Bile Esculin Test- Principle, procedure, results, uses, limitations

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Last Updated on October 5, 2020 by Sagar Aryal

What is the Bile Esculin Test?

The bile-esculin test is a biochemical test performed to differentiate Enterococci and group D Streptococci from non-group D viridans group Streptococci on the basis of their ability to hydrolyze esculin.

  • Many organisms are capable of hydrolyzing esculin, but only a few of them can do so in the presence of bile (4% bile salts or 40% bile.). Thus, this property is utilized to identify organisms of a particular group.
  • The Bile-esculin test is performed on a selective differential agar; bile esculin agar, which consists of bile as well as esculin.
  • The agar contains different bile salts that inhibit the growth of other Gram-positive organisms and allows the selective isolation of Enterococci and Group D Streptococci.
  • Esculin is a glycosidic coumarin derivative (6-beta-glucoside-7-hydroxy-coumarin) which is a fluorescent compound, and its hydrolysis can also be observed by the loss of the fluorescence.
  • The Bile-esculin test has been modified through the years and has been made more rapid. Nowadays, bile-esculin disks are available that are commonly used for the rapid differentiation between Group D Streptococci and non-Group D Streptococci.

Objectives of Bile Esculin Test

  • To identify Enterococci and Group D Streptococci on the basis of their ability to hydrolyze esculin in the presence of bile.
  • To differentiate members of Enterococci and Group D Streptococci from other viridans or non-Group D Streptococci.

Principle of Bile Esculin Test

  • The basis of the esculin test is the hydrolysis of esculin in the presence of bile salt as a result of the enzymatic action of esculinase.
  • Esculin is a glucoside consisting of glucose and hydroxycoumarin linked together by an ester bond through oxygen.
  • The bile esculin test selects organisms first on the basis of their ability to grow in a medium with 4% bile salts followed by the selection based on their ability to hydrolyze esculin.
  • The hydrolysis of esculin results in glucose and a compound called esculetin.
  • After the degradation of esculin, the esculetin produced by the hydrolysis of esculin reacts with iron ions (from ferric citrate) in the medium to form a phenolic iron complex, resulting in a dark brown or black color.
  • Alternatively, esculin is a fluorescent compound, and its hydrolysis can be observed by a loss of fluorescence.
  • If bile is added to the medium, the microorganism must be able to grow in its presence in order to hydrolyze esculin. The bile inhibits the growth of other Gram-positive organisms and makes the medium more selective.
  • The 40% bile (equivalent to 4% oxgall) in the bile esculin medium inhibits most strains of Streptococci, other than Streptococcus bovis, but does not inhibit Enterococci or Listeria.

Microorganism tested

  • Gram-positive cocci in chains, which are catalase-negative and morphologically identified as presumptive S. bovis.
  • Isolates of alpha- or gamma-hemolytic, Gram-positive cocci as part of differentiation of enterococci from other pyrrolidonyl-β-naphthylamide (PYR)-positive organisms
  • Non-spore-forming, hemolytic, Gram-positive rods that are catalase-positive and morphologically identified as presumptive Listeria
  • Positive blood cultures with Gram-positive cocci in chains or Gram-positive rods, to rapidly (within 4 hours) identify enterococci and Listeria
  • Esculin without bile for the identification of oxidase-positive aerobic Gramnegative rods, including Aeromonas and yellow-pigmented non-glucose-fermenting rods

Media, Reagents, and Supplies Used

Media Used

  • Bile-esculin agar slants with iron(III) citrate. Agar plate media, such as Enterococcosel agar, have a similar formulation.
  • Bile-esculin-azide agar or broth with iron(III) citrate and azide. Azide will inhibit most Gram-negative bacteria.
  • Peptose-yeast-esculin broth (usually in the anaerobic atmosphere).
  • Esculin agar (0.1% esculin in heart infusion basal medium) without bile or azide but with iron(III) citrate.
  • The composition of Bile Esculin Agar is given below:
S.N.Ingredients Gram/liter
1.Peptic digest of animal tissue5.0
2.Beef extract3.0
4.Bile Salts40.0
5.Ferric citrate0.5
6.Bacteriological agar15.0
Final pH at 25°C: 6.6 ±0.2
Store at 2°C to 8°C.

