BILE SOLUBILITY REAGENT
- For in vitro use only - Catalogue No. RB60
Our Bile Solubility Reagent can be used to
differentiate bile-soluble Streptococcus pneumoniae
from other bile-insoluble α-hemolytic streptococci.
S. pneumoniae possesses an autolytic enzyme
that causes the organism to breakdown its own cell
wall causing lysis of the cell. In the presence of the
bile salt, sodium deoxycholate, the autolytic process
is accelerated. On a solid medium, colonies of S.
pneumoniae disintegrate and disappear, or if growth
is suspended in saline the turbid solution exhibits a
clearing effect. In both cases, the reactions are
classified as soluble. Bile (Oxgall) and other bile
salts, such as sodium taurocholate can also be used to
perform the test but often give variable results. Other
α-hemolytic streptococci do not possess active
autolytic enzymes and will not dissolve in bile, and
are classified as bile insoluble.
It should be noted that only 80% of Streptococcus
pneumoniae strains will lyse completely, and
additional tests may be required to differentiate those
strains that show only partial or incomplete lysis.
Formulation per 100 mL
Sodium Deoxycholate ....................................... 10.0 g
Sterile Deionized Water.............................. 100.0 mL
Recommended Procedure
Plate Procedure
1. Perform a four-quadrant streak of the test
organism onto a blood agar plate to obtain
well-isolated colonies. (The test organism
should be an α-hemolytic, catalase-negative,
gram-positive cocci arranged in chains and this
determination should be made prior to
performing the test)
2. Incubate plates in an inverted position for 18 to
24 hours at 35°C.
3. Select a characteristic S. pneumoniae colony
that is well-isolated. Mark the location of the
colony on the bottom of the petri dish using an
wax pencil or permanent felt marker.
4. Place a loopful or a drop of Bile Solubility
Reagent onto the colony of interest.
5. Incubate plate aerobically, agar-side down and
lid-side up, at 35°C. Do not invert the plate.
6. After 30 minutes check the plate for appearance
of the suspect colony.
Tube Procedure
1. Obtain a pure, overnight culture of the
streptococci of interest from a blood agar plate
or Todd-Hewitt Broth.
2. Prepare a heavy suspension of the organism in
1.0-mL of physiologic saline solution (Dalynn
BS85 or TS85).
3. Add 1 drop of phenol red indicator and, if
required, adjust the pH to 7.0 using 0.1 N sodium
hydroxide. The solution should appear pink.
(This step can be skipped but the pH must be
above 6.8 to obtain accurate results)
4. Divide the saline suspension equally into two
tubes (0.5-mL per tube). Label one tube test and
the other control.
5. Add 0.5-mL (4 drops) of Bile Solubility Reagent
to the tube marked test.
6. Add 0.5-mL (4 drops) of sterile physiologic
saline to the tube marked control.
7. Gently agitate both tubes to ensure that the
suspensions are homogenous.
8. Incubate tubes at 35 to 37°C.
9. Check tubes hourly and make a final
interpretation after 3 hours. Observe for clearing
of the broth.
