Bile solubility test - Principle, Reagents, Procedure, Modified Bile Solubility Test
Introduction to Bile solubility test
The purpose of the bile solubility test is to differentiate Streptococcus pneumoniae from other α-hemolytic Streptococcus spp. S. pneumoniae is bile soluble while other species of α-hemolytic Streptococcus spp are not lysed by bile.
Principle of Bile solubility test
The principle of Bile or solution of bile salt (eg: sodium desoxycholate) is based on rapidly lysing pneumococcal colonies. Bile salt lowers the surface tension between the bacterial cell membrane and the medium.
Thus, acceleration of the organism's natural autolytic process by intracellular autolytic enzyme amidase occurs. Thus, Bile or a solution of bile salt rapidly lyses pneumococcal colonies.
S. pneumoniae produces an autolytic enzyme. If a broth culture is incubated for 25 hours, the broth turns turbid. But after a few days, if the broth is left undisturbed, the medium will once again become clear.
Reagents of Bile solubility test
The reagents used for the bile solubility test are
For tube method: Two grams of sodium deoxycholate are dissolved into 100 ml sterile distilled water to make a 2% sodium deoxycholate (bile salt) solution.
For plate method: Similarly, ten grams of sodium deoxycholate is dissolved into 100 ml sterile distilled water to make a 10% sodium deoxycholate (bile salt) solution.
Procedure of Bile solubility test
The procedures for the bile solubility test include the tube method and plate method.
Tube method
A heavy suspension of pure test culture is made in 2 ml of 0.85% saline and divided equally into two test tubes.
The turbidity of each tube is adjusted to that of 0.5-1 McFarland standard.
Two drops of 2% sodium desoxycholate are added to one test tube and mixed well
In the other one (control tube), 2 drops of sterile distilled water are added.
Both tubes are left at 35-37°C for 10-15 minutes
Clearing of turbidity in a tube with 2% sodium deoxycholate is noted
If the tube with 2% sodium deoxycholate remains turbid, incubate for three more hours
Result
Bile solubility test positive
In the tube test, a clear broth is observed
Bile solubility test negative
In the tube test, a turbid broth is observed
Fig: control tube (left), tube with S. pneumoniae (Source: RearchGate)
Plate method
Inoculate and incubate test organism on 5% sheep blood agar for
12-24 hours.
One or two drops of 10% sodium desoxycholate are added to the isolated colony
The solution is gently washed over the colony without dislodging it from the agar surface
Incubate at 35-37°C in ambient air for 30 mins
Examine for lysis/disappearance of the colony
Result
Bile solubility test positive
Colony disintegrates/flattens in the plate method, leaving α-hemolysis where the colony used to be
Bile solubility test negative
The colony remains intact in the plate method
Fig: intact colony (B), disintegrated colony (A) (Source: microbiology info)
Modified Bile Solubility Test (Rapid Test)
The modified Bile Solubility Test is a rapid test that can provide quick information about the source of the infection and proceed to accurate therapeutic treatment.
Process
Into each bottle containing tryptic soy broth and Thiol broth under vacuum with C02 and 0.025% sodium polyanethole sulfonate (SPS), blood (10%, vol/vol) was inoculated.
A clean grease-free slide is virtually divided into two blood smears- on one half-blood mixed with 1 drop of 2% sodium deoxycholate in place while on the other half, one drop of blood mixed with 1 drop of water is placed.
Following air drying at ambient temperature, gram staining was performed and examined for gram-positive cocci.
The number of micro-organisms in blood with 2% sodium deoxycholate and untreated blood (control) are compared
Result
Positive
Gram-positive cocci are not observed in both the test smear or the control smear.
Negative
Gram-positive cocci are seen in both the test smear and control smear.