|Enterococcus faecium colonies on Bile-Esculin Agar: dark
brown areas around colonies due to esculin hydrolysis
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 (variants commercially 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
Harvest a well isolated colony and inoculate a bile-esculin tube by stabbing the
medium then zig-zag streaking on the surface of the slant. Incubate at 37 ºC, 24 to
Reaction is considered positive if the slant turned blue-black / brown 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.
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