Reagents and Supplies Used

  • Long-wave (360-nm) UV light
  • 1% ferric [iron(III)] ammonium citrate if iron(III) is not incorporated into the medium

The procedure of Bile Esculin Test

Preparation of media

  • In a beaker, 64.5 grams of the dehydrated powder or lab-prepared media is added to 1000 milliliters of deionized or distilled water.
  • The medium is then heated up to boiling to dissolve the powder completely.
  • The dissolved medium is then distributed into tubes and sterilized in an autoclave at 15 lbs pressure (121°C) for 15 minutes.
  • Once the autoclaving process is complete, the tubes are taken out and cooled at a slanted position to a temperature of about 40-45°C. The position should be maintained in order to obtain butts of 1.5 – 2.0 cm depth.

Esculin Hydrolysis

  • Esculin hydrolysis can be observed either through a tube test or a disk test. A disk test is a rapid test.

Tube test

  • A well-isolated colony is taken from an 18-24 hour culture with a sterile inoculating needle.
  • The bile esculin agar tubes are inoculated by streaking the surface of the slant with either the light inoculum picked from the culture plate.
  • For enterococcus and S. bovis identification, 40% bile is used, and the tubes are inoculated with a 10-µl calibrated loopful of a 0.5 McFarland standard suspension prepared in sterile water.
  • The cap of the test tubes should be left loosened to ensure adequate aeration.
  • The tubes are then incubated aerobically at 35-37°C for 24 hours (or up to 7 days for slow-growing Gram-negative rods and anaerobes), and the color change is observed.
  • For esculin broth without iron (III) citrate, the tubes are observed daily for loss of fluorescence.
  • In the absence of fluorescence, 2 or 3 drops of 1.0% ferric ammonium citrate are added to the esculin tube, and the color change is observed.

Disk test

  • The esculin disk is moistened with a single drop of distilled or deionized water. The disk, however, should not be saturated.
  • Using a sterile loop, two or three well-isolated colonies are picked from an overnight (18- to 24-h) culture.
  • The disk is observed for the development of a dark brown or black color after about 10 minutes at room temperature.

Result Interpretation of Bile Esculin Test

Bile Esculin Test

  • A positive tube test in medium containing ferric ammonium citrate is indicated by the blackening of the medium.
  • A negative tube test is indicated by a lack of color change. The medium will fluoresce under UV light (366 nm).
  • For esculin broth without iron (III) citrate, a positive test is demonstrated either by blackening of the broth after addition of the ferric [iron(III)] reagent or by the loss of fluorescence of the medium.
  • A negative test result also occurs in the bile-esculin medium if the organism cannot grow in the presence of bile, regardless of the ability to hydrolyze esculin.
  • A positive disk test is indicated by the development of a dark brown or black color.
  • A negative disk test remains colorless.

The following table demonstrates the growth of some bacteria and their bile esculin hydrolysis test :

S.N.OrganismGrowth Bile esculin hydrolysis
1.Enterococcus faecalisGoodPositive reaction; Blackening of medium
2.Escherichia coliGoodNegative reaction
3.Enterococcus faeciumLuxuriantPositive reaction; Blackening of the medium around the growth.
4.Yersinia enterocolitica Good-luxuriantPositive reaction; Blackening of the medium.

Uses of Bile Esculin Test

  • Bile esculin test is performed as a biochemical test for the isolation of Enterococci and Group D Streptococci.
  • It can also be used to differentiate these organisms from viridans Streptococci and other Gram-positive microorganisms.
  • Bile Esculin Agar is a selective differential medium for the growth of organisms like Enterococcia, Listeria, and Yersinia enterocolitica.

Limitations of Bile Esculin Test

  • If a large inoculum is used or if the concentration of bile is less than 40%, viridans group streptococci other than S. bovis might give a positive reaction on bile-esculin agar.
  • Esculin tests without bile cannot be used to separate S. bovis (previously referred to as group D streptococci) from other viridans group streptococci.
  • Several organisms might produce H2S during metabolism that might react with iron and produce a black complex, which interferes with the results of the esculin hydrolysis test and might give a false-positive result.
  • Some microorganisms, such as E. coli that have β-glucosidase and will give a positive result in this test only after prolonged incubation. However, prolonged incubation should not be used if the test is being used to detect β-glucosidase in other organisms.

References and Sources

  • Bile esculin agar. M1225. HiMedia Laboratories.
  • Biochemical Tests for the Identification of Aerobic Bacteria. (2016). Clinical Microbiology Procedures Handbook,–
  • C. Chuard, L. B. Reller. Bile-Esculin Test for Presumptive Identification of Enterococci and Streptococci: Effects of Bile Concentration, Inoculation Technique, and Incubation Time. Journal of Clinical Microbiology Apr 1998, 36 (4) 1135-1136; DOI: 10.1128/JCM.36.4.1135-1136.1998.
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