Desulfotomaculum nigrificans (Werkman and Weaver) Campbell and Postgate
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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. Under anaerobic conditions, withdraw 0.5 ml of the #1249 broth from a single test tube (5 to 6 ml) and rehydrate the entire vial contents.
3. Aseptically transfer this aliquot back into the broth tube and inoculate a #260 (blood agar) plate to be incubated anaerobically. An aerobic blood plate may also be streaked to check for purity.
4. Incubate tubes and plate under an anaerobic atmosphere at 55oC. Incubate blood plate aerobically at 37oC.
5. Within 24 to 48 hours, growth is evident by moderate to good turbidity in the broth with sediment in the bottom of the tube. No growth occurs on the blood agar plate incubated aerobically. Once growth is achieved, transfer the culture to fresh tubes of #1249 broth. This culture does not grow well on agar.
Anaerobic conditions for transfer may be obtained by the following:
·Placement of test tubes under a gassing cannula system hooked to anaerobic gas.
Anaerobic conditions for incubation may be obtained by any of the following:
·Loose screw caps on test tubes in an activated anaerobic gas pack jar, or
·Use of sterile butyl rubber stoppers on test tubes so that an anaerobic gas headspace is retained.
The best results have been obtained using the gassing cannula system. For reviving the cultures initially, an anaerobic jar is not recommended. Once the culture has been established, an anaerobic jar can be used if the inoculum is 20% or greater.
Either 100% N2 or 80% N2-10% CO2-10% H2 can be used as the anaerobic gas for culturing this organism.
Once growth has been obtained, this culture is fairly easy to maintain if transferred every other day. A culture that has good growth and is fresh can be maintained at 4oC for up to a week. The cells can be stored at 70 to 80oC by growing a large volume in ATCC Medium #1249, harvesting the cells and then mixing the cell pellet in an equal volume of fresh #1249 and 20% glycerol (10% final concentration). Dispense the cells into vials (approximately 0.5 to 1.0 ml per vial) and freeze rapidly. Both the #1249 broth and glycerol need to be pre-reduced. This may be accomplished by adding 0.1 ml (for each 5 to 6 ml medium) of a 1.5% sodium sulfide solution.
Additional information on this culture is available on the ATCC® web site at www.atcc.org.
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Campbell LL, Postgate JR. Classification of the spore-forming sulfate-reducing bacteria. Bacteriol. Rev. 29: 359-363, 1965. PubMed: 5826606
Skerman VB, et al. Approved lists of bacterial names. Int J Syst Bacteriol 30: 225-420, 1980.
fresh water, Delft