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