Helicobacter bilis Fox et al.

1. This organism is shipped frozen in dry ice.  Just prior to use, thaw vial in water at approximately 37°C.  When thawed, a drop of the suspension may be used to do an immediate wet mount to observe the unique morphology of this organism and verify its viability by checking for motility.
2. Aseptically transfer the entire thawed suspension into a fresh #1705 broth (3-5 mL).  Mix well.  This suspension can now be used to inoculate agar slant(s), plate(s), or the preferred biphasic culture.  Two #1705 plates should be inoculated, one for microaerophilic growth and the second for aerobic growth.  No growth should occur on the plate incubated aerobically.
3. To obtain a biphasic culture, add 0.4 mL of the suspension to a #1705 slant.  The resulting pool at the bottom of the slant is where the best, most rapid growth will occur.  Add 0.1 mL of the suspension to a #1705 plate and streak for isolation.
4. Incubate at 37°C under microaerophilic conditions using an anaerobe jar with an active catalyst and a microaerophilic gas generator pack, or other acceptable method, to obtain microaerophilic conditions.  Incubate slant with cap loose.
5. Within 2-3 days of incubation, good growth should be obtained in the broth pool at the bottom of the slant.  Additional incubation may be required for colonies to appear on the plate.  Further subcultures can be made using broth pool as the inoculum source.

For best growth, pre-reduce all media overnight; recommend use of Anoxomat or an anaerobe jar with an active catalyst and a microaerophilic gas generator pack, or other acceptable method, to obtain microaerophilic conditions for reducing and incubation.

This is a slow growing organism that requires moist conditions for best growth. Growth at the broth/agar interface of the biphasic slant should occur within 7 days, but little turbidity will be seen. To observe growth, examine a wet mount of the broth under phase microscopy. 

The cells do not Gram stain well using traditional procedures. To obtain the best results, use a basic fuchsin counterstain in place of the safranin.

Once good growth is obtained, transfer or freeze the culture. Adding an equal amount of 20% sterile glycerol to pooled broth from several biphasic slants, followed by freezing in liquid nitrogen or “ultra-low temperature” freezer is recommended.

Additional information on this culture is available on the ATCC^® web site at www.atcc.org.

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