Esculin sesquihydrate is primarily used as a differential agent in microbiological media. It functions as a chromogenic substrate for detecting β-glucosidase activity; upon hydrolysis by bacteria, it produces esculetin, which reacts with ferric ions to form a black precipitate, facilitating the identification of pathogens such as Listeria monocytogenes and enterococci. It is commonly incorporated into selective-differential media like bile esculin agar to isolate and differentiate gram-positive bacteria while suppressing some unwanted flora through the medium's formulation. Additionally, it is employed in certain anticoagulant assays to evaluate clotting mechanisms due to its derivative's inhibitory effects on coagulation. Esculin also serves as a fluorescent compound in biochemical detection methods under UV light.