Bile solubility test for Streptococcus spp.

Hardy Diagnostics Bile Spot Reagent is recommended for use in presumptively differentiating bile soluble Streptococcus pneumoniae from bile insoluble alpha-hemolytic Streptococcus spp. The bile solubility test may be performed using a cell suspension or by applying the Bile Spot Reagent directly to the colony.


The bile solubility test is a qualitative procedure for determining the ability of bacterial cells to lyse in the presence of bile salts under specific conditions of time and temperature. The test is primarily used to differentiate bile soluble Streptococcus pneumoniae from bile insoluble alpha-hemolytic streptococci. The working mechanism of the test is not clearly understood, however, one theory is that the bile salts facilitate lysis of pneumococcal cells by activating an intracellular autolytic enzyme. 


Specimen Collection: This product is not intended for primary isolation of patient specimens. This product is used in conjunction with other biochemical tests to identify cultures of isolated organisms.

Test Tube Method:

1. Dispense 1ml of sterile 0.85% saline into a small test tube.

2. Prepare a heavy suspension of the organism in the saline. Shake or vortex to form a uniform suspension.

3. Divide the suspension into two tubes (0.5ml each), one labeled "TEST," the other labeled "CONTROL."

4. Dispense 10 drops of Bile Spot Reagent into the tube marked "TEST". Add 0.5ml of saline to the tube marked "CONTROL". Gently mix each tube.

5. Incubate the tubes for 3 hours at 35 degrees C., checking hourly.

Direct Plate Method:

1. Place a drop of Bile Spot Reagent near a suspected 18-24 hour old colony, gently roll the drop over several representative colonies. Take care not to dislodge the colonies.

2. Keep the plate right side up and incubate at 35 degrees C. for 30 minutes.


Test Tube Method:

Examine the "TEST" tube for clearing or loss of turbidity as compared with the "CONTROL" tube. Bile solubility is demonstrated as a clearing or loss of turbidity, relative to the "CONTROL" tube within three hours.

Direct Plate Method:

Bile solubility is demonstrated as a disintegration of the colony and/or the appearance of a hemolytic zone in the medium at the sight where the colony was located within thirty minutes. Insolubility is demonstrated when there is no change in the integrity of the colony within 30 minutes.  

Cat. no. HDZ61
Size: 15mL


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