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Introduction of Esculin hydrolysis Test 

The Esculin hydrolysis test is useful for those organisms which are able to hydrolyze the substrate, esculin by the enzyme, esculinase release by them. The list of  positive and negative bacteria are as follows:

 Positive bacteria

Enterococcus spp.

Pediococcus spp.

Streptococcus group D (non-Enterococci)

Negative bacteria

Gemella haemolysans

Gemella morbillorum

Streptococcus agalactiae
(group B)

Variable result

Aerococcus viridans

Aerococcus urinae

V(+) = Variable reactions where the majority of isolates are positive (>80%)

Leuconostoc spp.

V(-) = Variable reactions where the majority of isolates are negative (>80%)

Streptococcus pyogenes
(group A)

Streptococcus pneumoniae

Streptococcus viridans (non-group D)

Principle of Esculin hydrolysis Test 

Esculin and ferric ammonium citrate are contained within the ESC disk. Esculin is hydrolyzed by organisms capable of producing the enzyme esculinase. Esculetin, the end product of the hydrolysis reaction, complexes with ferric ions to produce a light gray to gray color indicating esculin hydrolysis test positive whereas other than gray negative.

Requirements for Esculin hydrolysis Test 

• Test organisms -Fresh growth less than 24 hours old of catalase-negative, gram-positive cocci. If growth is poor, a culture that has been incubated for up to 72 hours may be used.
• ESC Disk
• Petri dish or slide
• Control strains

Positive Control (PC): Enterococcus faecalis ATCC 29212

Negative Control (NC): Aerococcus viridans ATCC 11563

Procedure of Esculin hydrolysis test 

1. Moisten the disk with distilled or deionized water( but Do not saturate).
2. Using a sterile loop, pick 2-3 well isolated, 18-24 hour colonies and rub them into a small area of the disk so that there is a visible paste.
3. Observe for a light gray to gray color. Any darkening at all should be considered positive. Allow 10-15 minutes for full-color development.

Result Interpretation

Positive: Light gray to gray color development

Negative: Any color other than gray

Enterococcus faecalis ATCC 29212:Positive

Aerococcus viridans ATCC 11563: Negative

Limitation

1. It is recommended that biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for complete identification.
2. A Gram stain and catalase test should be performed to confirm the presence of gram-positive, catalase-negative cocci.
3. Only pure cultures or isolated colonies of gram-positive, catalase-negative cocci should be tested; mixed cultures may give false-positive reactions.
4. False-negative results can occur for any of the reactions if inadequate inoculum is used.
5. Bile tolerance is not included in the esculin hydrolysis test, so results obtained with a disk may not correlate with results obtained from Bile Esculin media.

Bibliography

• Cowan and Steel’s, manual for the identification of medical bacteria
• Lynne S. Garcia , Clinical Microbiology Procedures Handbook
• https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/StrepQuick.htm