Bile-Solubility Test- Scope, Principle, Procedure, Results and Interpretation

 

Scope

Bile- soluble test has been used to differentiate the Streptococcus pneumoniae from other alpha-hemolytic streptococci based on ability to lysis in the presence of bile salt. Streptococcus pneumoniae is bile soluble whereas all other alpha-hemolytic streptococci are bile insoluble.

Principle

Bile salts, specifically sodium deoxycholate and sodium taurocholate, have the capability to lyse Streptococcus pneumoniae selectively when added to actively growing bacteria in agar or broth media. S. pneumoniae produces autolytic enzymes (autolysins) that account for the central depression or umbilication characteristic of older pneumococcal colonies on agar media. The addition of bile salts activates the autolysins and accelerates the natural lytic reaction observed with cultures or pneumococci.

The bile-solubility test can be performed either with a broth culture of the organism or with colonies growing on agar media. The turbidity of a broth suspension visibly clears on addition of bile salts if the organism is soluble. On agar medium, bile-soluble colonies dissolve when drops of the reagent are placed on them. Because sodium deoxycholate may precipitate at a pH of 6.5 or less, the broth culture medium used must be adjusted to pH 7.0 to prevent false-negative reactions.

Media and Reagents

1. A pure culture of the test organism grown at 35°C for 18–24 hours in Todd–Hewitt broth (or equivalent)
2. Sheep blood agar plate
3. Sodium deoxycholate (10% for tube test, 2% for plate test)
4.  Phenol red solution (1% aqueous)
5. Sodium hydroxide (NaOH) solution, 0.10 N

Quality Control Procedure

Positive control (Bile-soluble): Streptococcus pneumoniae

Negative control (Bile-insoluble): Any of the viridans group streptococci

Test Procedure

Broth Test

1. Transfer approximately 0.5 ml of an 18- to 24-hour broth culture to two clean test tubes. Alternatively, a suspension of the organism may be prepared from growth on agar media in phosphate buffered saline, pH 7.0. If the latter is done, the pH need not be readjusted.
2. Add one drop of the phenol red indicator to each tube.
3. Add 0.10 N NaOH to adjust the pH to 7.0 (indicator a light pink color).
4. Add 0.5 ml of 10% sodium deoxycholate to one of the tubes labelled as test.
5. Add 0.5 ml sterile normal saline to the other tube labelled as control.
6. Gently agitate both test tubes and place them in an incubator or a water bath at 35°C for 3 hours, checking hourly.

Plate Test

To a few well-isolated colonies on the test organism growing on sheep blood agar, place a drop of 2% sodium deoxycholate. Without inverting the plate, place it in a 35°C incubator for 30 minutes.

Results and Interpretation

Broth Test

Positive Result: There is visible clearing of the suspension in the tube containing the sodium deoxycholate, with no change in the saline control suspension.

Negative Result: There is no change in the turbidity of the sodium deoxycholate– containing tube relative to the control saline suspension.

Bile solubility Broth test

Plate Test

Positive Result: Colonies on which the reagent was placed dissolve, leaving a partially hemolyzed area where the colony had been.

Negative Result: Colonies where the reagent was placed remain intact and visible.

Bile solubility Plate test: (A) Colony lysed (Positive). (B) Intact colony (Negative).

Limitations of Test

Only 86% of pneumococcal strains will lyse completely. Optochin susceptibility or other studies are useful for the identification of incompletely lysed strains.

Reference

1. Forbes BA, Sahm DF, Weissfeld AS. Bailey & Scott’s Diagnostic Microbiology, 12th Ed., Mosby Elsevier.
2. Winn et al. Koneman’s Color Atlas and Text book of Diagnistic Microbiology. 6th Ed., Lippincott Williams & Wilkins, Wolters Kluwer.