Bile-Esculin test for Enterococcus species: Principle, Procedure and results

Bile-esculin test is widely used to differentiate Enterococci and nonenterococcus group D streptococci, which are bile tolerant and can hydrolyze esculin to esculetin, from non-group D viridans group streptococci, which grow poorly on bile.   It is a low cost, rapid test with good sensitivity and specificity  (>90%).

Bile Esculin Test Results: Control, Positive and Negative
Bile Esculin Test Results: Control, Positive and Negative


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).

Note: Many bacteria can hydrolyze esculin, but few can do so in the presence of bile. 

Esculin is a glycosidic coumarin derivative (6-beta-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, first of all, able to grow in the presence of bile salts. Hydrolysis of the esculin in the medium results in the formation of glucose and a compound called esculetin.

Esculin Hydrolysis; Chemical reaction
                                                                                      Esculin Hydrolysis test 

Esculetin, in turn, reacts with ferric ions (supplied by the inorganic medium component ferric citrate) to form a black diffusible complex. Group D streptococci and enterococci include opportunistic pathogens such as Enterococcus faecalis, Enterococcus faecium, and Streptococcus bovis.

Fig: Chemical Reaction of the Bile Esculin Test
Fig: Chemical Reaction of the Bile Esculin Test

Bile-esculin agar medium is prepared as agar slants or plates. The constituent of Bile-esculin agar medium are: Peptone, Beef extract, Oxgall (Bile), Esculin, Ferric citrate and Agar. Bile esculin medium contains esculin and peptone for nutrition and bile to inhibit Gram-positive bacteria other than Group D streptococci and enterococci. Ferric citrate is added as a color indicator.


  1. With an inoculating wire or loop, touch two or three morphologically similar streptococcal colonies and inoculate the slant of the bile esculin medium with an S-shaped motion, or streak the surface of a bile esculin plate for isolation.  (Note: There is no need to stab the medium.)
  2. The inoculated tube is incubated at 35-37 degree Celsius  for 24 hours and the results are determined.

Results and Interpretation

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.

Quality Control

  1. Positive control: Enterococcus species (e.g. E. faecalis)
  2. Negative control: Viridans streptococcus, not group D

Positive Test Result: 

Both Group D Streptococci; i.e. D(Enterococcus) & D (nonenterococcus) gives positive Bile Esculin Test.

Limitation of the Test

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

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