium. Notably, HWP1 expression increased 100-fold at 4 h and 85-fold at 6 h (p < 0.05), while HGC1 showed a 7.09-fold and 6.07-fold upregulation at the same time points (p < 0.05). Additionally, there was an initial shift toward alkalinity at 1 and 2 h time point and a drop to acidic pH at 4 h and 6 h time point suggesting hyphal formation and upregulation of hyphal specific genes induced with the lowering of pH in medium.
