# 480a: DESULFURELLA MEDIUM (BRACKISH WATER) ## Main sol. 480a 10 g NaCl 0.33 g NH4Cl 0.33 g CaCl2 x 2 H2O 0.33 g MgCl2 x 6 H2O 0.33 g KCl 0.33 g KH2PO4 1 ml **Trace element solution SL-10** 0.1 g Yeast extract 5 g Na-acetate 0.5 ml Sodium resazurin (0.1% w/v) 10 g Sulfur (powder) 1 g Na2CO3 1 ml **Wolin's vitamin solution (10x)** 0.5 g Na2S x 9 H2O 1000 ml Distilled water 1. Dissolve ingredients (except sulfur, carbonate, vitamins and sulfide) and sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Adjust pH to 5.9, dispense medium under 80% N2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials containing already the appropriate amount of sulfur and sterilize medium by heating in a boiling water bath for 2- 3 hours on each of 3 successive days. Add vitamins (sterilized by filtration) and sulfide 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. Prior to use adjust pH of complete medium to 6.8 - 7.0, if necessary. ## 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. ## 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