# 311a: SPOROMUSA MEDIUM (H2/CO2) ## Main sol. 311a 0.5 g NH4Cl 0.5 g MgSO4 x 7 H2O 0.25 g CaCl2 x 2 H2O 2.25 g NaCl 2 ml FeSO4 x 7 H2O (0.1% w/v in 0.1 N H2SO4) 1 ml **Trace element solution SL-10** 1 ml **Selenite-tungstate solution** 2 g Yeast extract 2 g Casitone 0.5 ml Sodium resazurin (0.1% w/v) 0.35 g K2HPO4 0.23 g KH2PO4 1 g Na2CO3 1 ml **Wolin's vitamin solution (10x)** 0.15 g DL-Dithiothreitol 1000 ml Distilled water 1. Dissolve ingredients (except phosphates, carbonate, vitamins, and dithiothreitol) and sparge medium with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense under the same gas atmosphere in anoxic Hungate-type tubes or serum vials to 30% of their volume and autoclave. Add phosphates to the medium after autoclaving from sterile stock solutions prepared under 100% N2 gas and carbonate from a sterile stock solution prepared under 80% N2 and 20% CO2 gas mixture. Vitamins and dithiothreitol should be prepared under N2 gas and sterilized by filtration. Adjust the pH of the completed medium to pH 7.0. ## 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. ## 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