# 335: EUBACTERIUM OXIDOREDUCENS MEDIUM ## Main sol. 335 892 ml **Solution A** 100 ml **Solution B** 10 ml **Solution C** 1 ml **Solution D** 1. Prepare solution A under 80% N2 and 20% CO2 gas mixture, distribute 8.9 ml per Hungate-type tube under same gas atmosphere and autoclave. Filter sterilize NaHCO3 solution and gas for 20 min with 80% N2 and 20% CO2 gas mixture. Autoclave cysteine and sulfide under 100% N2 gas. Complete medium by adding the solutions B, C, and D with a syringe to solution A. Adjust final pH of medium to 7.0 - 7.5. ## Solution A 50 ml **Mineral solution** 1 ml **Trace element solution SL-10** 2 g Yeast extract 5 g Crotonic acid 1 ml **Seven vitamins solution** 1 mg Resazurin 840 ml Distilled water 1. Adjust pH to 6.9 before autoclaving. ## Solution B 5 g NaHCO3 100 ml Distilled water ## Solution C 0.24 g L-Cysteine HCl x H2O 10 ml Distilled water ## Solution D 78 mg Na2S x 9 H2O 1 ml Distilled water ## Mineral solution 10 g KH2PO4 6.6 g MgCl2 x 6 H2O 8 g NaCl 8 g NH4Cl 1 g CaCl2 x 2 H2O 1000 ml Distilled water ## Trace element solution SL-10 10 ml HCl (25%) 1.5 g FeCl2 x 4 H2O 70 mg ZnCl2 100 mg MnCl2 x 4 H2O 6 mg H3BO3 190 mg CoCl2 x 6 H2O 2 mg CuCl2 x 2 H2O 24 mg NiCl2 x 6 H2O 36 mg Na2MoO4 x 2 H2O 990 ml Distilled water 1. First dissolve FeCl2 in the HCl, then dilute in water, add and dissolve the other salts. Finally make up to 1000.00 ml. ## Seven vitamins solution 100 mg Vitamin B12 80 mg p-Aminobenzoic acid 20 mg D-(+)-biotin 200 mg Nicotinic acid 100 mg Calcium pantothenate 300 mg Pyridoxine hydrochloride 200 mg Thiamine-HCl x 2 H2O 1000 ml Distilled water