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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 test tube for 80% N2-20% CO2
3. Add 0.1 ml of sodium sulfide per each 10 ml of medium. Let the medium sit at room temperature for 30 minutes.
4. When the Balch tube is ready to inoculate, open the vial according to enclosed instructions. Use an anaerobic 1.0 ml syringe tipped with 22-gauge needle to withdraw 0.5 ml #1249 broth and use to rehydrate the entire pellet.
5. Using the same syringe, transfer the rehydrated cell suspension back to a tube of #1249 broth. Mix well. Additional broth tubes may be inoculated from this culture.
6. Plate 0.1 ml on a non-selective medium to check for aerobic and anaerobic contamination.
7. Incubate tubes and one plate under an anaerobic atmosphere at 37oC. Incubate non-selective plate aerobically at 37oC to check for purity.
8. In 48 hours, growth should be evident by turbidity in the broth. No growth should occur on the non-selective plate incubated aerobically.
a. Balch tubes (available from Bellco Glass, Vineland, NJ) are specially designed for anaerobic work and use an aluminum crimp cap to hold a rubber stopper in place. Needles can easily be inserted through the stopper, and the tubes can be pressurized to 2 atm. Alternatively, serum vials may be used, or screw cap tubes with butyl rubber stoppers, in the latter case the stopper may be removed and the tube placed under a cannula system that dispenses sterile, oxygen free gas for addition of reducing agents or inoculation.
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.
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Tebo BM, Obraztsova AY. Sulfate-reducing bacterium grows with Cr(VI), U(VI), Mn(IV), and Fe(III) as electron acceptors. FEMS Microbiol. Lett. 162: 193-198, 1998.