ATCC determines the biosafety level of a material based on our risk assessment as guided by the current edition of Biosafety in Microbiological and Biomedical Laboratories (BMBL), U.S. Department of Health and Human Services. It is your responsibility to understand the hazards associated with the material per your organization’s policies and procedures as well as any other applicable regulations as enforced by your local or national agencies.
ATCC highly recommends that appropriate personal protective equipment is always used when handling vials. For cultures that require storage in liquid nitrogen, it is important to note that some vials may leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vial exploding or blowing off its cap with dangerous force creating flying debris. Unless necessary, ATCC recommends that these cultures be stored in the vapor phase of liquid nitrogen rather than submersed in liquid nitrogen.
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 37oC under microaerophilic conditions. This organism requires additional free hydrogen for best growth. To obtain this, use an anaerobe jar WITHOUT an active catalyst and with an ANAEROBIC gas generator pack, or other acceptable method, to obtain the desired gas mixture. Incubate slant with cap loose.
4. 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.
Growth on agar takes longer than with the biphasic culture. Colonies are gray or clear forming a spreading film over the surface of the agar. 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. 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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Lee A, et al. Helicobacter muridarum sp. nov., a microaerophilic helical bacterium with a novel ultrastructure isolated from the intestinal mucosa of rodents. Int. J. Syst. Bacteriol. 42: 27-36, 1992. PubMed: 1736969
Mendes EN, et al. Helicobacter trogontum sp. nov., isolated from the rat intestine. Int. J. Syst. Bacteriol. 46: 916-921, 1996. PubMed: 8863417