Principle of Bile Solubility test:

Bile solubility test is used to differentiate Streptococcus pneumoniae from other alpha-hemolytic Streptococci. Streptococcus pneumoniae is bile soluble whereas all other alpha-hemolytic streptococci are insoluble. Bile or a solution of bile salt, like sodium desoxycholate rapidly lyses the pneumococcal colonies. This lysis depends on the presence of intracellular autolytic enzyme. Bile salts help to lower the surface tension between the bacterial cell and the medium, thus enhancing the organism’s natural autolytic process. 

Procedure of Bile Solubility test:

There are two ways to perform this test.

1)     Plate method

2)     Tube method

 Plate method

  • Put one to two drops of 10% sodium desoxycholate to the side of a freshly isolated colony (18 -24 hrs) on 5% sheep blood agar.
  • Gently wash the solution over the colony with dislodging the colony from the medium.
  • Incubate the culture plate at 35-37oC for 30 minutes.

Expected results

Positive: Colonies disintegrate. There might be an imprint of lysed colonies.

Negative: No change i.e. colonies remain intact

Tube method

  • Prepare a suspension by adding few colonies from freshly isolated bacterial growth on 5% sheep blood agar to 1.0 ml of normal saline to achieve turbidity in the range of a 0.5-1.0 McFarland standard.
  • Divide the cell suspension equally into 2 tubes (0.5 ml per tube).
  • Add 0.5 ml of 2% sodium desoxycholate to one tube. Add 0.5 ml of normal saline to the other tube. Mix each tube well.
  • Incubate the tubes at 35-37°C in ambient air.
  • Vortex both the tubes.
  • Observe the tubes for any clearing of turbidity after 10 minutes.  If negative, continue to incubate the tubes for up to 2 hours. Observe again for clearing.

Expected results

Positive: Clearing of the turbidity in the tube in which bile is added but not in the tube to which saline is added Negative: Cell suspension remains turbid even after incubation for 2 hours

Quality control:

Positive: Streptococcus pneumoniae

Negative: Enterococcus faecalis

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