Online Monitoring of U. maydis Utilizing Fluorescence and Scattered Light Measurements in Microtiter Plate Cultivations.

High-throughput cultivation at the microtiter scale enables rapid, parameter-rich screening of various organisms, including Ustilago maydis. This chapter presents a detailed protocol for online monitoring methods, which integrate scattered light (SL) and fluorescence readouts. This chapter outlines the preparation of microtiter plates and medium, inoculation, plate sealing, and software setup. It also covers the selection of SL wavelengths and appropriate fluorescence filter cassettes. In addition, robust normalization strategies using uninoculated controls or first-measurement baselines are presented. The chapter examines how SL measurements can be calibrated and used to resolve growth phases, detect nutrient limitations relevant to product formation, and point to rare events such as spore formation. Complementary fluorescence channels expand observability. Naturally metabolic fluorophores (e.g., NADH, FAD) support biomass tracking when SL is unsuitable, while tagged strains or dye-based approaches enable pathway- and product-level readouts. As an application case, a real-time monitoring of intracellular lipid accumulation by staining with BODIPY 493/503 is demonstrated. Step-by-step procedures are paired with data-interpretation guidelines, including control design, reference selection, and pitfalls when media composition or strain background alter optical properties.