Bile Solubility Test

Bile solubility test is a biochemical test that distinguishes bile soluble and bile resistant α-haemolytic streptococci. Streptococcus pneumoniae is the only strain that emulsifies on reaction with bile solubility reagent, while the other α-haemolytic streptococci do not undergo such reaction. The reason for the dissolution of the Streptococcus pneumoniae is due to the presence of an autolytic enzyme (Autolysins), which activates in response to the bile spot reagent.

The autolysin amidase causes autolysis of bacterial cell by disintegrating the peptidoglycan backbone. An intracellular autolytic amidase gets activated on reaction with a surface reagent like bile salts and cleaves the bond between an alanine and muramic acid of peptidoglycan.

Content: Bile Solubility Test

  1. Definition
  2. Principle
  3. Test Reagent
  4. Procedure
  5. Limitations
  6. Conclusion


Bile solubility test can define as the qualitative method that primarily isolates the bile sensitive Streptococcus pneumoniae and bile resistant α-haemolytic Streptococci, based on the stimulation of autolysin amidase by the bile salt reagent. Streptococcus pneumoniae undergoes cell-lysis by the action of bile salt reagent and refers as “Bile soluble organisms” while the others are not.

It involves direct and indirect method, where the bile salt reagent can be added directly on to the isolated test organism and indirectly added to the medium containing saline and heavy suspension of the test organism.

Principle of Bile Solubility Test

The mechanism of the bile solubility test depends upon the “Autolysis reaction”. Streptococcus pneumoniae differs from the other alpha-haemolytic streptococci, by giving favourable bile solubility and Optochin susceptibility test. The bile solubility test relies upon the activity of cell-bound Autolysin amidase Lyt-A enzyme, capable of lysing the cell. The enzyme activates in reaction with the surface-active reagents like bile salt. Bile solubility test makes the use of 2% and 10% bile spot reagent of sodium deoxycholate solution.

principle of bile solubility test

The sodium deoxycholate solution reagent carries out the sequential degradation of the bacterial cell, by first lowering the surface tension between the medium and the cell membrane interface. It results in the cell disruption of Streptococcus pneumoniae, after which an intracellular autolytic enzyme activates. This enzyme will break the bond between an alanine and muramic acid of the peptidoglycan, and finally cause autolysis of the bacterial cell.

Bile solubility reagent

It is prepared by dissolving bile salt like sodium deoxycholate in sterile water.
To prepare 2% of bile solubility reagent: Take 2 grams of sodium deoxycholate bile salt in 100 ml of sterile water.
To prepare 10% of bile solubility reagent: Take 10 grams of sodium deoxycholate bile salt in 100 ml of sterile water.

The procedure of Bile Solubility Test

The bile solubility test includes the following three methods.
methods for bile solubility test

Tube Method

It is an indirect method, which involves the following steps:

  1. Take sterile test tubes and pour 2ml of 0.85% saline solution.
  2. Then, inoculate heavy inoculum from the blood agar via a sterile inoculating loop.
  3. Thoroughly mix the bacterial suspension with the saline solution.
  4. Then equally, partition the solution containing saline and bacterial suspension into two separate test tubes, and label them as control and test culture. The turbidity of both the test tubes should be in between 0.5-1.
  5. In a test culture tube, add two drops of bile salt reagent, whereas in control tube adds two drops of saline.
  6. Vigorously shake the test tubes to ensure uniform mixing of the cell suspension with the reagent.
  7. After that, incubate the tubes for 18-24 hours at 35 degrees Celsius.
  8. At last, observe the tubes for the turbidity clearance.

Test Results
Test result of tube bile solubility test
Positive result: Clearing of turbidity represents a positive test result.
Negative result: Here, the suspension remains turbid.

Plate Method

It is a direct method, which involves the following steps:

  1. Add two drops of 10% bile salt reagent over the isolates test colony grown on blood agar media.
  2. Mix the reagent, by rotating the Petri plate back and forth.
  3. Then, subject the test plates into an incubator at 35-37 degrees Celsius for a maximum of 30 minutes.
  4. Examine the plates for the appearance of disintegrated colony.

Test Results
Test result of bile solubility test by plate method
Positive result: Appearance of the disintegrated colony after 30 minutes represents a positive test result.
Negative result: Appearance of unlysed colonies indicates a negative result.

Slide Method

It is also a direct method to perform the bile solubility test and includes the following steps:

  1. Add one drop of bacterial suspension over the clean, grease-free glass slide.
  2. Then add a drop of bile spot reagent on a glass slide.
  3. After that, air dry the glass slide.
  4. At last, perform gram staining procedure to examine for the appearance of cocci.

Test Results
test result of bile solubility test by slide method
Positive result: Cocci will not appear on microscopic examination.
Negative result: Cocci appears on the microscopic examination.


The bile solubility test includes the following limitations:

  • Bile solubility can only detect the presence of Streptococcus pneumoniae from the other alpha haemolytic streptococci.
  • If a test organism undergoes incomplete or partial autolysis, then bile solubility test will consider as a possible method, which identifies the presence or absence of pathogenic Streptococcus pneumoniae. But confirmed after doing another test, named as “Optochin susceptibility test”.
  • The higher concentration of bile salt may also restrain the autolysis of Streptococcus pneumoniae.
  • It is not reliable to test the old bacterial culture, as it may lose the metabolically active enzyme, and the saline and broth should have neutral ph-7.


Therefore, we can conclude that the bile solubility test helps in the identification and differentiation of Streptococcus pneumoniae from other streptococci, based on the property of bile solubility. S.pneumoniae is bile sensitive, as it emulsifies by the reaction of bile salt reagent with its intracellular autolytic enzyme, whereas other strains are bile resistant.

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