Bile Solubility Test- Principle, Procedure and Result Interpretation

Objective of Bile Solubility Test

This test differentiates Streptococcus pneumoniae (positive–soluble) from alpha-hemolytic streptococci (negative–insoluble).

Principle of Bile Solubility Test

Bile or a solution of a bile salt (e.g., sodium deoxycholate) 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.

Procedure of Bile Solubility Test

After 12 to 24 hours of incubation on 5% sheep blood agar, place 1 to 2 drops of 10% sodium deoxycholate on a well isolated colony. Note: A tube test is performed with 2% sodium deoxycholate.

Gently wash liquid over the colony without dislodging the colony from the agar.

Incubate the plate at 35°-37°C in ambient air for 30 minutes.

Examine for lysis of colony.

Result Interpretation of Bile Solubility Test

Positive: Colony disintegrates; an imprint of the lysed colony may remain in the zone.

Negative: Intact colonies.

Limitations of Bile Solubility Test

Enzyme activity may be reduced in old cultures. Therefore, negative results with colonies resembling S. pneumoniae should be further tested for identification with alternate methods.

Quality Control

Positive: Streptococcus pneumoniae (ATCC49619)—bile soluble

Negative: Enterococcus faecalis (ATCC29212)—bile insoluble