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

This post was most recently updated on April 20th, 2019

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

Materials
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.

Procedure

  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) gives positive Bile Esculin Test.

Limitation of the Test

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

About tankeshwar 386 Articles
Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion, I am working as an Asst. Professor and Microbiologist at Department of Microbiology and Immunology, Patan Academy of Health Sciences, Nepal. If you want me to write about any posts that you found confusing/difficult, please mention in the comments below.

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