Desulfurispirillum indicum Rauschenbach et al. 

1. Sterilize the top of the Balch tube by spraying it with 70% ethanol and then flaming the top.
2. If needed exchange the gas in the test tube for 80% N₂ - 20% CO₂.
3. Add 0.1 ml of 1 M selenate and 0.1 ml of 2 M pyruvate per each 10 ml of medium.  Add 0.1 ml of reducing agent (3% cysteine, stock solution) per each 10 ml of medium.  When reducing media, also add the sterile additions before inoculating.  Let the medium sit at room temperature for 1 hour. 
4. Allow the frozen vial to thaw under anaerobic conditions.  Once thawed, take a gassed 1.0 ml syringe tipped with 22-gauge needle and withdraw the entire contents of the thawed vial and immediately transfer it to the prepared Balch tube of #1439 broth.  Mix well.
5. Plate 0.1 ml on a non-selective medium to check for aerobic and anaerobic contamination.
6. Incubate tubes and one plate under an anaerobic atmosphere at 26°C.  Incubate non-selective plate aerobically at 26°C to check for purity.  In two weeks time, there should be good growth in broth.  No growth should occur on the aerobic plates.

ANAEROBIC CONDITIONS:

1. 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.
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.

Cells are vibroid in shape.

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

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