Esculin Hydrolysis Test- Principle, Procedure, Results

Objective of Esculin Hydrolysis Test

This test is used for the presumptive identification and differentiation of Enterobacteriaceae.

Principle of Esculin Hydrolysis Test

This test is used to determine whether an organism is able to hydrolyze the glycoside esculin. Esculin is hydrolyzed to esculetin, which reacts with Fe3+ and forms a dark brown to black precipitate.


 NaCl (8 g), K2HPO4 (0.4 g), KH2PO4 (0.1 g), esculin (5 g), ferric ammonium citrate (0.5 g), agar (15 g), per 1000 mL, pH 7.0.

Procedure of Esculin Hydrolysis Test

  1. Inoculate the medium with 1 drop of 24-hour broth culture.
  2. Incubate at 35°-37°C for up to 7 days.
  3. Examine the slants for blackening and, under the ultraviolet rays of a Wood’s lamp, for esculin hydrolysis.

Result Interpretation of Esculin Hydrolysis Test

Positive: Blackened medium, which would also show a loss of fluorescence under Wood’s lamp.
Negative: No blackening and no loss of fluorescence under the Wood’s lamp, or slight blackening with no loss of fluorescence under the Wood’s lamp.


This medium is a non-selective agar. The bile esculin hydrolysis test is a selective differential method.

Quality Control

Positive: Enterococcus faecalis (ATCC29212)
Negative: Escherichia coli (ATCC25922)

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