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

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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 tissue 5.0
2. Beef extract 3.0
3. Esculin 1.0
4. Bile Salts 40.0
5. Ferric citrate 0.5
6. Bacteriological agar 15.0
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. Organism Growth Bile esculin hydrolysis
1. Enterococcus faecalis Good Positive reaction; Blackening of medium
2. Escherichia coli Good Negative reaction
3. Enterococcus faecium Luxuriant Positive reaction; Blackening of the medium around the growth.
4. Yersinia enterocolitica Good-luxuriant Positive 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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About Author

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Anupama Sapkota

Anupama Sapkota has a bachelor’s degree (B.Sc.) in Microbiology from St. Xavier's College, Kathmandu, Nepal. She is particularly interested in studies regarding antibiotic resistance with a focus on drug discovery.

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