# 557: CLOSTRIDIUM SP. MEDIUM (CREATININE) ## Main sol. 557 0.05 g CaSO4 x 2 H2O 0.5 g MgSO4 x 7 H2O 0.01 g FeSO4 x 7 H2O 0.06 g MnCl2 x 4 H2O 1 g NaCl 1 ml **Trace element solution SL-11** 1 ml **Selenite-tungstate solution** 5 g Yeast extract 0.5 ml Sodium resazurin (0.1% w/v) 2.64 g KH2PO4 5.33 g K2HPO4 1 g NaHCO3 5.5 g Creatinine 1 ml **Wolin's vitamin solution (10x)** 0.5 g L-Cysteine HCl x H2O 0.5 g Na2S x 9 H2O 1000 ml Distilled water 1. Dissolve ingredients except hydrogenphosphates, bicarbonate, creatinine, vitamins, cysteine and sulfide, adjust pH to 7.0 and sparge medium with 100% N2 gas for 30 - 45 min to make it anoxic. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Complete medium by adding hydrogenphosphates, creatinine, vitamins, cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas atmosphere and bicarbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas atmosphere. Stock solutions of creatinine (5% w/v) and vitamins should be sterilized by filtration. Adjust pH of complete medium to 7.0, if necessary. ## Trace element solution SL-11 5.2 g Na2-EDTA x 2 H2O 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 1000 ml Distilled water 1. Dissolve EDTA in 800 ml distilled water, adjust pH to 7 using 2 N NaOH and add ferrous chloride. After ferrous chloride has dissolved add other compounds. Finally adjust pH to 6.0 and bring volume to 1000 ml. ## Selenite-tungstate solution 0.5 g NaOH 3 mg Na2SeO3 x 5 H2O 4 mg Na2WO4 x 2 H2O 1000 ml Distilled water ## Wolin's vitamin solution (10x) 20 mg Biotin 20 mg Folic acid 100 mg Pyridoxine hydrochloride 50 mg Thiamine HCl 50 mg Riboflavin 50 mg Nicotinic acid 50 mg Calcium D-(+)-pantothenate 1 mg Vitamin B12 50 mg p-Aminobenzoic acid 50 mg (DL)-alpha-Lipoic acid 1000 ml Distilled water