Desulfobacula phenolica (Bak and Widdel) Kuever 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. If the medium is pink (see discussion about resazurin) add 2.0 mL of reducing agent (1.5% sodium sulfide, stock solution) per 100 mL of medium.  Let the medium sit at room temperature for 10 to 20 minutes until the resazurin becomes colorless 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 a 1.0 mL syringe tipped with 22-gauge needle, withdraw the entire cell suspension from the vial and transfer it to the broth. Using the same needle withdraw 0.5 mL from the broth and inoculate a second Balch tube.  Plate 0.1 mL of the inoculated culture onto a non-selective medium and incubate aerobically at 30^oC. 
6. Growth should be detected in the #1628 broth within 2 to 4 days.  There should be no growth detected on the aerobic plate or broth.

NOTE: Addition of 10-20 mg sodium dithionite per liter (e.g. from 5% w/v solution freshly prepared under N₂ and filter-sterilized) may stimulate growth of all strains at the beginning.  For transfers use 5-10% inoculum.  Incubate all strains in the dark.    

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. 

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

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