# 558a: CLOSTRIDIUM (RB-9) MEDIUM ## Main sol. 558a 1.5 g KH2PO4 4.2 g Na2HPO4 x 12 H2O 0.5 g NH4Cl 0.36 g MgCl2 x 6 H2O 1 g Na2SO4 10 ml **Modified Wolin's mineral solution** 0.5 ml Sodium resazurin (0.1% w/v) 10 g D-Glucose 2 g Yeast extract 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 glucose, yeast extract, vitamin solution and reducing agents), adjust pH to 5 - 6 using HCl 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. After autoclaving add glucose, yeast extract, vitamin solution, cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas. The vitamin solution should be sterilized by filtration. Adjust pH of complete medium to 5.0 - 6.0, if necessary. ## Modified Wolin's mineral solution 1.5 g Nitrilotriacetic acid 3 g MgSO4 x 7 H2O 0.5 g MnSO4 x H2O 1 g NaCl 0.1 g FeSO4 x 7 H2O 0.18 g CoSO4 x 7 H2O 0.1 g CaCl2 x 2 H2O 0.18 g ZnSO4 x 7 H2O 0.01 g CuSO4 x 5 H2O 0.02 g AlK(SO4)2 x 12 H2O 0.01 g H3BO3 0.01 g Na2MoO4 x 2 H2O 0.03 g NiCl2 x 6 H2O 0.3 mg Na2SeO3 x 5 H2O 0.4 mg Na2WO4 x 2 H2O 1000 ml Distilled water 1. First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Adjust final to pH 7.0 with KOH. ## 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