Objective: To determine the ability of an organism to hydrolyze the glycoside esculin to esculetin and glucose. Esculetin reacts with iron to form a dark brown to black complex. The medium contains 40% bile. Some streptococci that are capable of splitting esculin cannot tolerate an increased concentration of bile. So this is basically two tests: (a) growth on 40% bile and (b) hydrolysis of esculin.
Obtain a bile esculin slant. With a sterile loop, touch a colony of your pure culture to obtain a light inoculum. Uncap the tube and flame the lip of the tube. Insert the loop to the bottom surface of the agar. Touch the agar and gently slide the loop in a zigzag fashion along the surface of the agar as you pull the loop out. Flame the lip of the tube again and put the cap back on. Incubate the slant at 37°C for 48 hrs and check the results. If the results seem negative, continue incubation for up to 96 hrs before reporting the results as negative.
-- Positive: Half or more of the medium is blackened (black to dark brown) in any time interval.
-- Negative: Less than half of the tube is blackened after 96 hrs.