Organisms that split the esculin molecules and use the liberated glucose to
supply energy needs release esculetin into the medium. The free esculetin reacts with
ferric citrate in the medium to form a phenolic iron complex, which turns the agar slant
dark brown to black. An agar slant that is more than half darkened after no more than
48 hours’ incubation is bile-esculin positive. If less than half the slant has darkened, the
result is negative.