# 1277: SULFURICELLA MEDIUM ## Main sol. 1277 0.25 g NaCl 0.2 g MgCl2 x 6 H2O 0.1 g CaCl2 x 2 H2O 1.7 g NaNO3 0.1 g (NH4)2SO4 0.1 g KH2PO4 0.1 g KCl 1 ml **Trace element solution SL-10** 1 ml **Selenite-tungstate solution** 1.5 g Na2CO3 2.5 g Na2S2O3 x 5 H2O 1 ml **Wolin's vitamin solution (10x)** 1000 ml Distilled water 1. Dissolve ingredients (except carbonate, thiosulfate 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 to 30% volume and autoclave. Add thiosulfate and vitamins from sterile anoxic stock solutions prepared under 100% N2 gas and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Stock solutions of thiosulfate and vitamins should be sterilized by filtration. Adjust pH of complete medium to 7.5, if necessary. 2. After inoculation add sterile air in an amount equivalent to 1% (v/v) of the total volume of the culture vessel. ## 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