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Bile Esculin Test Introduction

Generally Biochemical tests are used to identify the species of bacteria by differentiating the species on the basis of their biochemical activities.

The factors such as protein and fat metabolisms and the enzyme production and ability of the organism to utilize the compounds in the mediums helps us in differentiating and identifying the specific species of the bacteria.

Bile Esculin test is one of the biochemical tests which is used widely used for differentiating the Enterococci from the group D viridians group of Streptococci.

What is Bile Esculin Test?

Bile esculin test is one of the biochemical tests which is performed in order to differentiate Enterococci and the group D streptococci from non-group-D viridians group Streptococci, based on their ability to hydrolyze esculin.

Many organisms have the capability to hydrolyze esculin, but only few species have the capability to hydrolase esculin in the presence of bile.

This property is used in the biochemical tests to differentiate the organisms.

The bile esculin test is usually performed in a medium of a selective agar known as bile esculin test that contains bile and esculin as their vital substances.

As agar consists of various types of bile salts, that inhibits the growth of other Growth-positive organisms and it also allows the selective isolation of the Enterococci and the Group D Streptococci.

Esculin is one of the derivatives of the glycoside-coumarin, which is also considered as the fluorescent compound as its hydrolysis, and it can be differentiated on the absence of fluorescence.

As years passed, bile esculin tests are modified accordingly and in recent times they are performed using bile esculin disks and they are performed more widely.

Bile-esculin disks are usually used for rapid differentiation of the Group D Streptococci and the non-Group D streptococci.

Bile Esculin Test Procedure

 The main aim of the test is to identify the Enterococci and the Group D Streptococci on the basis of their ability to hydrolyze esculin in the presence of the bile.

 To detect the difference between the members of the family Enterococci and Group D streptococci from other species of Non-Group D Streptococci.

Bile Esculin Test Principle

The basis of the esculin test is to hydrolyze the esculin that is hydrolyze at the presence of bile salts due to the enzymatic activity of enzyme esculinase.

Esculin is a glucoside containing glucose and hydroxycoumarin which are linked together by an ester bond with the help of an oxygen.

This test helps us to selects the organisms initially on the basis of their ability to grow in the medium containing about 4% of bile salts and they are detected on the basis of their ability to hydrolyze esculin.

Thus, the hydrolysis of esculin results in glucose and another compound known as esculetin.

On degrading esculin, Esculetin is produced which on hydrolysis reacts with the iron ions present in the medium and forms a compound known as phenolic iron complex, and results in the formation of dark brown or black color.

On the other hand, Esculin is a fluorescent compound, which on hydrolysis can be observed as a loss of fluorescence.

Once a bile is added into a medium, the micro-organisms get the capability to grow and it starts hydrolyzing the esculin.

The bile in turn inhibits the growth or production of the other Gram-positive organisms which in turn makes the medium more selective.

Availability of 4% of bile in the esculin medium inhibits most of the strains of the Streptococci excluding the Species S. bovis. Whereas it does not inhibit Enterococci or Listeria.

Bile Esculin Test Reagent


 Bile esculin agar that slants with iron citrate is used.

 Agar plate media is usually composed of Enterococcosel agar, or any other similar formulation.

 Bile-esculin aide agar or a broth with iron citrate and azide is used. Azide inhibits most of the Gram-negative bacteria.

 Peptone-yeast esculin broth, but it is used mostly in the anaerobic atmosphere.

 Esculin agar is used without bile or aside but it usually contains iron citrate. 

Lab Supplies:

 Long wave-UV light

 1% of ferric ammonium citrate.

Bile Esculin Test Procedure

About 64.5 grams of the dehydrated powder is added in a beaker containing 1000 milli liters of the distilled or deionized water.

The prepared solution is heated till it boils to dissolve the solution completely.

The dissolved medium is then dispensed into the tubes.

Further the tubes are sterilized in an autoclave at a temperature of about 121ºC for about 15 minutes.

After the completion of the autoclave processing the tubes are taken out and they are cooled in a slanted position at a temperature of about 40 to 45ºC.

The same position is maintained until the butts are formed at a depth of about 1.5 to 2.0 cm.

Esculate Hydrolysis

Esculate hydrolysis is detected either by using tube test or by disk test. Disk test is considered as one of the rapid tests.

I. Bile Esculin Tube Test

An isolated colony is taken from an 18 to 24-hour culture using a sterile inoculating needle.

The bile inoculating tubes are then inoculated by streaking the surface of the slant by using light inoculum from the agar plate and the tubes are inoculated with 10µl loopful of 0.5 McFarland standard suspension using a sterilized water.

Then the cap of the test tubes is left loosened in order to ensure aeration.

Then the tubes are left to incubate aerobically at a temperature of about 35 to 37ºC for about 24 hours and for a maximum of 7 days until the color changes are observed.

For esculin broth without iron citrate, the tubes are observed daily for the decent of fluorescence.

In the absence of the fluorescence 2 to 3 drops of 1.0% ferric ammonium citrate is added to the esculin tube and the color change is observed.

I. Bile Esculin Disk Test

 In disk test, esculin test is moistened with the one drop of distilled water.

 And it is ensures, that it is not saturated. By using a sterile loop, two to three isolated colonies are picked from 18 to 24-hour culture.

 Then the disk is observed for the development of a dark brown or black color for about 10 minutes in a room temperature.

Bile Esculin Test Result: Tube Test

 A positive result in a tube test is determined in the medium containing ferric ammonium citrate is usually detected by blackening of the medium.

 A negative result in the tube test is detected by absence of change in colors. On the other hand, the medium will fluorescence under the UV light.

 For esculin broth without iron citrate, a positive test is usually observed by blackening of the medium on addition of ferric reagent or by the loss of fluorescence in the medium.

 Whereas here, the negative tests occurs when the bile esculin medium losses its ability to let grow the organisms on the medium containing bile.

Bile Esculin Test Result: Disk Test

 In disk test, the positive result is determined by development of dark color or black color

 Where as the negative disk test is determined by absence of color.

Bile Esculin Test Citations


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