Development of a Cost-Effective and Low-Toxicity Safranin-Based Assay for Discovering Candida albicans Biofilm Formation Inhibitors in Large-Scale Screening Campaigns

Screening assays for discovering Candida albicans biofilm inhibitors typically measure either metabolic activity or attached biomass. Although biomass staining with Crystal Violet or Safranin is affordable and easy to perform, standard protocols often require toxic solvents for biofilm fixation and dye extraction. We developed and evaluated an alternative Safranin staining method that uses heat fixation and direct absorbance measurement of stained biofilms without a dye-extraction step. Under the experimental conditions tested, the proposed method reduced solvent use and plate handling and minimized reagent use while offering assay consistency and reliability for primary screening campaigns. The assay quality was verified using positive and negative inhibition controls, with acceptable Z′-factor values supporting its use for primary drug screening. The protocol was further evaluated by screening 1520 compounds from the Prestwick Chemical Library to identify inhibitors of C. albicans biofilm formation. The proposed Safranin method provides a simplified, extraction-free alternative for biofilm-inhibitor screening under the experimental conditions evaluated in this study.