Desulfotignum toluenicum
BAA-1460 ™
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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. If needed, exchange the gas in the media test tube for 80% N2 - 20% CO2 or 100% N2.
3. If the medium is pink indicating non-reducing conditions add 0.2 ml Co-enzyme M (5% stock solution) per 10 ml of medium. Let the medium sit at room temperature for 30 to 40 minutes until the resazurin becomes colorless before inoculating.
4. When the medium is ready to inoculate, open the vial as described in the instructions.
5. For inoculation, use an anaerobic (see c below) 1.0 ml syringe tipped with 22-gauge needle, withdraw 0.5 ml of medium #2679 with which to rehydrate the entire cell pellet (perform this rehydration by holding the vial under a cannula while the medium is being added). The cannula should have a gentle stream of oxygen free gas flowing through it. When the pellet has dissolved draw it into the 1 ml syringe and inoculate the broth tube. Transfer 0.5 ml of the inoculated culture to a second anaerobic tube of #2679. Plate 0.1 ml of the inoculated culture onto a non-selective medium and incubate aerobically at 30oC. Use 0.1 ml of the inoculated culture to inoculate a nonselective aerobic broth and plate. Incubate at 30oC.
6. Growth should be detected in the modified #2679 broth within 4 to 7 days. There should be no growth detected on the aerobic plate or in the aerobic broth.
ANAEROBIC CONDITIONS:
a. Resazurin is a commonly used redox indicator that is pink when the redox potential is above 50 mv., and colorless when the redox potential is below 110 mv. i.e. highly reducing. Most strict anaerobes require this low redox potential for optimum growth.
b. To obtain a fully reduced medium, it is necessary that the medium be anoxic and that a reducing agent be added. Common reducing agents are sodium sulfide, cysteine, dithiothreitol, and titanium citrate.
1000X:
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Ommedal H, Torsvik T. Desulfotignum toluenicum sp. nov., a novel toluene-degrading, sulphate-reducing bacterium isolated from an oil-reservoir model column. Int. J. Syst. Evol. Microbiol. 57(Pt 12): 2865-2869, 2007. PubMed: 18048740