Bile Esculin Test – Purpose, Principle, Procedure, Uses, Results and Interpretation

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Purpose:

The test used to identify the Group D Streptococci and Enteroccocci from non–group D viridans streptococci based on ability of organisms to hydrolyze esculin in the presence of bile. 

Principle

The bile esculin test is based on the ability of certain bacteria, notably the group D streptococci and Enterococcus species, to hydrolyze esculin in the presence of bile (4% bile salts or 40% bile). Esculin is a glycosidic coumarin derivative (6-β-glucoside-7-hydroxy-coumarin). The two moieties of the molecule (glucose and 7-hydroxycoumarin) are linked together by an ester bond through oxygen. For this test, esculin is incorporated into a medium containing 4% bile salts. Bacteria that are bile esculin–positive are able to grow in the presence of bile salts. Subsequent hydrolysis of the esculin in the medium results in the formation of glucose and a compound called esculetin. Esculetin, in turn, reacts with ferric ions (supplied by the inorganic medium component ferric citrate) to form a black diffusible complex.

Medium

Bile esculin agar medium is prepared as agar slants or plates. The medium compositions are:

Compositions

Peptone

5 g

Beef extract

3 g

Oxgall (bile)

40 g

Esculin

1 g

Ferric citrate

0.5 g

Agar

15 g

Distilled water

1 L

pH

7.0

Quality Controls

Positive control: Enterococcus species (e.g., E. faecalis)

Negative control: Viridans group streptococci, not group D

Test Procedure

Three morphologically similar streptococcal colonies touch with loop and inoculate the slant or plate of the bile esculin medium with an S-shaped motion, or streak the surface of a bile esculin plate for isolation.

Incubate the tube or plate at 37°C for 24–48 hours in an ambient air incubator.

Results and Interpretation

Positive: Growth and blackening of the medium

Negative: Growth and No blackening of the medium

Diffuse blackening of more than half of the slant within 24–48 hours indicates esculin hydrolysis. On plates, black haloes will be observed around isolated colonies and any blackening is considered positive.

All group D streptococci will be bile esculin–positive within 48 hours.

Bile Esculin Test

Uses

The test commonly used for presumptive identification of Group D Streptococci and Enteroccocci.

Limitations of Test

Some viridans streptococci (approximately 3%) may also hydrolyze esculin in the presence of bile.

Reference

Koneman’s Color Atlas and Text book of Diagnistic Microbiology.

Bailey and Scott’s Diagnostic Microbiology.

Mackie and McCartney Practical Medical Microbiology.


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