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
- Ox bile 10 g (10 g ox bile is equal to 100 g bile)
- Nutrient agar
- 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
- Peptone :. 15 g
- Yeast extract: 0.05 g
- Nacl: 0.05 g
- Agar :. 0.01 g
- Thioglycolic acid: 0.01 g
- Glucose: 5 g
- Methylene Blue: 0.02 ml
- 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
- Inoculate Bile Agar with the test organisms. Inoculate the test organisms on a Blood Agar plate as well.
- Incubate at 37°C for 24-48 hours.
- 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.
- Add 0.5 ml of 40% Oxgall Solution in 10 ml of slightly warm Thioglycollate Broth.
- Inoculate this broth and one Thioglycollate Broth (without bile) with 1 to 2 colonies of test.
- Incubate aerobically for 24-48 hours with tight caps.
- 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.