Enterococcus: Identification Related Tests and Details

Enterococcus: Identification Related Tests and Details

Enterococcus Identification Chart


Pinpoint colony on growth  medium

Gram Stain: Gram-positive cocci in chains

Catalase test: Negative


Bile Esculin Test:  Positive

Group D

Growth in 6.5% NaCl ( may also grow @ 10, 45 and even 60°C)


Pyruvate Fermentation Test

Positive: Enterococcus faecalis

Negative: Enterococcus faecium

Gamma  Hemolytic Colony on Blood Agar of Enterococcus

5% sheep blood agar (BAP) is with gamma (γ) hemolytic i.e. non-hemolytic colony of Enterococcus faecalis as shown above picture. Enterococci are small (less than 1 mm) or pinpoint colony and catalase test negative.

Classification of Enterococcus

Scientific classification

Domain: Bacteria

Phylum: Firmicutes

Class: Bacilli

Order: Lactobacillales

Family: Enterococcaceae

Genus: Enterococcus

Species: Enterococcus faecalis and E. faecium

Introduction of Enterococcus

Streptococci belonging to group D are classified as enterococci and they are usually non-hemolytic while some strains may be alpha or beta-hemolytic. This enterococci group has been classified in 1984 as a separate genus called Enterococcus based on nucleic acid hybridization studies that showed a more distant relationship to streptococci. The genus Enterococcus contains over 50 species. They are normal flora of the human intestinal tract.  E. faecalis and E. faecium are medically important species.

Morphology of Enterococcus

Gram-positive cocci in pairs and short chains. They are non-motile (except, E. gallinarum and E. casseliflavus) non-capsulated, and non- sporing.


Blood agar: They are usually non-hemolytic or gamma ( as shown above image) whereas some strains may be alpha or beta-hemolytic.

MacConkey agar: On MacConkey agar without crystal violet they grow as tiny deep pink colonies.

Note: Types of MacConkey agar are as follows-

MacConkey agar without bile salt and crystal violet: for both Gram-positive  and  negative bacteria

MacConkey medium with bile salt: For mainly Gram-negative bacteria but Enterococci may also be grown.

MacConkey medium with both bile salt and crystal violet: Only for Gram-negative rods

Biochemical Reactions

  • They are able to grow in the presence of 40% bile and 6.5% sodium chloride.
  • Similarly, their ability to grow at 45°C and at pH 9.6.
  • They are PYR test positive.
  • E. faecalis is the most commonly isolated Enterococcus from human sources. Other enterococci are E. faecium and E. durans.  E. faeclais can be identified by fermentation of mannitol, sucrose, sorbitol, and aesculin and by producing black colonies when grown on tellurite blood agar.


They have the ability to grow over wide-temperature and pH ranges, survive desiccation and grow in the presence of 6.5% NaCl and 40% bile salts.


Their ability to cause –

  • Bacteremia
  • Urinary Tract Infection
  • Endocarditis
  • Surgical site infections
  • Root canal failure
  • Immune evasion
  • Tissue Damage
  • Toxic Metabolites


Lab Diagnosis of Enterococci

Enterococci can be isolated as follow-

  1. Pinpoint  gamma i.e. non-hemolytic colonies on blood agar except some
  2. Catalase test : Negative
  3. Bile esculin test: Positive
  4. Growth in 6.5% sodium chloride and at pH 9.6
  5. Growth at 10°C and 45°C
  6. Pyruvate fermentation test
  7. Acetoin production test
  8. Litmus milk decolorization test
  9. PYRase test
  10. Reduces potassium tellurite test
  11. Reduces triphenyl tetrazolium chloride (TTC) test


Enterococci resistance to penicillin and other antibiotics occurs frequently. Vancomycin is the primary alternative drug to penicillin for treating enterococcal infections. Vancomycin-Resistant Enterococcus  (VRE) has also been isolated. Resistance is most common in E. faecium but vanocomycin resistant strains of E. faecalis also occur.

Further Reading

  1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6629438/
  2. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
  3. 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.
  4. Colour Atlas and Textbook of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr and Sommers H.M.
  5. 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.
  6. 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.
  7.  Manual of Clinical Microbiology. Editors: P.R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover and R. H. Yolken, 7th ed 2005, Publisher ASM, USA
  8.  Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
  9. Topley & Wilsons Principle of Bacteriology, Virology and immunology Editors: M.T. Parker & L.H. Collier, 8th ed 1990, Publisher Edward Arnold publication, London.
  10. Medical Microbiology-The Practice of Medical Microbiology Vol-2-12th Edn. –Robert Cruickshank
  11. District Laboratory Practice in  Tropical Countries  –  Part-2-   Monica Cheesebrough-   2nd Edn Update
  12. https://en.wikipedia.org/wiki/Enterococcus
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