Desulfovibrio salexigens Postgate and Campbell

1. Open vial according to enclosed instructions.
2. Perform all steps under anaerobic conditions. (see below).  Exchange the gas in the head space for a fresh anaerobic gas, either 80% N₂ – 20% CO₂ or 100% N₂.  To insure that the media is anaerobic add 0.1 mL 1.5% sodium sulfide (stock concentration) for each 5 to 10 mL of medium. 
3. Using an anaerobic 1 mL syringe (see below) aseptically transfer 0.5 mL of ATCC Medium #1250 to the vial and rehydrate the entire freeze-dried pellet.  Transfer the entire suspension back into the primary tube.   The primary tube should not contain more than 8 to 10 mL of #1250 broth.  Secondary tube(s) can be inoculated with 0.5 mL of the primary broth.  Inoculate a plate of non-selective medium with 0.1 of the culture.
4. Incubate the tubes anaerobically at 37°C with gentle shaking.  Incubate the plate(s) aerobically as a purity check.
5. After two or three days, growth should be evident as indicated by turbidity throughout the broth.  Once growth has been established, the culture should be transferred to fresh broth every 24 to 48 hours.
6. This culture is sensitive to oxygen, therefore steps should be taken to avoid exposure to oxygen.  When the culture exhibits good growth it will remain viable for up to 1 week if stored at 4°C under anaerobic condition.

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. 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 the 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, titanium citrate and Co-enzyme M (see D). If component IV is added to the medium sodium sulfide, dithiothreitol and titanium citrate will cause the ferrous ammonium sulfate to precipitate even without growth. 
3. We suggest adding the reducing agent to the medium at least one hour before the medium is to be inoculated. 
4. 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. 

The cells typically appear as comma-shaped rods that are motile.

Once growth has been established the culture should be transferred every 24 hours when maintained at 37°C.  The culture can be maintained at 4°C for up to 1 week.

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

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