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Bile Solubility Test

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Bile Solubility Test

Bile Solubility Test is the test which differentiate Streptococcus pneumoniae (positive- soluble) from alpha-hemolytic streptococci (negative- insoluble). Streptococcus pneumoniae is bile soluble whereas all other alpha-hemolytic streptococci are bile resistant.
Principle 
S. pneumoniae has an autolytic enzyme which can be demonstrated by allowing a broth culture to age in the incubator; at 24 hours the broth is turbid; after a few days the medium will become clear.
Bile or a solution of a bile salt (e.g., sodium desoxycholate) rapidly lyses pneumococcal colonies. Lysis depends on the presence of an intracellular autolytic enzyme, amidase. Bile salts lower the surface tension between the bacterial cell membrane and the medium, thus accelerating the organism’s natural autolytic process. Bile salts activate the autolytic enzyme which induces clearing of the culture.
Reagents 
Bile salt preparation

1-Prepare 10% bile salt solution for plate method: Dissolve 10 gram of sodium desoxycholate in 100 ml distilled water.

2-Prepare 2% bile salt solution for test tube method. Dissolve 2 gram of sodium desoxycholate in 100 ml distilled water.

Blood agar plat 5% isolate grow 18-24h


Image result for 2 sodium deoxycholate reagent
Procedure 
Tube Method

1.    Prepare a heavy suspension of a pure culture in 2 ml of 0.9% saline.
2.    Divide the organism suspension into two tubes.
3.    Adjust the turbidity to that of 0.5-1 McFarland standard.
4.    To one tube (test tube), add 2 drops of 2% sodium desoxycholate and mix.
5.    To the other tube (control tube), add 2 drops of sterile water distilled water and mix.
6.    Leave both tubes for 10-15 minutes at 35-37°C.
7.    Observe for a clearing of turbidity in the tube containing 2% sodium deoxycholate.
8.    If negative, continue to incubate up to 3 hours. Observe again for clearing.

Plate Method

1.    Incubate the sample on 5% sheep blood agar for 12 to 24 hours.
2.    Place one to two drops of 10% sodium desoxycholate to the side of a freshly isolated colony (18 -24 hours) on 5% sheep blood agar.
3.    Gently wash the solution over the colony with dislodging the colony from the medium.
4.    Incubate the culture plate at 35-37°C for 30 minutes.
5.    Examine for lysis of colony (Disappearance of the colony).

Result Interpretation 
Tube Method
Positive Result: Suspension clears in tube labelled test and remains turbid in control tube.
Negative Result: Suspension remains turbid.
Note: Partial clearing (partial solubility) is not considered positive for S. pneumoniae identification.

Plate Method

Positive Result: Colony disintegration or flattening of the colony within 30 minutes, leaving an alpha-hemolytic where colony may be located.
Negative Result: No change i.e. colonies remain intact.

Quality Control 
Positive Control: Streptococcus pneumoniae 
Negative Control: Enterococcus faecalis

References
www.time2026end.com
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