Esculin Hydrolysis Test- Principle, Procedure, Results, Uses

Esculin hydrolysis test is a biochemical test that can be used to differentiate bacteria based on their ability to hydrolyze esculin, a compound derived from the horse chestnut tree (Aesculus hippocastanum). Esculin hydrolysis is a common characteristic of many bacteria, including gram-positive and gram-negative bacteria, aerobes, facultative anaerobes, and anaerobes. The test can be used as a taxonomic tool in the identification of various microorganisms, such as Enterobacteriaceae, Streptococcus, Listeria, Aeromonas, and others.

The main objective of the esculin hydrolysis test is to detect the ability of an organism to hydrolyze esculin by the production of esculinase enzyme. This enzyme is present in some bacteria that can break down the glycosidic bond between glucose and esculetin in esculin. The resulting esculetin reacts with ferric ions in the medium to form a dark brown or black complex that indicates a positive test.

Another objective of the esculin hydrolysis test is to differentiate and identify members of the Enterobacteriaceae family. This family includes many gram-negative bacilli that are commonly found in the intestinal tract of humans and animals. Some of these bacteria can cause infections such as diarrhea, dysentery, typhoid fever, and urinary tract infections. The esculin hydrolysis test can help to distinguish between different genera and species of Enterobacteriaceae based on their ability to hydrolyze esculin.

A third objective of the esculin hydrolysis test is to identify other microorganisms that can hydrolyze esculin, such as Streptococcus, Listeria, Aeromonas, and some anaerobes. These microorganisms have different clinical significance and require different treatment options. For example, Streptococcus bovis is associated with endocarditis and colorectal cancer, Listeria monocytogenes can cause meningitis and septicemia in immunocompromised patients, Aeromonas hydrophila can cause wound infections and gastroenteritis, and some anaerobes can cause abscesses and gangrene. The esculin hydrolysis test can help to confirm or rule out the presence of these microorganisms in clinical specimens.

The esculin hydrolysis test is a simple, rapid, and inexpensive method that can provide useful information for the identification and diagnosis of various bacterial infections. It can also be used as a screening test or a confirmatory test depending on the type of medium and reagent used. The test can be performed in solid or liquid media, with or without bile salts, and with or without ferric ammonium citrate. The results can be observed visually or under UV light depending on the method used. The test can be performed in the laboratory or at the point of care using commercially available kits or reagents.

The principle of the esculin hydrolysis test is based on the ability of some microorganisms to produce an enzyme called esculinase or β-glucosidase that can break down esculin, a compound derived from the horse chestnut tree (Aesculus hippocastanum). Esculin is a glucoside consisting of glucose and hydroxycoumarin linked together by an ester bond through oxygen.

When esculin is hydrolyzed by esculinase, it yields two products: glucose and esculetin. Esculetin is a phenolic compound that can react with ferric ions (Fe3+) to form a dark brown to black complex. Ferric ions are usually supplied by the medium component ferric ammonium citrate. The formation of the black complex indicates a positive test for esculin hydrolysis.

Alternatively, esculin is a fluorescent compound that can be detected by its loss of fluorescence when hydrolyzed by esculinase. This method does not require ferric ions and can be performed by using a 0.02% esculin solution and a UV light source.

The esculin hydrolysis test can be performed in different media, such as esculin agar, bile-esculin agar, or Pfizer selective enterococcus medium. The addition of bile salts to the medium makes it selective for microorganisms that can grow in the presence of bile, such as enterococci and group D streptococci. The test can also be performed by using esculin disks or spot tests that are rapid and convenient methods for differentiation of microorganisms.

The esculin hydrolysis test can be used to identify and differentiate various microorganisms based on their ability to hydrolyze esculin in the presence or absence of bile. Some of the microorganisms that are commonly tested by this method are:

• Enterococci and Group D Streptococci: These are Gram-positive cocci that are catalase-negative and can grow in the presence of bile and hydrolyze esculin. They can be differentiated from other non-group D viridans streptococci by the bile-esculin test. The test is especially useful for the presumptive identification of Enterococcus faecalis and Streptococcus bovis.
• Listeria monocytogenes: This is a Gram-positive rod that is catalase-positive and can cause serious infections in humans and animals. It can also grow in the presence of bile and hydrolyze esculin, but it forms minute black colonies on the bile-esculin agar. It can be differentiated from enterococci and group D streptococci by other biochemical tests, such as motility and hemolysis.
• Aeromonas spp.: These are oxidase-positive, facultative anaerobic Gram-negative rods that are found in aquatic environments and can cause gastroenteritis and wound infections in humans. They can hydrolyze esculin without bile and produce a black precipitate on the esculin agar or a loss of fluorescence on the esculin spot test.
• Non-glucose-fermenting Gram-negative rods: These are oxidase-positive, aerobic Gram-negative rods that do not ferment glucose and are often resistant to antibiotics. Some of them, such as Pseudomonas aeruginosa, Burkholderia cepacia, and Stenotrophomonas maltophilia, can hydrolyze esculin without bile and produce a black precipitate on the esculin agar or a loss of fluorescence on the esculin spot test. They can be differentiated from Aeromonas spp. by other biochemical tests, such as growth on MacConkey agar and pigment production.

