Used as a chromogenic substrate in molecular biology and microbiology for the detection of β-glucuronidase (GUS) enzyme activity. Its primary application is in genetic engineering, especially in plant transformation studies, where the GUS reporter gene is introduced into organisms. When the enzyme β-glucuronidase is expressed, it cleaves the glucuronide bond, releasing the insoluble blue indigo dye 6-chloro-3-hydroxyindole, which rapidly dimerizes and oxidizes to form a visible blue precipitate. This allows for easy visualization of gene expression patterns in tissues. The cyclohexylammonium salt form improves solubility and stability in aqueous solutions, making it suitable for histochemical staining. Also used in rapid detection of pathogenic bacteria such as Escherichia coli O157:H7 in food and clinical samples, where GUS activity serves as a marker for identification.