Bile Tolerance test procedure, principle, and Reagents

The Bile Tolerance test is used to differentiate streptococcus agalactia from other gram-positive cocci.

Certain streptococci (S.agalactiae) and enterococci are resistant to 10-40% bile and this test is used to differentiate these from other Gram-positive cocci.

Anaerobic bacteria also vary in their ability to grow in the presence of 20% bile. Bile tolerance is most helpful in separating the Bacteroides fragilis group from other Bacteroides sp. and in separating fusubacterium mortiferum varium from most other clinically-significant fusobacteria.

Bile Tolerance Test Reagents and Media

Following are the Reagents.

• Bile Agar
  1. Ox bile 10 g (10 g ox bile is equal to 100 g bile)
• Nutrient agar
  1. Melt the Nutrient Agar. Add the bile, mix, and dissolve. Sterilize at 115 C for 20 minutes. Cool to about 55°C and distribute.
• Thioglycollate Broth
  1. Peptone :. 15 g
  2. Yeast extract: 0.05 g
  3. Nacl: 0.05 g
  4. Agar :. 0.01 g
  5. Thioglycolic acid: 0.01 g
  6. Glucose: 5 g
  7. Methylene Blue: 0.02 ml
  8. water: 1000 ml
• Oxgall Solution (20%)
  • Prepare 40 % oxgall solution. Sterilize it by autoclaving and store it at 2 to 8 C temperature.

How to prepare Bile Tolerance test media?

Dissolve the solids in the water with gentle heat. Add Thioglycolic Acid and adjust to pH 8.5 with N-NaOH and autoclave at 115°C for 10 minutes.

(to prevent a darkening of the medium, the screw caps should be loosened during autoclaving).

Adjust to pH 7.2, add glucose and dye solution, mix well, and sterilize at 115°C for 10 minutes.

Bile Tolerance test procedure

They’re two types of procedures. Type 1 and type 2.

Type 1 test procedures

Following are the steps

1. Inoculate Bile Agar with the test organisms. Inoculate the test organisms on a Blood Agar plate as well.
2. Incubate at 37°C for 24-48 hours.
3. Examine for growth on both of the plates and, if growth appears on both of the plates, then it is bile-tolerant.

Type 2 test procedure

Following are the steps in the second type of bile Tolerance test.

1. Add 0.5 ml of 40% Oxgall Solution in 10 ml of slightly warm Thioglycollate Broth.
2. Inoculate this broth and one Thioglycollate Broth (without bile) with 1 to 2 colonies of test.
3. Incubate aerobically for 24-48 hours with tight caps.
4. Now compare both of the tubes for growth. If the bile tube shows growth, it means the test organism is bile tolerant.

Bile Tolerance Test Results

• Bile Tolerance tests positive bacteria (with 40%): Enterococcus faeccalis, strep agalactia.
• With 10%: Strep Salvarius
• With 20%: Bacteroid fragilis, beggerthi, B. splanchnic

Bile Tolerance tests negative bacteria:

All other streptococci Other than Bacteroides are bile Tolerance test negative.