Esculin in the medium is hydrolyzed to esculetin and other compounds. The esculetin (released from esculin by
beta-glucosidase) reacts with ferric chloride in the medium to form a black-brown color.
Prepare bile-esculin agar medium (commercial variants available)
containing 0.1% esculin, 4% oxgall & 0.05% ferric citrate, adjust pH to 6.6 .
Pour the medium filling the bottom half of the tube and creating a slant in
the upper half.
Harvest a well isolated colony and inoculate a bile-esculin tube by
stabbing the medium then zigzag streaking on the surface of the slant.
Incubate at 37 °C, 24 to 120 hours.
Reaction is considered positive if the slant turned blue-black and negative
if no change from the original color occurred.
The determination of esculin hydrolysis by fluorescence loss, visualized with a 365 nm UV lamp.
Prepare 0.02% esculin solution. Add few drops of esculin solution on filter paper, using a microscope slide as
support. Pick a colony and dispense on filter paper. Incubate 30 minutes at 37 °C then observe with ultraviolet light.
Darkening indicates esculin hydrolysis; no hydrolysis if fluorescence persists.
Bile-esculin is a selective and differential medium used in the identification of catalase-negative bacteria.
The selective agent bile, inhibits most Gram positive bacteria.
1. Murray, P.R., Baron, E. J., Jorgensen, J.J., Pfaller, M.A., and Yolken, R.H. Manual of Clinical Microbiology, 8th
ed. ASM Press: Washington, DC, 2003.
2. Stephen C. Edberg, Sample Pittman & Jacques M. Singer. Esculin Hydrolysis by Enterobacteriaceae. JOURNAL
OF CLINICAL MICROBIOLOGY, Aug. 1977, P. 111-116.
3. Ki-Sun Kwon, Jaehoon Lee, Hyung Gyoo Kang, and Yung Chil Hah. Detection of β-Glucosidase Activity in
Polyacrylamide Gels with Esculin as Substrate. Appl Environ Microbiol. 1994 December; 60(12): 4584–4586.
(c) Costin Stoica