# 846a: AMINOBACTERIUM MEDIUM ## Main sol. 846a 0.3 g NH4Cl 0.2 g K2HPO4 0.3 g KH2PO4 0.15 g CaCl2 x 2 H2O 0.5 g KCl 20 g NaCl 1 ml **Trace element solution SL-10** 1 ml **Selenite-tungstate solution** 2 g Yeast extract 0.5 ml Sodium resazurin (0.1% w/v) 3 g MgCl2 x 6 H2O 1.5 g Na2CO3 1.05 g L-Serine 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 magnesium chloride, carbonate, serine, vitamins, cysteine, and sulfide), then sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense medium under the same gas atmosphere into anoxic Hungate-type tubes or serum vials to 30% of their volume and autoclave. Add magnesium chloride, serine, vitamins, cysteine, 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 atmosphere. The vitamin solution should be sterilized by filtration. Adjust the pH of the complete medium to 7.2, 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. ## 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