Bile Esculin Test

Bile Esculin Test

Last Updated on January 12, 2020 by Sagar Aryal

Bile Esculin Test

Objectives of Bile Esculin Test

  • To identify Enterococci and group D Streptococci based on the ability of an organism to hydrolyze esculin in presence of bile.
  • To differentiates enterococci and group D streptococci from non–group D viridans streptococci.

Principle of Bile Esculin Test

Esculin is a glycoside (a sugar molecule bonded by an acetyl linkage to an alcohol) composed of glucose and esculetin. These linkages are easily hydrolyzed under acidic conditions. Many bacteria can hydrolyze esculin, but few can do so in the presence of bile. Bile esculin medium contains esculin and peptone for nutrition and bile to inhibit Gram-positive bacteria other than Group D streptococci and enterococci.  Organisms capable of growth in the presence of 4% bile 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.

Media Used for Bile Esculin Test

Bile esculin agar base: Peptone 5.0gm/L, Meat(beef) extract  3.0gm/L, Bile 40.0gm/L, Ferric citrate 0.50gm/L, Agar 15.0gm/L,  Final pH ( at 25°C) 6.6±0.2

Procedure of Bile Esculin Test

  1. Inoculate one to two colonies from an 18- to 24-hour culture onto the surface of the slant.
  2. Incubate the tubes at 35°-37°C in ambient air for 48hours.
  3. Examine tube for the growth and colour change.

Result Interpretation of Bile Esculin Test

Bile Esculin Test

Positive test: Growth and blackening of the agar slant

Negative test: Growth and no blackening of medium or No growth

Limitations of Bile Esculin Test

  • As a result of nutritional requirements, some organisms may grow poorly or not at all on this medium.
  • Some strains of StaphylococcusAerococcus, and Listeria monocytogenes may grow in the presence of bile and hydrolyze esculin.  monocytogenes will form minute black colonies.
  • A heavy inoculum on BEA may cause interpretation of the bile esculin test difficult to read. Excess inoculum decreases the ability of the bile to inhibit growth of other gram-positive organisms that may hydrolyze esculin.
  • There are a few streptococci that do not hydrolyze esculin but will grow in the presence of bile. Growth without blackening of this medium does not constitute a positive test.

Quality Control of Bile Esculin Test

 Positive: Enterococcus faecalis (ATCC29212) — growth; black precipitate

Negative: Escherichia coli (ATCC25922) — growth; no color change, Streptococcus pyogenes (ATCC19615) —no growth; no color change


  1. Tille P.M (2014)Bailey and Scott’s diagnostic microbiology, Thirteen edition, Mosby, Inc., an affiliate of Elsevier Inc., 3251 Riverport Lane, St. Louis, Missouri 63043
  2. Facklam RR, Padula JF, Wortham EC, Cooksey RC and Rountree HA.1979. Presumptive Identification of Group A, B, and D Streptococci on Agar Plate Media. Journal of clinical microbiology. p. 665-672 0095-1137/79/06-0665/08$02.00/0.
  3. Lindell, S. S. and Quinn, P.1975. Use of bile-esculin agar for rapid differentiation of Enterobacteriaceae. Journal of Clinical Microbiology1(5), 440–443.
  4. Chuard C and Reller L.B. 1998. Bile-Esculin Test for Presumptive Identification of Enterococci and Streptococci: Effects of Bile Concentration, Inoculation Technique, and Incubation Time. Journal of Clinical Microbiology. 1135–1136

Bile Esculin Test

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