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
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
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