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Bile Solubility test: Principle, Procedure, expected result and quality control

Principle: The bile (sodium deoxycholate) solubility test distinguishes Streptococcus pneumoniae from all other alpha-hemolytic streptococci.  Streptococcus pneumoniae is bile soluble whereas all other alpha-hemolytic streptococci are bile resistant. Sodium deoxycholate (2% in water) will lyse the pneumococcal cell wall.

Procedure of bile solubility test:

Preparation of 2% sodium deoxycholate (bile salt) solution

Performing the bile solubility test

  1. Grow the isolate(s) to be tested for 18-24 hours on a blood agar plate (BAP) at 35-37°C with ~5% CO2 (or in a candle-jar).
  2. Add bacterial growth from the overnight blood agar plate to 1.0 ml of 0.85% saline to achieve turbidity in the range of a 0.5-1.0 McFarland standard.
  3. Divide the cell suspension equally into 2 tubes (0.5 ml per tube).
  4. Add 0.5 ml of 2% sodium deoxycholate (bile salts) to one tube. Add 0.5 ml of 0.85% saline to the other tube. Mix each tube well.
  5. Incubate the tubes at 35-37°C in CO2.
  6. Vortex the tubes.
  7. Observe the tubes for any clearing of turbidity after 10 minutes. Continue to incubate the tubes for up to 2 hours at 35-37°C in CO2 if negative after 10 minutes. Observe again for clearing.
Bile Solubility Test

Reading the bile solubility test results

A clearing of the turbidity in the bile tube but not in the saline control tube indicates a positive test.

Troubleshooting in Bile solubility test:

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.

Quality control in Bile Solubility test:

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.


Remember the sentence “Streptococcus pnuemoniae is A BOSS” i.e.

Streptococcus pneumoniae is

  1. Alpha hemolysis
  2. Bile Soluble
  3. Optochin Sensitive