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2. 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.
3. 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.
Individual colonies will not be observed. Growth appears as a film on the surface of fresh, moist plates, extending out from original inoculation site to fill entire plate, usually within 48 to 72 hours.
The cells do not Gram stain well using traditional procedures. For 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.