What substance does the ONPG test use to detect β-galactosidase activity?

The ONPG test utilizes Ortho-Nitrophenyl-β-galactoside as the substrate to detect β-galactosidase activity. This compound is a synthetic substrate that, when cleaved by β-galactosidase, releases ortho-nitrophenol, which can then be detected colorimetrically. The presence of the yellow color indicates that the enzyme is active, thus confirming the organism's ability to produce β-galactosidase. This testing is particularly useful in microbiology for differentiating members of the Enterobacteriaceae family and other bacteria based on their enzymatic capabilities. The other choices listed—deoxyribonucleic acid, phenylalanine, and glucose—do not serve as substrates for this specific enzymatic reaction, and therefore, are not relevant in the context of this test.