Helicobacter fennelliae (Totten et al.) Vandamme et al.

1. Open vial according to enclosed instructions or visit www.atcc.org for instructions.
2. Rehydrate the entire pellet with approximately 0.5 mL of #18 broth.
3. Aseptically transfer the entire contents to a 5-6 mL tube of #18 broth. Additional test tubes can be inoculated by transferring 0.5 mL of the primary broth tube to these secondary broth tubes.
4. Use several drops of the primary broth tube to inoculate a #260 plate and/or #260 agar slant.
5. Or, to obtain a biphasic culture, add several drops of the primary broth tube to a #260 agar slant. Best practice is to incubate these slants at an angle.
6. Incubate at 37°C under microaerophilic conditions for 3-5 days. Use an anaerobe jar with an active catalyst and a microaerophilic gas generator pack or other acceptable method. All tubes and slants should be incubated with caps loosened.
7. Within 3-5 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. Subcultures will require only 24 to 48 hours of 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 three to five days, but little turbidity will be seen. To observe growth, examine a wet mount of the broth under phase microscopy.  Organisms are small thin spiral rods.  Cells from old cultures may be spherical.  The presence of spheroid cells indicates that viability is being lost either due to age or too much exposure to oxygen.

Growth on agar takes longer than with the biphasic culture.  Colonies on Brucella agar plate are smaller, entire, glistening, circular, and smooth.  Spreading may occur with continued incubation. 

Once good growth is present, these organisms tend to lose viability, especially if exposed to air for lengthy periods.  Viability also decreases with repeated sub-culturing.  Therefore, transfer or freeze the culture when optimal growth is achieved.  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.

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.

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

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