Helicobacter winghamensis Melito et al.

1. Open thawed vial according to enclosed instructions or visit www.atcc.org for instructions.
2. 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 tubes.
3. To obtain a biphasic culture, add 0.4 mL of the suspension to a #260 slant.  Add remaining 0.1 mL of the suspension to a #260 plate and streak for isolation.
4. Incubate at 37°C under microaerophilic conditions.  This organism requires additional free hydrogen for best growth.  To obtain this, use an anaerobe jar WITHOUT an active catalyst and an ANAEROBIC gas generator pack, or other acceptable method, to obtain the desired gas mixture.  Incubate slant with cap loose.
5. 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.  

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. This organism does not readily grow on agar surfaces. 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.

Once good growth is present, these organisms tend to lose viability, especially if exposed to air for lengthy periods.  Viability also decreases with repeated subculturing.  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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