Bile Solubility Test

Generally Biochemical tests are used to identify the species of bacteria by differentiating the species on the basis of their biochemical activities.

The factors such as protein and fat metabolisms and the enzyme production and ability of the organism to utilize the compounds in the mediums helps us in differentiating and identifying the specific species of the bacteria.

Bile solubility test is one such biochemical test, which helps us to identify and differentiate the species of bacteria namely, Streptococcus pneumonia from another species named Alpha-hemolytic Streptococci.

Whereas the Streptococcus pneumoniae is soluble in bile, where as the other species of Alpha-hemolytic streptococci are resistant to the bile and it does not dissolve.

Here let us discuss briefly about the objectives and the preparations of this test.

What is Bile Solubility Test?

Bile solubility test is a biochemical test which is used for differentiation and confirmation of the species of Streptococcus namely S. pneumoniae from all other alpha-hemolytic Streptococci.

This bile solubility test is used as one of the vital tests for differentiating S. pneumoniae as it allows the differentiation between the two species of the Streptococcus namely S. pneumonia and the S. pseudopneumoniae, this is considered as the most challenging task.

This test is usually based on the lysis of the bacterial cells in the presence of a few bile salts like Sodium deoxycholate under specific conditions.

The organisms which lyse in the presence of these specific salts are considered as positive and the others which do not lyse in such medium is known as negative.

However, the exact mechanisms of these specific tests are not clearly understood but it has been detected that the lysis is brought about by inducing the autolytic enzymes.

This test is generally considered as an accurate test for identifying Streptococcus pneumoniae from other species of Streptococci.

Anyhow the results are difficult to interpret as it is based on the evaluation of subjective human.

Bile Solubility Test Principle

Bile solubility test us usually sed for identifying and distinguishing the species of Pneumonia from the Alpha-hemolytic streptococcus species.

This test is usually performed by using a suspension of cell on slide or in a tube depending upon the method we use, and adding the reagent directly to the colony.

The basic principle of the bile solubility test is the lysis of the pneumococcal cells when the sodium deoxycholate is applied on the colony under some of the specific conditions depending on the time and temperature where as the other species do not lyse.

As pneumococcus has an intracellular autolytic enzyme, known as amidase and causes the organisms to undergo an autolysis when the species is cultivated on an artificial membrane.

The bile salts alter the surface tension of the medium which causes the rearrangement of the cell-membrane.

Bile Solubility Test Objective

 To identify and to detect the difference between S. pneumoniae and Alpha-hemolytic streptococcus.

 To observe the capability of the organism to undergo lysis on the presence of bile salts.

Bile Solubility Test Reagents

Bile salts:

• About 10 % of bile solution is either purchased commercially or prepared in the laboratory. Bile salts is prepared usually by adding 10 grams of Sodium deoxycholate in a beaker containing 100 ml of distilled water.

• Then the prepared solution is dispensed in a small amount in the test tubes to minimize the contamination.

• The shelf life of the bile salts which was prepared in the laboratory is usually 270 days.

• It should be stores at a temperature of about 15 to 30ºC.

• If it is stored less than the defined temperature, the solution becomes thickened.

• 0.85% of sodium chloride • Broth culture medium

Reagents:

• Loops

• Slides

• Test tubes

• Pipettes

Bile Solubility Test Procedure

Bile solubility test can be done in two ways, either in the test tube method or by using Direct plate method.

1. Test Tube Method

About 0.5 ml of sterile saline or the suitable broth is dispensed into a small tube.

A heavy suspension of the organism is prepared in the saline and the suspension is then shaken by hand or by using a vortex to form the uniform suspension.

The suspension is further divided into two tubes and it is labelled as TEST and the other as CONTROL.

Then two to five drops of the reagent are dispensed into the both the tubes and they are mixed thoroughly.

The tubes are then incubated at a temperature of 35ºC, for about 3 hours. And the tubes are checked at an interval of each hour and they are examined by adding a gram stain of methyl blue wet mount for lysis of cells at an interval of about 15 minutes.

2. Direct Plate Method

About 2 drops of bile reagent is placed near a suspected colony from the 18 to 24-hour culture.

Then the drops are rolled gently over several colonies by tilting the plate gently, we must be awarded that colonies should not be dislodged against the bile reagent.

Then the plate is placed in the upper right side and it is incubated at a temperature of about 35ºC for a period of about 15 to 30 minutes or in some cases it can also be incubated till it evaporates.

Here instead of incubator, heat block can also be used.

The flattening of the colony is seen.

The colony is observed properly and it should ensure that colony simply float away.

3. Direct Slide Blood Culture Test

A drop of blood culture broth is added to the drop of a bile reagent on a glass slide and it is allowed to let dry.

Then one drop of the blood culture is added to one drop of water and it is let to dry away.

Further, the resulting suspension is gram stained and they are examined if there is presence of any cocci.

Bile Solubility Test Result

Test Tube method: Generally, bile solubility test is detected by the test tube methods a clear zone or a loss of turbidity can be observed when compared to the CONTROL tube, within 3hours. This results in the lysis of the cells and it can be observed under the microscope.

Direct slide blood culture method: The organism is considered as bile soluble if all the cocci in the smear are lysed completely and the Control smear shows an intact bacterium.

Direct plate method: Here the bile solubility is determined by observing the integrity or flattening of the colony within 30 minutes. In case of negative results, there will be no change in the integrity of the colony within half an hour.