Bile-Esculin test for Enterococcus species

Bile-esculin test is widely used to differentiate Enterococci and non-enterococcus 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

Principle of Bile-esculin test

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 constituents 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. Incubate the inoculated tube at 35-37°C for 24 hours and then observe the results.

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 (Non-enterococcus) give positive bile esculin test.

Limitation of the Test

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


  1. Chuard, C., & Reller, L. B. (1998). Bile-esculin test for presumptive identification of enterococci and streptococci: effects of bile concentration, inoculation technique, and incubation time. Journal of clinical microbiology, 36(4), 1135–1136. 
  2. Facklam, R. R., & Moody, M. D. (1970). Presumptive identification of group D streptococci: the bile-esculin test. Applied microbiology, 20(2), 245–250.
  3. Forbes, S., Sahm, D. F., & Weissfeld, A. S. (2002). Bailey & Scott’s Diagnostic Microbiology. Mosby.

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

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