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Medium No.	812
Medium	Rhodomicrobium Medium
Composition
Yeast extract 0.2 g   Disodium succinate 1 g   Ammonium acetate 0.5 g   Ferric citrate solution* (0.1%) 5 ml   KH2PO4 0.5 g   MgSO4·7H2O 0.4 g   NaCl 0.4 g   NH4Cl 0.4 g   CaCl2·2H2O 0.05 g   Vitamin B12 solution (0.01%) 0.4 ml   Trace element solution SL-6** 1 ml   Neutralized sulfide solution*** 5 ml   Distilled water 1 L   pH 5.7       Mix ingredients except vitamin B12 solution and neutralized sulfide solution, and adjust pH to 5.7 with 6 N H2SO4. Dispense the medium into suitable culture vessels (e.g. 20 ml of the medium in 120 ml serum bottles) under a stream of nitrogen gas, seal with butyl rubber stoppers, and autoclave at 121°C for 15 min. Separately autoclave neutralized sulfide solution under a N2 atmosphere. Sterilize vitamin B12 by filtration. Sterile syringes are used to inoculate and remove samples. Prior to inoculation, aseptically and anaerobically add vitamin B12 solution and neutralized sulfide solution.                   *Soluble in hot water.                   **Trace element solution SL-6       ZnSO4·7H2O 0.1 g       MnCl2·4H2O 0.03 g       H3BO3 0.3 g       CoCl2·6H2O 0.2 g       CuCl2·2H2O 0.01 g       NiCl2·6H2O 0.02 g       Na2MoO4·2H2O 0.03 g       Distilled water 1 L                   ***Neutralized sulfide solution       Na2S·9H2O 1.5 g       Distilled water 100 ml     The sulfide solution is prepared in a vessel sealed with a butyl rubber stopper. Autoclave at 121℃ for 15 min under a N2 atmosphere. After cooling to room temperature, the pH is adjusted to about 7.3 by adding of sterile 2 N H2SO4 drop-wise with a syringe without opening the bottle. Appearance of yellow colour indicates the pH is about 8. The vessel should be shaken continuously to avoid precipitation of elemental sulfur. The final solution should be transparent yellow.
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