Bile Solubility Test: Introduction, Principle, Test Requirements, Procedure, Result-Interpretation, and Keynotes

Introduction of Bile Solubility Test

Bile solubility test uses for presumptive identification of Streptococcus pneumoniae.

Principle of Bile Solubility Test

Bile salts, specifically sodium deoxycholate and sodium taurocholate, have the capacity to selectively lyse Streptococcus pneumoniae when added to actively growing bacteria in agar or broth media. S. pneumoniae produces autolytic enzymes that account for the central depression or umbilication characteristic of older pneumococcal colonies on agar media. The addition of bile salts activates the autolysins and accelerates the natural lytic reactions observed with cultures of pneumococci. The turbidity of a broth suspension visibly clears on addition of bile salts if the organism is soluble.

Requirements for Bile Solubility Test

• Test organism (α -haemolytic streptococci)

• Test tube
• 10% sodium deoxycholate
• 1 McFarland standard or McFarland Densitometer
• Phosphate buffered saline
• Bunsen burner
• Inoculating loop
• Quality control strains  for positive control Streptococcus pneumoniae strain ATCC 49619  and Streptococcus mitis strain ATCC 49456 as a negative control

Procedure of Bile Solubility Test

1. Prepare a heavy suspension of the organism (equivalent to 1 McFarland standard) from growth on agar media in 0.5 ml of phosphate buffered saline, pH 7.0.
2. Then, add  5 drops of 10% sodium deoxycholate to one of the tubes (labeled ‘test’) and 5 drops of sterile normal saline was added to other tube (labeled’ control’).
3. Agitate both the tubes gently  and place in a water bath at 35°C for 3 hours, checking hourly for clearing.

Result and Interpretations of Bile Solubility Test

Positive Bile solubility test : Clearance of turbidity

Negative bile solubility test: No clearance of turbidity

Streptococcus pneumoniae  strain ATCC 49619 : Positive (as shown in figure)

Streptococcus mitis strain ATCC 49456 :Negative (as shown in figure)

Keynotes

• Partial clearing i.e partial solubility is not  positive for Streptococcus pneumoniae identification. Partially soluble strains that have optochin zones of inhibition of less than 14 mm are other than S. pneumoniae.
• S. pneumoniae have following features-

1. Alpha haemoltyic colony on blood agar
2. Bile soluble and Optochin sensitivity test is positive.

Further Readings

1. Cowan & Steel’s Manual for identification of Medical Bacteria. Editors: G.I. Barron & R.K. Felthani, 3rd ed 1993, Publisher Cambridge University press.
2. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
3. Clinical Microbiology Procedure Hand book Vol. I & II, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
4. Colour Atlas and Text book of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr and Sommers H.M.
5. Jawetz, Melnick and Adelberg’s Medical Microbiology. Editors: Geo. F. Brook, Janet S. Butel & Stephen A. Morse, 21st ed 1998, Publisher Appleton & Lance, Co Stamford Connecticut.
6. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
7.  Text book of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.