Desulfobacter curvatus Widdel
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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. Perform all steps under anaerobic conditions.
3. Aseptically transfer 0.5 ml of #1648 broth to the vial and rehydrate the pellet. Transfer the suspension back into the broth tube. Inoculate a plate of a non-selective medium such as Trypticase Soy, Nutrient, or blood agar with
0.1 ml of the cell suspension.
4. Seal the tube with a rubber stopper and incubate anaerobically at 30oC. Incubate the plate(s) aerobically as a purity check.
5. After 2 or 4 days, growth should be evident as indicated by turbidity through out the broth. Once growth has been established the culture should be transferred to fresh broth every 48 hours.
6. This culture is very sensitive to oxygen when initially rehydrated, therefore steps should be taken to avoid exposure to oxygen. When the culture exhibits good growth it will remain viable for up to 1 week if stored at 4oC under anaerobic condition.
· Tubes of media are placed under a gassing cannula system hooked to a source of oxygen free gas.
· All transfers are performed while the test tubes are on the cannula system with a gentle stream of oxygen free gas flowing through the system.
· As the test tubes are removed from the cannula system each is sealed with butyl rubber stopper thus maintaining the anaerobic headspace.
· 100% nitrogen or 80% nitrogen-10% carbon dioxide-10% hydrogen gas mixture is typically employed as the oxygen free gas source.
Always use freshly prepared anaerobic medium. If there is any question about the anaerobic condition of the medium, the medium can be reduced with the addition of 1.5% cysteine
(2.0 ml per 100 ml of medium).
Other commonly used reducing agents are sodium sulfide, cysteine, dithiothreitol, and titanium citrate. Cysteine is the reducing agent of choice since it does not cause the ferrous ammonium sulfate to precipitate.
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Widdel F. New types of acetate-oxidizing sulfate-reducing Desulfobacter species Desulfobacter hydrogenophilus new species Desulfobacter latus new species and Desulfobacter curvatus new species. Arch. Microbiol. 148: 286-291, 1987.
Validation list no. 26. Int. J. Syst. Bacteriol. 38: 328-329, 1988.