Bile Esculin Discs are used for detection of esculin hydrolysis in the presence of bile, for differentiating Group D streptococci from other Streptococcal groups. Group D streptococci hydrolyze esculin to esculetin and dextrose. Esculetin reacts with an iron salt such as ferric citrate to form a blackish brown coloured complex. Bile esculin test as a reliable means of identifying Group D streptococci and differentiating them from other streptococci groups. Enterococcus faecalis ATCC 29212 positive: blackening of media around the disc ; Streptococcus agalactiae ATCC 13813: negative no blackening