Esculin Hydrolysis Test: Principle, Procedure, Purpose, and Results

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Esculin Hydrolysis Test

Biochemical tests are considered as the most important method to detect the micro-organisms and other pathogens. One such biochemical test is Esculin hydrolysis test.

It is one of the differential tests, which helps in differentiating the bacteria depending on the ability of an organism to hydrolyse esculin. This test is generally based on the hydrolysis of the esculin, a glucoside into a glucose and esculetin by a micro-organism which has constitutive β-glucosidase or esculinase enzyme.

What is Esculin Hydrolysis Test?

Esculin hydrolysis test is used to differentiate the type of bacterial species like the gram-positive and gram-negative bacteria, which also includes along with it a a broad spectrum of aerobes, anaerobes and facultative anaerobes.

This test is generally used as a taxonomic tool in identifying the variety of microbes, including Enterobacteriaceae family, streptococcus genera and Listeria, non-fermentative gram negative-bacilli and anaerobes.

Esculin hydrolysis test thus helps in differentiating the bacteria based on their ability to hydrolyse esculin.

This test is usually done selectively by adding bile in the medium, and it is commonly called as bile esculin test.

Hydrolysis of esculin by bacteria can be done by detecting the growth in the supporting media such as Vaugh-Levine, bile-esculin, or Pfizer selective enterococcus media or by non-growth supporting media like Patho Tech or Rapid spot tests.

This test fully depends upon the hydrolysis of esculin into glucose and esculetin with the help of microorganism which constitutes β-glucosidase or an enzyme esculin’s.

Objective of Esculin Hydrolysis Test

• This test helps in detecting the ability of an organism to hydrolyse esculin by producing the enzyme esculinase.

• This test also helps in differentiating the members of the family Enterobacteriaceae.

Esculin Hydrolysis Test Principle

The basis of esculin test is to hydrolyse the esculin during the presence of bile salts, by the enzyme esculinase.

Esculin is a glucosidase consisting of glucose and the hydroxycoumarin which is linked together by an ester bond through oxygen.

The bile esculin test selects an organism on the basis of their ability to grow in a medium containing 4% of salts which is followed by selection on the basis of their ability to hydrolysis of the esculin, which results in the formation of the compound called esculetin.

After the degradation of esculetin it reacts with the iron ions in the medium to form phenolic iron complex which results in a dark brown or black colour.

In the other sense, esculin is a fluorescent compound and on hydrolysis can be observed by loss of fluorescence.

When bile is added to the medium, micro-organisms grow in order to hydrolyse esculin. Bile inhibits the growth of other gram-positive organisms and makes the medium selective.

Esculin Hydrolysis Test Materials

Media: Esculin agar is used for detecting the hydrolysis of esculin. This medium is a differential medium and they can be made selective by adding bile.

Composition of Esculin agar is listed below;

Casein enzymic hydrolysate13.0
Yeast extract5.0
Beef heart infusion in a solid state2.0
Sodium chloride5.0
Ferric chloride0.5

Reagents: In esculin spot test, 0.02% of esculin solution is prepared using distilled water.


• Long wave UV light up to 360nm

• Sterilised sticks, needles, or inoculating loops

• Pasteur pipettes

• Boiling heat block

Esculin Hydrolysis Test Procedure
1. Preparation of the media

About 41.5 grams of dehydrated powder is added in 1000 millilitres of distilled or deionised water and it is mixed thoroughly, after mixing the solution is heated till it reaches the boiling point and the medium to let to dissolve completely.

The solution is then dispensed into a tube having screw caps and it is sterilised in the autoclave at 121 degree Celsius for about 15 minutes.

Then the tubes are taken out from the autoclave and they are cooled by placing them in a slanted position up to a temperature of about 40 to 45º.

This position is maintained at the same angle to achieve the butts of about 1.5 to 2,0 cm depth.

2. Esculin Hydrolysis Test

Esculin hydrolysis test is usually observed in two methods namely tube test or esculin spot test. The spot test is often known as rapid test.

I. Esculin Hydrolysis Tube Test

In tube test, first the light inoculum is taken from the 18 to 24-hour culture using a sterile inoculating needle from the centre of the well-isolated colony.

Then the esculin agar tubes are inoculating by streaking a slant on the surface using the light inoculum which has been picked up from the culture.

Then the caps of the tube are checked for adequate aeration. Then further the tubes are incubated in the air at 35 to 37ºC for about 24 hours and any change is colour is observed up to 7 days of incubating.

In case, if esculin broths are used the tubes are observed daily and checked whether there is any loss in fluorescence of UV light. If there is loss of fluorescence, 1% of ferric ammonium id added in drops into the tube and colour changes are observed.

II. Esculin Hydrolysis Spot Test

In rapid spot test, the prepared 0.2% of esculin solution is autoclaved and it is placed on the filter paper; then the filter paper is placed on a standard microscopic slide and they are positioned on the supporting rods.

Esculin solution is pipetted over the filter paper and over saturation must be avoided. Then the inoculum is taken from a 24-hour colony and streaked in middle of the filter paper.

Then the prepared filter paper along with a slide is placed in an inoculum at 37ºC for about 10 to 15 minutes.

Klebsiella generally gives a positive result within 15 minutes, but it should be holded for 30 minutes to check the proper result.

Hand-held wood lamp is used in the subdued light to observe the loss of fluorescence.

Esculin Hydrolysis Test Result

Esculin Hydrolysis TubeTest: Blackening of the medium detects the positive result and lack of colour change detects the negative result.

Esculin Hydrolysis Spot Test: Loss of fluorescence and black colour under a UV light demonstrates a positive result and bright fluorescence indicates the negative result.

Esculin Hydrolysis Test Citations


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