Bile Esculin Test - Introduction, Composition, Principle, Procedure, Result, Interpretation

Introduction to Bile Esculin Test

Bile Esculin Test (BET) uses the Bile Esculin agar (BEA), which is a selective and differential medium. This test is used in the presumptive identification of Enterococci and group D Streptococci and is based on the test organism’s ability to hydrolyze esculin. Aside from Enterococci, streptococci are the only gram-positive bacteria that are not inhibited by the bile salts in this medium.

Composition of Bile Esculin agar (BEA)

BEA contains oxygall which inhibits the growth of other gram-positive organisms except for Enterococci and group D Streptococci. The selective and differential growth media also contains azide which functions by inhibiting the growth of gram-negative organisms. It also contains ferric citrate and esculin.

Esculin, which is a differential ingredient, is a glycosidic coumarin derivative (6-beta-glucoside-7-hydroxy-coumarin). Its glucose and 7-hydroxycoumarin, the two moieties of the molecule are linked by an ester bond through oxygen. Moreover, it is fluorescent in nature and its hydrolysis can be detected by the loss in fluorescence.

For rapid tests to detect esculin hydrolysis in the presence of bile, a bile esculin disk can also be used to differentiate between non-group D Streptococci and group D Streptococci.

List of ingredients of Bile Esculin agar (BEA)

The list of the composition of BEA per 1000 ml:

1. Beef extract (11 g)

2. enzymatic digest of gelatin (34.5 g)

3. esculin (1 g)

4. ox bile (2 g)

5. ferric ammonium citrate (0.5 g)

6. agar (15 g)

7. pH 6.6

Principle of Bile Esculin Test

Bile Esculin Test is based on the ability of the test microorganism to hydrolyze esculin into glucose and esculetin (6, 7-dihydroxy-coumarin) by the production and action of enzyme esculinase in the presence of bile (4% bile salts or 40% bile). If esculetin has been produced, it reacts with an iron salt (ferric citrate) in the medium which in turn will result in the formation of a phenolic iron complex, resulting in the production of a dark brown or black color.

Esculin + Acid → β-D-glucose + Esculetin

Esculetin + Fe3+ → Dark Brown Color

Many microorganisms can hydrolyze esculin but only a select few can do so in the presence of bile.

Procedure of Bile Esculin Test

The Bile Esculin Test can be performed by the tube test or the disk test.

Procedure for Tube Test for Bile Esculin Test

1. With the help of a sterile loop, pick one or two isolated colonies of test microorganisms from an 18-24 hours culture.

2. Inoculate the test organism onto the surface of the slant of the bile esculin medium by following an S-shaped path

3. Incubate the inoculated test tube for 24 hours at 35-37°C

4. Observe the change in color of the BEA medium

Procedure for Disk Test for Bile Esculin Test

1. Add a drop of distilled water to the bile esculin disk to moisten it

2. With the help of a sterile loop, pick two to three isolated colonies of test microorganisms from an 18-24 hours culture.

3. Inoculate the test organism onto the surface of the moist disk.

4. Incubate at room temperature for 10 minutes.

5. Observe the change in color of the disk around which the test organisms have been inoculated.

Result and Interpretation of Bile Esculin Test

The rest and interpretation of the Bile Esculin Test are as follows:

For Tube Test of Bile Esculin Test

Positive:

If the medium contains ferric ammonium citrate, more than half of the agar slant is blackened.

Negative:

The inoculated BEA does not develop any black color i.e. remains unchanged.

For Disk Test of Bile Esculin Test

Positive:

The color of the disk around which the test organism has been inoculated changes to dark brown or black.

Negative:

The color of the disk around which the test organism has been inoculated remains colorless after 10 minutes.

Limitation of Bile Esculin Test

The limitation of the Bile Esculin Test is as follows:

1. Microorganisms such as Escherichia coli produces β-glucosidase which will react after prolonged incubation

2. H2S, which readily reacts with iron to produce a black complex, is produced by several gram-negative organisms. It can hinder the interpretation of the esculin hydrolysis test.

3. If the bile concentration is less than 40% and the inoculum is too high in number, viridans group streptococci (except S. bovis) may give positive resulting

4. The amount of inoculum used for the Bile Esculin Test is also a defining factor. If heavy inoculum is used, it decreases the ability of bile to inhibit the growth of other gram-positive organisms capable of hydrolyzing esculin.

5. Some strains of Staphylococcus, Aerococcus, and Listeria monocytogenes are able to grow in the presence of bile as well as hydrolyze esculin.

6. Since some streptococci are able to grow in the presence of bile but are not able to hydrolyze esculin, they are reported to be negative for Bile Esculin Test

7. If BEA lacks azide in its composition, gram-negative rods may be able to grow on this medium. Such microorganisms may be able to hydrolyze esculin.