Bile Esculin Test: Introduction, Principle, Test Requirements, Procedure, Result Interpretation, Uses, Limitation, Keynotes, and Related Footages

Introduction of Bile Esculin test

Bile esculin test is widely used to differentiate Enterococci from streptococci, other than streptococcus bovis as well as Yersinia enterocolitica from food and animal feeding stuff and Listeria monocytogenes which are bile tolerant and can hydrolyze esculin to esculetin, it reacts with an iron salt in the medium to form a phenolic iron complex which produces a dark brown or black color. This test is based on the ability of Enterococcus species, to hydrolyze esculin in the presence of bile (4% bile salts or 40% bile). Esculin is a glycosidic compound. For this test, esculin is incorporated into a medium containing 4% bile salts, and a test having sensitivity and specificity for Enterococcus (>90%).

Principle of Bile Esculin test

Bacteria that are bile-esculin positive, 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 esculetin. Esculetin reacts with ferric ions present ferric citrate in the medium to form a phenolic iron complex which produces dark brown or black color.

Requirements for the Bile Esculin test

• Bile-esculin agar-tube or plate depening on the purpose of test organisms
• Inoculating straight wire
• Bunsen burner
• Test organisms/ specimen
• Control strains-Positive control (E. faecalis ATCC 29212) and negative control (Escherichia coli ATCC25923)

• BOD Incubator

Composition of Bile Esculin Agar

Ingredients	Gms/Litre
Peptic digest of animal tissue (PDAT)	5.0
Beef extract	3.0
Esculin	1.0
Bile salts	40.0
Ferric citrate	0.5
Agar	15.0
Final pH (at 25°C)	6.6±0.2

Note: The constituent of the bile-esculin agar medium is peptone, beef extract, ox gall (bile), esculin, ferric citrate, and agar. Bile esculin medium contains esculin and peptone for nutrition and bile to inhibit Gram-positive bacteria other than enterococci and Streptococcus bovis. Ferric citrate is included as a color indicator.

Preparation of Bile Esculin Agar

1. Suspend 6.45 g of medium in 100 ml of distilled water and mix thoroughly.
2. Heat with frequent agitation and boil for 1 minute to completely dissolve the components.
3. Autoclave at 121°C for 15 minutes.
4. Cool to 45-50°C.
5. Mix well before pouring into sterile tubes or Petri plates.
6. Allow the tubed medium to solidify in a slanted position with a butt of 2.5 cm deep or pour into sterile Petri plates.

Storage and Shelf life of MacConkey agar

• Store at 2-8ºC  and away from direct light.
• Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination.
• The product is light and temperature-sensitive; protects from light, excessive heat, moisture, and freezing.

Procedure of bile esculin test

• 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 while for suspected Yersinia enterocolitica from food, inoculate food into plate bile esculin agar .
• The inoculated tube/plate is incubated at 35-37°C for 24 hours and the results are determined.

Results and Interpretation of Bile Esculin test

• Blackening of the medium within 24-48 hours indicates esculin hydrolysis.
• Quality Control-Positive control, E. faecalis ATCC 29212:Positive and negative control, Escherichia coli ATCC25923: Negative

• Test: Positive/Negative

Colony Morphology of Bacteria on Bile Esculin Agar (BEA)

Colony morphology of various bacteria on bile esculin agar (BEA) are as follows-

Organisms	Growth	Bile Esculin Hydrolysis Test
Enterococcus faecalis	Good-luxuriant	Positive reaction (blackening of medium)
E. coli	Good	Negative reaction (no blackening of medium)
Enterococcus faecium	Good-luxuriant	Positive reaction (blackening of medium)
Yersinia enterocolitica	Good-luxuriant	Positive reaction (blackening of medium)

Uses of Bile Esculin Test

1. Bile esculin test is applied as a biochemical assay for the isolation of enterococci and group D streptococci.
2. It can also be used to test the organisms of viridans streptococci and other Gram-positive microbes.
3. BEA is a selective differential medium for the growth of organisms like enterococci, Yersinia enterocolitica, and Listeria monoctogenes.

Keynotes

• A variety of bacteria can hydrolyze esculin, but few can do so in the presence of bile. e.g. Streptococcus bovis (previously referred to as group D streptococci) and Listeria.
• Bile esculin agar (BEA) is recommended for the isolation and identification of Yersinia enterocolitica from food and animal feeding stuffs.
• Bile escilin disk is vaialble for rapid testing.
• Bile Esculin Agar (BEA) is used for the presumptive identification of Enterobacter, Klebsiella, and Serratia among the Enterobacteriaceae.
• Esculin hydrolysis was first described by Rochaix in 1924 while Swan first introduced the use of BEA in 1954 and it is a selective differential agar applied to isolate and identify members of the genus Enterococcus, formerly part of the “group D streptococci”.
• The ability to hydrolyze esculin in the presence of bile is a feature of enterococci and group D streptococci.
• In 1970, Facklam and Moody determined that the use of the bile esculin test is a reliable method of identifying different groups of bacteria like Klebsiella and Serratia among the Enterobacteriaceae.

Limitations of Bile Esculin test

• Some viridians streptococci (approximately 3%) may also hydrolyze esculin in the presence of bile. 
• Streptococcus bovis cannot be distinguished from other viridans group streptococci using esculin tests without bile.
• Several species create H₂S during metabolism, which reacts with iron to form a black complex,in-terfering with the results of the esculin hydrolysis test and potentially leading to a false-positive result.

Related Footages

Enterococcus non-hemolytic colony on blood agar

Gram-positive cocci in singles, pairs and short chains of Enterococcus in Gram staining of culture

Bile Esculin Test Negative and Positive Tubes Demonstration

Antibiogram of Enterococcus on modified MHA agar (blood agar)

Listeria monocytogenes colony morphology on blood agar

Small, grey, translucent drop-like colonies surrounded by a small zone of indistinct beta hemolysis of Listeria monocytogenes

Listeria monocytogenes in Gram staining of culture

Further Readings

• Cowan & Steel’s Manual for identification of Medical Bacteria. Editors: G.I. Barron & R.K. Felthani, 3rd ed 1993, Publisher Cambridge University Press.
• https://himedialabs.com/TD/M972I.pdf
• Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
• Clinical Microbiology Procedure Handbook Vol. I & II, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
• Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
• Colour Atlas and Textbook of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr, and Sommers H.M.
• https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/604/416/80507dat.pdf
• Jawetz, Melnick and Adelberg’s Medical Microbiology. Editors: Geo. F. Brook, Janet S. Butel & Stephen A. Morse, 21st ed 1998, Publisher Appleton & Lance, Co Stamford Connecticut.