Desulfitobacterium metallireducens Finneran et al.

1. Sterilize the top of the Balch tube by spraying it with 70% ethanol and then flame the top.
2. If needed exchange the gas in the test tube for 80% N₂ 20% CO₂.
3. Add 0.2 ml Cysteine 3.0% stock solution per 10 ml of medium.  Let the medium sit at room temperature for 30 to 40 minutes before inoculating.
4. When the Balch tube is ready to inoculate, thaw the frozen vial at room temperature under a gentle stream of oxygen-free gas.
5. For inoculation, use an anaerobic (see "c" below) 1.0 ml syringe tipped with 22-gauge needle, withdraw the cell suspension from the vial and transfer it to the broth.  Plate 0.1 ml of the inoculated culture onto a non-selective medium and incubate aerobically at 30^oC.  Use 0.5 ml of the inoculated culture to inoculate a nonselective aerobic broth and an additional tube of #2550 broth. Incubate the non-selective aerobic broth tubes at 30^oC.  Incubate the anaerobic tube at 30^oC.
6. Growth should be detected in the #2550 broth within 24 to 48 hours. There should be no growth detected on the aerobic plate or in the aerobic broth. Once growth has been established the culture should be transferred to fresh broth every 2-4 days.
7. This culture is very sensitive to oxygen when initially rehydrated, therefore steps should be taken to avoid exposure to oxygen.

ANAEROBIC CONDITIONS:

1. 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.
2. 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.
3. Syringes can be made anaerobic by one of two methods.

1. Displace the dead space in the syringe with a sterile oxygen-free gas.  
2. Displace the dead space in the syringe with a reducing agent.  

When examined microscopically, the cells appear as slightly curved rods.

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).  Cysteine is the reducing agent of choice since it does not cause the ferrous ammonium sulfate to precipitate. 

Additional information on this culture is available on the ATCC web site at www.atcc.org.

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