Bile Solubility Test on Sheep Blood Agar:Principle, Method and Interpretation

Principle:

The bile solubility test differentiates Streptococcus pneumoniae (positive) from other alpha- hemolytic streptococci, also known as "Viridans Streptococci" (negative). Pneumococcal colonies are rapidy lysed by bile or a solution of a bile salt, such as sodium desoxycholate. Lysis depends on the presence of an intracellular autolytic enzyme. Bile salts lower the surface tension between the bacterial cell membrane and the medium, thus accelerating the organism's natural autolytic process.

Method:

1. Place 1 to 2 drops of 10% sodium desoxycholate to the side of a young (18 to 24- hour) , well isolated colony growing on 5% sheep blood agar. Note: A tube test is performed with 2% sodium desoxycholate.
2. Gently wash liquid over colony, without dislodging colony from agar.
3. Incubate plate at 35 degree celsius in ambient air for 30 minutes.
4. Examine for lysis of colony.

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Result and Interpretation:

Positive- Colony disintegrates; an imprint of the lysed colony may remain wihtin the zone.

Negative- Intact colonies

Bile Solubility Test Troubleshooting:

Partial clearing (partial solubility) is not considered positive for pneumococcal identification. Partially soluble strains that have optochin zones of inhibition of less than 14 mm are not considered pneumococci.

Bile Solubility Test Quality Control:

Each new lot of sodium deoxycholate should be tested with positive and negative Quality control strains. S. pneumoniae strain ATCC 49619 can be used as a positive control and S. mitis strain ATCC 49456 can be used as a negative control.