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Bile Esculin Test Principle, Procedure, Result, and application.

Bile Esculin test

  • The Bile Esculin test is a biological test that is used to distinguish Enterococci from Group D Streptococci from non-group D viridans group Streptococci by their capacity to hydrolyze esculin.
  • A variety of organisms are capable hydrolyzing esculin. But only a handful of them can do it when they are surrounded by the bile (4 percent bile salts or 40 percent the bile.). This property can be utilized to determine the species of specific groups.
  • The Bile-esculin test is carried out on a differential agar that is a selective bile esculin-based agar which is composed of bile and the esculin.
  • The agar is enriched with Bile salts that hinder the growth of Gram-positive organisms as well as allows for the specific removal from Enterococci as well as the Group D Streptococci.
  • Esculin is a glycosidic coumarin derivative (6-beta-glucoside-7-hydroxy-coumarin) which is a fluorescent compound, and its hydrolysis can also be observed by the loss of the fluorescence.
  • The Bile-esculin test has undergone several modifications throughout the years and has been improved to be more efficient. Today, there are bile-esculin disks readily available and are utilized to make a quick distinction between the Group D Streptococci and non-Group D Streptococci.

Purpose of Bile Esculin test

  • To identify and isolate bacteria capable of hydrolyzing the esculin, in the presence of Bile.
  • To determine the group D streptococci as well as enterococci (All are Bile-esculin test positive). The group D streptococci, as well as the enterococci, include pathogens that are opportunistic, such as Enterococcus falciparum, Enterococcus flavius and Streptococcus bovis.

Principle of Bile Esculin test

Bile esculin media contains peptone and esculin for nutrition, as well as bile for the purpose of inhibiting Gram-positive bacteria, other than Enterococci and streptococci from Group D. Bile esculin azide Agar makes use of sodium azide to prevent Gram-negative bacteria. Since you’ll use isolated Gram positive strains this isn’t necessary. Ferric citrate is used to provide a color indication.

Esculin is glycoside (a sugar molecule linked through an acetyl bond to an alcohol) made up of esculetin and glucose. These linkages are readily hydrolyzed when under conditions of acidity. Numerous bacteria are able to hydrolyze esculin however, only a few are able to perform this feat when bile is present.

Organisms that break the esculin molecules and utilize the free glucose to satisfy energy requirements release esculetin into the medium. It reacts with ferric acid in the medium and forms the phenolic iron complex that turns the agar slant dark from brown to black. Agar slants that have at or near the half-darkness within 48 hours of incubation time is considered to be bile-esculin positive. If less than half of the surface has been darkened it is negative.

Microorganism tested in Bile Esculin test

  • Chains of Gram-positive cocci that are catalase negative and can be morphologically identified as presumed S. bovis.
  • Isolates of alpha- or gamma-hemolytic, Gram-positive cocci as part of differentiation of enterococci from other pyrrolidonyl-b-naphthylamide (PYR)-positive organisms
  • Hemolytic, non-spore-forming rods with Gram-positive morphology that are catalase positive and morphologically classified as probable Listeria.
  • Positive blood tests with Gram-positive cocci in chains or Gram positive rods to quickly (within less than 4 hours) detect enterococci as well as Listeria
  • Esculin without bile for the identification of oxidase-positive aerobic Gramnegative rods, including Aeromonas and yellow-pigmented non-glucose-fermenting rods

Requirement for Bile Esculin test

A. Media Used

  1. Bile-esculin agar slants with iron(III) citrate. Agar plate media, such as Enterococcosel agar, have a similar formulation.
  2. Bile-esculin-azide agar or broth with iron(III) citrate and azide. Azide will inhibit most Gram-negative bacteria.
  3. Peptose-yeast-esculin broth (normally in the anaerobic atmosphere).
  4. Esculin agar (0.1% esculin in heart infusion basal medium) without bile or azide but with iron(III) citrate.

Composition of Bile Esculin test

Peptic digest of animal tissue5.0
Beef extract3.0
Bile Salts40.0
Ferric citrate0.5
Bacteriological agar15.0

Note: Final pH at 25°C: 6.6 ±0.2 / Store at 2°C to 8°C.

