Rapid spot test for the determination of esculin hydrolysis

Esculin hydrolysis is a useful test in the differentiation of both gram-positive and gram-negative bacteria covering a wide spectrum of aerobes, facultative anaerobes, and anaerobes. Commonly utilized methods require a minimum of 18 h of incubation in broth or agar medium and utilize the production of a brown-black compound, due to the combination of ferric ions with the hydrolysis product esculetin, as indicator. A procedure is presented that requires 15 to 30 min for completion and utilizes fluorescence loss as the indicator of hydrolysis. Esculin fluoresces at 366 nm, whereas the hydrolysis product esculetin does not. Over 1,400 strains of gram-positive and gram-negative bacteria were tested. There was 98.4% of correlation between the spot test and esculin broth and 97% correlation with the bile-esculin agar.