Esculin Hydrolysis Test- Principle, Procedure, Results, Uses

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What is the Esculin Hydrolysis Test?

Esculin hydrolysis is a useful test in the differentiation of both gram-positive and gram-negative bacteria covering a broad spectrum of aerobes, facultative anaerobes, and anaerobes. Esculin hydrolysis is utilized as a taxonomic tool in the identification of a wide variety of microorganisms, including the family Enterobacteriaceae, genera Streptococcus and Listeria, non-fermentative gram-negative bacilli, and anaerobes. Esculin hydrolysis is a differential test that differentiates bacteria on the basis of their ability to hydrolyze esculin.

  • This test can be made selective by adding bile to the medium, which is called the bile esculin test.
  • Hydrolysis of esculin by bacteria can be determined in growth-supporting media like Vaughn-Levine, bile-esculin, or Pfizer selective enterococcus media or by non-growth-supporting methods like PathoTec and rapid spot tests.
  • The esculin hydrolysis test is based on the hydrolysis of esculin (a glucoside) into glucose and esculetin by a microorganism that has constitutive β-glucosidase or esculinase enzyme.

Objectives of Esculin Hydrolysis Test

  • To detect the ability of an organism to hydrolyze esculin by the production of esculinase enzyme.
  • To differentiate and identify members of the Enterobacteriaceae family.

Principle of Esculin Hydrolysis Test

  • Esculin is a β-glucose-6,7-dihydroxycoumarin, a compound derived from the horse chestnut tree (Aesculus hippocastanum).
  • The compound can be enzymatically hydrolyzed at the 8-glucose linkage to yield two products, esculetin, and glucose.
  • Esculin hydrolysis is commonly determined by detecting the end product esculetin.
  • Esculetin combines with ferric ions, generally incorporated in the medium as ferric ammonium citrate, to produce a brown-black colored compound.
  • Alternatively, the end product glucose can be determined by detecting the change in pH resulting from its fermentation. This method is limited to those organisms that ferment glucose.
  • In the esculin hydrolysis test performed in the laboratory, the reaction between esculetin and ferric ions forms a black-colored compound that can be detected on the esculin agar or via the Esculin spot test.

Microorganisms Tested

  • Gram-positive cocci in chains, which are catalase-negative and morphologically identified as presumptive Streptococcus bovis.
  • Microorganisms that are alpha- or gamma-hemolytic, Gram-positive cocci as part of differentiation of enterococci from other pyrrolidonyl-β-naphthylamide (PYR)-positive organism.
  • Non-spore-forming, hemolytic, Gram-positive rods that are catalase-positive and morphologically presumed as Listeria.
  • Positive blood cultures with Gram-positive cocci in chains or Gram-positive rods, to rapidly (4 hours) identify enterococci and Listeria.
  • Esculin hydrolysis for the identification of oxidase-positive aerobic Gram-negative rods, including Aeromonas spp. and yellow-pigmented non-glucose-fermenting rods.

Media, Reagent, and Supplies Used

Media Used

  • Esculin Agar is used for the detection of the hydrolysis of esculin. The medium is a differential medium and can be made selective by adding bile.
  • The composition of the Esculin Agar is given below:
S.N Ingredients Gram/liter
1. Casein enzymic hydrolysate 13.0
2. Yeast extract 5.0
3. Beef heart infusion (solids) 2.0
4. Sodium chloride 5.0
5. Ferric citrate 0.5
6. Agar 15.0
Final pH at 25°C: 7.3 ±0.2

Reagent Used

  • For the esculin spot test, 0.02% esculin solution is prepared in distilled water.

Supplies Used

  • Long-wave (360-nm) UV light
  • Sterile sticks, needles, or inoculating loops
  • Pasteur pipettes or drinking straws
  • Boiling heat block
  • Incubators at 35 and 30°C

Procedure of Esculin Hydrolysis Test

A. Preparation of the media

  • In a beaker, 41.5 grams of the dehydrated powder or lab-prepared media is added to 1000 milliliters of distilled or deionized water.
  • The mixing is followed by heating with agitation up to boiling to dissolve the medium completely.
  • The solution is then dispensed into screw-capped tubes (about 3 ml each) and sterilized in an autoclave at 15 lbs pressure (121°C) for 15 minutes.
  • The tubes are taken out after autoclaving and cooled at a slanted position to a temperature of about 40-45°C. The position should be maintained at an angle to achieve butts of 1.5 – 2.0 cm depth.

