Bile Esculin Test: Introduction,Principle, Procedure, Result Interpretation and Limitations

Bile Esculin Test -Positive

Introduction of Bile Esculin test

Bile esculin test is widely used to differentiate Enterococci from streptococci, other than streptococcus bovis 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  (>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 medium is prepared as agar slants. The constituent of the Bile-esculin agar medium is 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 enterococci and Streptococcus bovis. Ferric citrate is included as a color indicator.
  • Inoculating straight wire
  • Bunsen burner
  • Test and control organisms

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.

The inoculated tube 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)

Negative control: Escherichia coli (ATCC25923)

  • Test: Positive

Note: 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

Limitations of Bile Esculin test

  • Some viridians streptococci (approx. 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 H2during metabolism, which reacts with iron to form black complex, interfering with the results of the esculin hydrolysis test and potentially leading to false-positive result.

Further Readings

  • 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.
  • Colour Atlas and Textbook of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr, and Sommers H.M.
  • Cowan & Steel’s Manual for identification of Medical Bacteria. Editors: G.I. Barron & R.K. Felthani, 3rd ed 1993, Publisher Cambridge University Press.
  • 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.
  • 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.
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