Cat. no. Z7091 Bile Differentiation Disks 50 disks/cartridge


HardyDisk™ Bile Differentiation Disks are used for the differentiation of Bacteroides fragilis group and for identification of other gram-negative anaerobic bacilli.


Anaerobic gram-negative bacilli are the most commonly encountered anaerobes in clinical specimens, with Bacteroides fragilis group isolated more frequently than any other anaerobe. (4) Organisms in the Bacteroides genus have received notoriety due to their frequent involvement in infectious disease and their resistance to antimicrobial agents. Penicillin-resistant strains of the B. fragilis group are common, however, there are recent reports of new resistance to cefotetan and clindamycin and occasional resistance to piperacillin-tazobactam, imipenem and quinolones.(6) HardyDisk™ Bile Differentiation Disks are instrumental in differentiating the B. fragilis group from otherBacteroides and Prevotellaspecies.(2,6)

The HardyDisk™ Bile Differentiation Disk is used to determine an organism's ability to grow in the presence of high concentrations of bile. Other indicators of bile resistance, including significant growth on BBE (Bacteroides Bile Esculin) media and growth in 20% bile broth, require the inoculation of additional media. In 1983, it was demonstrated that comparable results were obtained using a 15mg bile disk and traditional bile tolerance methods. This study confirmed that the bile disk method is capable of clearly distinguishing between bile-resistant and bile-sensitive anaerobic organisms.(2) A bile disk is a rapid and cost effective method for detecting bile resistance. The disk can be conveniently added to a Brucella subculture plate when a gram stain reveals an isolate to be an anaerobic gram-negative rod.

HardyDisk™ Bile Differentiation Disks can be used to determine bile sensitivity in a variety of organisms. An anaerobic, gram-negative rod that is bile-tolerant and resistant to vancomycin, kanamycin, and colistin can be identified as a member of the Bacteroides fragilis group. Additionally, Bilophila which are phenotypically similar to B. ureolyticus, can be differentiated by bile tolerance and a strong catalase reaction. Bile resistance is also useful in the presumptive differentiation of Fusobacterium mortiferum and Fusobacterium varium from other Fusobacterium species.(2,6)


Each HardyDisk™ Bile Differentiation Disk is prepared by impregnating carefully controlled concentrations of bile onto a high quality 6mm diameter filter paper disk.


Storage: Upon receipt store at -20 to +8ºC. away from direct light. Product should not be used if there are any signs of deterioration or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, and moisture.



1. Allow disks to equilibrate to room temperature before use. Prepare a suspension, equivalent to a 0.5 McFarland opacity standard, of the organism to be tested in Thioglycollate Broth (Cat. no. K21).

2. Dip a sterile non-toxic swab (Cat. no. Z5800R) the organism suspension. Rotate the swab several times, pressing firmly on the inside wall of the tube above the fluid level. This will remove excess inoculum from the swab. Evenly inoculate the dried surface of Brucella Agar with Hemin and Vitamin K (Cat. no. A30) to obtain heavy confluent growth.

3. Aseptically place a single bile disk on the media surface. With sterile forceps, gently tap each disk to the media surface to ensure uniform diffusion of the bile into the medium.

4. Incubate anaerobically at 35ºC. for 24-48 hours.

5. When adequate growth is present, examine the plate for a zone of inhibition.


An organism is considered sensitive to bile when a zone of inhibition is present around the disk

An organism is considered resistant to bile when there is no zone of inhibition surrounding the disk.


The presence of bile disks will cause a zone of hemolysis on blood based media, however, this is not an indication of organism growth.

Among the Bacteroides fragilis group, some B. uniformis strains may grow poorly in the presence of bile and will have a zone of inhibition around the disk.(6)

Some non- B. fragilis group are bile-resistant; morphology, biochemical tests and other special potency disks will differentiate these species from the B. fragilis group.


Standard microbiological supplies and equipment such as loops, other culture media (including Thioglycollate Broth and Brucella Agar with H and K), swabs, applicator sticks, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.


Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Bacteroides fragilis
ATCC ® 25285
F 24-48hr 35 o C Anaerobic Resistant; no zone of inhibition
Prevotella melaninogenica
ATCC ® 25845
F 24-48hr 35 o C Anaerobic Sensitive; zone of inhibition present

User Quality Control

Physical Appearance

HardyDisk™ Bile Differentiation Disks are 6mm (in diameter) filter paper disks with the letters BILE printed on both sides and should appear beige in color.


Bile-Resistant (no zone of inhibition)
Bacteriodes fragilis (ATCC® 25285) growing around a HardyDisk™ Bile Differentiation Disk (Cat. no. Z7901) on Brucella Agar with Hemin and Vitamin K (Cat. no. A30). Incubated anaerobically for 48 hours at 35ºC.


Prevotella melaninogencia (ATCC® 25845) growing with a zone of inhibition around a HardyDisk™ Bile Differentiation Disk (Cat. no. Z7901) on Brucella Agar with Hemin and Vitamin K (Cat. no. A30). Incubated anaerobically for 48 hours at 35 deg. C.


1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

2. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

3. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

4. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

5. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology, J.B. Lippincott Company, Philadelphia, PA.

6. Jousimies-Somer, H., et al. 2002. Wadsworth Anaerobic Bacteriology Manual, 6th ed. Star Publishing, Belmont, CA.

ATCC is a registered trademark of the American Type Culture Collection.