#6.

Name of the test: Bile Esculin Test

Purpose (1-3 sentences): To isolate and identify bacteria able to hydrolyze esculin in the presence of bile.
Commonly used for presumptive identification of Group D streptococci and enterococci. The medium contains
esculin and peptone for nutrition and bile to inhibit Gram-positive bacteria other than Group D streptococci and
enterococci due to their ability hydrolyze esculin in the presence of bile (4% bile salts or 40% bile).

Methods (brief overview on how the test is performed) If there is more than one method, provide the most
widely used.
Tube test
1. A well-isolated colony is taken from an 18–24-hour culture with a sterile inoculating needle.
2. The bile esculin agar tubes are inoculated by streaking the surface of the slant with the inoculum
picked from the culture plate.
3. For enterococcus and S. bovis identification, 40% bile is used, and the tubes are inoculated with a
10-µl calibrated loopful of a 0.5 McFarland standard suspension prepared in sterile water.
4. The cap of the test tubes should be left loosened to ensure adequate aeration.
5. The tubes are then incubated aerobically at 35-37°C for 24 hours (or up to 7 days for slow-growing
Gram-negative rods and anaerobes).
6. Observe for growth and blackening of the medium.

Expected Results (Positive and Negative Results); Provide an example of a positive and negative organism
(ex. Indole Test (+): Escherichia coli. Indole Test (-): Klebsiella pneumoniae)

Expected Results:
Positive: Growth and blackening of the agar slant; Dark brown or black media
Negative: Growth and no blackening of medium; No growth and no color change

Example Organism
Bile Esculin Test (+): Group D streptococci and Enterococcus spp. like Enterococcus faecalis, Enterococcus
faecium, and Streptococcus bovis (CHOOSE 1 ONLY)
Bile Esculin Test (-): Escherichia coli, Streptococcus agalactiae, Streptococcus equi, Streptococcus disgalactiae
(CHOOSE 1)

Sample Image of the Result (Positive and Negative Results) (CHOOSE 1)

https://microbiologyinfo.com/wp-content/uploads/2019/04/Bile-Esculin-Hydrolysis-test.jpg
https://microbenotes.com/wp-content/uploads/2018/08/Bile-Esculin-Test.jpg

References:
Aryal, S. (2019, June 14). Bile Esculin test - Principle, procedure, uses and interpretation. Microbiology
Info. https://microbiologyinfo.com/bile-esculin-test/
Austin Community College. (2008). Bile Esculin Test.
https://www.austincc.edu/microbugz/handouts/Bile%20Esculin%20Test%20Handout.pdf
Sapkota, A. (2018, August 1). Bile Esculin test- Principle, procedure, results, uses, limitations. Microbe
Notes. https://microbenotes.com/bile-esculin-test/
Tankeshwar, A. (2021, May 30). Bile-esculin test for Enterococcus species. Microbe Online.
https://microbeonline.com/bile-esculin-test-enterococcus-species-principle-procedure-results/