B. Reagents and Supplies Used

  1. Long-wave (360-nm) UV light
  2. 1% ferric [iron(III)] ammonium citrate if iron(III) is not incorporated into the medium

Bile Esculin test procedure 

Media Preparation

  1. In a beaker 64.5 grams of dehydrated powder or lab-prepared medium is added to 1000 milliliters deionized or distillate water.
  2. The solution is then heated until boiling point to melt the powder completely.
  3. The dissolving medium is dispersed into tubes, and then sterilized using an autoclave at 15lbs tension (121degC) over 15 mins.
  4. When the autoclaving process has been completed, the tubes can be removed out and then cooled to an slanted angle to temperatures of 40-45degC. It is important to maintain the slanted position in order to get butts that are 1.5 to 2.0 cm in depth.

Esculin Hydrolysis

  1. Esculin hydrolysis can be detected via tubes or an examination on a disk. Disk tests are an extremely fast test.

Tube test

  1. A colony that is well-isolated is harvested from an 18-24-hour culture with a needle for inoculation that is sterile.
  2. The bile esculin tubes are inoculated by streaking on the top of the slant using or using the light inoculum taken from the plate of culture.
  3. For the purpose of enterococcus as well as S. bovis detection for identification, the bile concentration of 40% is employed and the tubes are inoculated using 10 ul of calibrated loops of the 0.5 McFarland standard suspension prepared in the sterile water.
  4. The cap on the test tubes must be left loose in order to provide an adequate air flow.
  5. After that, the tubes will be incubated aerobically during 24-hours (or as long as 7 days in the case of slow-growing, Gram-negative anaerobes and rods) and the change in color is then seen.
  6. For broths containing esculin that do not contain ferrous (III) citrate The tubes are inspected each day for a loss of fluorescence.
  7. In the absence of fluorescence 2-3 drops 1.0 ferric ammonium citrate is added to the esculin tube and the change in color can be observed.

Disk test

  1. The disk of esculin is filled with just a drop of deionized or distillated water. The disk, however, must not be completely saturated.
  2. With a sterile loop two or three colonies that are well-isolated are harvested in an overnight (18up to 24 h) culture.
  3. The disk is examined to determine the appearance of dark brown or black hue after approximately 10 minutes at the temperature of room.

Bile Esculin Test Result

  • Positive bile esculin test: A positive test tube in the medium containing ferric ammonium citrate is characterized by the appearance of black spots on the medium.
  • Nagative bile esculin test: A negative result is characterized by the absence of color changes. The substance will light up in UV ultraviolet light (366 num).
  • In the case of esculin broth that is not enriched with the iron (III) citrate A positive test can be evident either through the darkening of the broth following the addition of ferric [iron(III)[iron(III)] reagent or by the disappearance of fluorescence from the medium.
  • The negative test result can be observed in the bile-esculin system when the organism is unable to grow in the presence bile regardless of its capability to hydrolyze esculin.
  • A positive test for disks is evident by the appearance of deep black or dark brown hue.
  • A negative test for disks remains in colorless.
Bile Esculin Test Result
Bile Esculin Test Result

Bile esculin hydrolysis test of Different Microorganisms

The table below demonstrates the growth of a few bacteria as well as their bile esculin hydrolysis test:

OrganismGrowthBile esculin hydrolysis
Enterococcus faecalisGoodPositive reaction; Blackening of medium
Escherichia coliGoodNegative reaction
Enterococcus faeciumLuxuriantPositive reaction; Blackening of the medium around the growth.
Yersinia enterocoliticaGood-luxuriantPositive reaction; Blackening of the medium.

Quality Control of Bile Esculin Test

  • Positive control of Bile Esculin Test: Enterococcus species (e.g. E. faecalis)
  • Negative control of Bile Esculin Test: Viridans streptococcus, not group D

Application of Bile Esculin Test

  • Bile Esculin test is used to determine the biochemical basis to determine the identification from Enterococci as well as the Group D Streptococci.
  • It may also be used to distinguish these organisms from viridans Streptococci as well as other Gram-positive microorganisms.
  • Bile Esculin Agar can be a differentiating medium that promotes the growth of organisms such as Enterococcia, Listeria, and Yersinia enterocolitica.

Limitations of Bile Esculin Test

  • If a large amount of inoculum is utilized or if the amount of bile is lower than 40percent, the viridans group streptococci different from S. bovis could produce a positive reaction to the bile-esculin-agar.
  • Esculin tests that do not contain bile can’t be used to differentiate S. bovis (previously known as the group streptococci D) from other viridans groups streptococci.
  • A variety of organisms could produce H2S in the course of metabolism, which may react with iron to result in a black compound, which could interfere in the result of an test for hydrolysis esculin and may give a false positive result.
  • Some microorganismslike E. coli that have b-glucosidase will give the positive results in this test only after long incubation. But, extended incubation is not recommended when the test is utilized to identify b-glucosidase from other organisms.

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