Other microorganisms that can hydrolyze esculin with or without bile include some species of Staphylococcus, Aerococcus, Bacillus, Clostridium, Bacteroides, and Fusobacterium. However, they are usually not tested by this method as they have other distinctive characteristics that allow their identification.

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:

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

There are two methods to perform the esculin hydrolysis test: the tube test and the spot test. The tube test uses esculin agar as the medium, while the spot test uses a filter paper soaked with esculin solution. Both methods require an inoculum from an 18-24 hour culture of the microorganism to be tested.

Tube Test

1. In a beaker, add 41.5 grams of dehydrated esculin agar powder to 1000 milliliters of distilled or deionized water and mix well.
2. Heat the solution with agitation until it boils and the medium dissolves completely.
3. Dispense the solution into screw-capped tubes (about 3 ml each) and sterilize them in an autoclave at 121°C for 15 minutes.
4. After autoclaving, cool the tubes 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.
5. Using a sterile inoculating needle, pick a light inoculum from the center of a well-isolated colony on a culture plate.
6. Streak the surface of the slant with the inoculum and cap the tube loosely to ensure adequate aeration.
7. Incubate the tube in air at 35-37°C for 24 hours (or up to 7 days for slow-growing Gram-negative rods and anaerobes).
8. Observe the color change of the medium. A blackening of the medium indicates a positive result, while no color change indicates a negative result.

Spot Test

1. Prepare a 0.02% esculin solution in distilled water and sterilize it by autoclaving or filter-sterilization.
2. Place a filter paper on a standard microscope slide and position it on supporting glass rods.
3. Pipette some esculin solution over the paper, but avoid over-saturation.
4. Using a sterile wooden stick, pick an inoculum from a 24-hour bacterial colony and rub it into the center of the filter paper.
5. Incubate the slide at 37°C for about 10-15 minutes. Some microorganisms may take up to 30 minutes to show a positive result.
6. Using a hand-held UV light in subdued light, observe the spot for the loss of fluorescence. A loss of fluorescence resulting in a black colored spot indicates a positive result, while a bright fluorescence indicates a negative result.

The result of the esculin hydrolysis test can be observed either by using the tube test or the spot test. The tube test is performed on esculin agar or broth, whereas the spot test is performed on filter paper with esculin solution.

Tube Test: The tube test is based on the formation of a black complex between esculetin and ferric ions in the medium. A positive result is indicated by the blackening of the medium or a significant portion of it, which demonstrates the breakdown of esculin to esculetin by the microorganism. A negative result is indicated by the lack of color change or only slight darkening of the medium, which demonstrates the inability of the microorganism to hydrolyze esculin.

Spot Test: The spot test is based on the loss of fluorescence of esculin under UV light after its hydrolysis. A positive result is indicated by the loss of fluorescence and a black color under a UV light, which demonstrates the decrease in esculin concentration due to its hydrolysis by the microorganism. A negative result is indicated by a bright fluorescence under a UV light, which demonstrates no decrease in esculin concentration due to the lack of hydrolysis by the microorganism.

Some examples of microorganisms that give positive and negative results in the esculin hydrolysis test are:

• Positive: Enterococcus faecalis, Streptococcus bovis, Listeria monocytogenes, Aeromonas spp., Klebsiella spp., etc.
• Negative: Escherichia coli, Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa, etc.

The esculin hydrolysis test is used for 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. This is especially useful for differentiating enterococci and group D streptococci from non–group D viridans streptococci. The test can also be used to rapidly identify enterococci and Listeria from positive blood cultures.

The test can also be used to identify oxidase-positive aerobic gram-negative rods, such as Aeromonas spp. and yellow-pigmented non-glucose-fermenting rods. These organisms are usually associated with waterborne infections and food poisoning.

The test can also be used as a taxonomic tool in the classification of bacteria based on their ability to hydrolyze esculin. The test can help to distinguish between different genera and species of bacteria based on their esculin hydrolysis patterns.

• Esculin hydrolysis test is not specific for esculinase enzyme, as some microorganisms can hydrolyze esculin by other mechanisms, such as β-glucosidase or β-galactosidase.
• 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.
• Some 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 viridans streptococci (approx. 3%) may also hydrolyze esculin in the presence of bile. Streptococcus bovis cannot be distinguished from other viridans group streptococci using esculin tests without bile.
• The test cannot differentiate between Enterococcus and Lactococcus strains, as both can hydrolyze esculin in the presence of bile. Other tests, such as AccuProbe- Enterococcus test, are required for further identification.

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