B. Hydrolysis Test

Esculin hydrolysis can be observed either through tube test or esculin spot test. The spot test is a rapid test.

1. Tube Test

  • A light inoculum is derived from an 18-24 hour culture with a sterile inoculating needle from the center of a well-isolated colony.
  • The esculin agar tubes are inoculated by streaking the surface of the slant with the light inoculum picked from the culture plate.
  • The caps of the test tubes should be capped loosely to ensure adequate aeration.
  • The inoculated tubes are then incubated in the air at 35-37°C for 24 hours (or up to 7 days for slow-growing Gram-negative rods and anaerobes), and the color change is observed.
  • If esculin broth without iron (III) citrate is used, the tubes are observed daily for loss of fluorescence with UV light.
  • In the case of loss of fluorescence, 2 or 3 drops of 1% ferric ammonium citrate are added to the tube, and the change in color is observed.

2. Esculin Spot Test

  • A 0.02% esculin solution is made in distilled water which is then sterile by autoclaving or by filter-sterilization.
  • A filter paper is placed on a standard microscope slide and positioned on supporting glass rods.
  • Esculin solution is pipetted over the paper while avoiding the over-saturation of the paper.
  • The inoculum is derived from a 24-h bacterial colony with a wooden stick that is rubbed in the center of the filter paper.
  • The slide is then incubated at 37°C for about 10-15 minutes. Although Klebsiella generally yielded a positive test within 10 to 15 min, one should hold the test 30 min before calling it negative.
  • Using a hand-held Wood lamp in subdued light, the spot is observed for the loss of fluorescence.

Control organisms

  • Positive: Enterococcus faecalis
  • Negative: Escherichia coli

Result Interpretation of Esculin Hydrolysis Test

Esculin Hydrolysis Test- Principle, Procedure and Result Interpretation

Figure: Result Interpretation of Esculin Hydrolysis Test. Image Source: Bailey and Scott’s Diagnostic Microbiology. Elsevier.

  • The blackening of the medium demonstrates a positive tube test in the esculin medium with ferric ammonium citrate.
  • The lack of color change demonstrates a negative tube test.
  • A positive spot test is demonstrated by the loss of fluorescence, resulting in a black colored spot under the UV light.
  • A negative test is demonstrated by a bright fluorescence, indicating no decrease in the esculin concentration.

Uses of Esculin Hydrolysis Test

  • Esculin Hydrolysis test is used in the identification of a wide variety of microorganisms, including the family Enterobacteriaceae, genera Streptococcus and Listeria, non-fermentative gram-negative bacilli, and anaerobes.
  • The test can be performed to determine an organism’s ability to hydrolyze esculin or to produce the esculinase enzyme.
  • The test can be made selective for Streptococcus species by the addition of the bile solution.

Limitations of Esculin Hydrolysis Test

  • Several organisms produce H2S during metabolism, which might react with iron to produce a black complex and interfere with the interpretation of the esculin hydrolysis test. Therefore, for Gram-negative rods, check tubes showing darkening after the addition of the reagent under UV light.
  • Some microorganisms, such as E. coli, have an inducible β-glucosidase and will give a positive result only after prolonged incubation (up to 7 days). However, prolonged incubation should not be used if the test is being used to detect only constitutive β-glucosidase.

References and Sources

  • Esculin agar. M1386. HiMedia Laboratories.
  • Biochemical Tests for the Identification of Aerobic Bacteria. (2016). Clinical Microbiology Procedures Handbook,– 
  • Edberg, S. C., Gam, K., Bottenbley, C. J., & Singer, J. M. (1976). Rapid spot test for the determination of esculin hydrolysis. Journal of clinical microbiology4(2), 180–184.
  • S C Edberg, K Gam, C J Bottenbley, J M Singer. Rapid spot test for the determination of esculin hydrolysis. Journal of Clinical Microbiology. Aug 1976, 4 (2) 180-184.
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About Author

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Anupama Sapkota

Anupama Sapkota has a bachelor’s degree (B.Sc.) in Microbiology from St. Xavier's College, Kathmandu, Nepal. She is particularly interested in studies regarding antibiotic resistance with a focus on drug discovery.

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