Open-source digital tools for filamentous fungi analysis

Reproducibility and standardization of methods are key approaches to microbiological research. However, laboratories differ widely in equipment, protocols, and operator expertise. These disparities are especially pronounced in fungal biology, since accurate spore counting and precise measurements of colony growth remain vulnerable to subjective bias and inconsistent technique. To standardize these foundational tasks, we developed two free, open-source image-analysis tools - SporeQuant and Orbis v2 - that automate hemocytometer spore counting and colony-area measurement, respectively. SporeQuant applies image thresholding and blob detection to identify individual spores and compute their concentration, while Orbis v2 segments colonies and calculates colony areas through thresholding and contour detection. Compared with manual analysis, SporeQuant and Orbis v2 markedly reduce analysis time by 63% and 76%, respectively, while achieving high measurement reliability, with MAE = 0 for SporeQuant and R² > 0.975 for Orbis v2. These tools reduce user-to-user variability, and achieve comparable or improved accuracy versus previously established methods. Furthermore, both tools can be accessed through a web page on both a computer or smartphone, requiring no specialized hardware, software, or prior knowledge. They provide accessible solutions for low-budget and high-throughput laboratories alike, enabling more consistent, scalable analysis of fungal growth and morphology in research and industrial settings.