# 1570: THIOGRANUM MEDIUM ## Main sol. 1570 30 g NaCl 0.15 g CaCl2 x 2 H2O 3.05 g MgCl2 x 6 H2O 0.33 g KCl 0.66 g (NH4)2SO4 10 ml **Modified Wolin's mineral solution** 5 ml **Phosphate buffer** 1.5 g Na2S2O3 x 5 H2O 1 g Na2CO3 1 ml **Wolin's vitamin solution (10x)** 1000 ml Distilled water 1. Dissolve ingredients (except phosphate buffer, thiosulfate, carbonate and vitamins) and sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials up to a volume of 20% and autoclave. Add phosphate buffer, thiosulfate, carbonate, and vitamin solution to the autoclaved medium from sterile anoxic stock solutions prepared under 100% N2 gas. Phosphate buffer, stock solutions of vitamins and thiosulfate are sterilized by filtration and stored under N2, whereas the solution of carbonate is prepared under 80% N2 and 20% CO2 gas mixture and autoclaved. Adjust pH of the complete medium to 6.7, if necessary. 2. After inoculation pressurize vessels to 0.5 bar overpressure with sterile 80% N2 and 20% CO2 gas mixture and add sterile air in an amount that is equivalent to a volume of 20% of the headspace. ## Phosphate buffer 119 g KH2PO4 21 g K2HPO4 1000 ml Distilled water ## 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