# 318c: PELOBACTER ACIDIGALLICI MEDIUM ## Main sol. 318c 0.3 g KH2PO4 0.6 g NaCl 0.1 g MgCl2 x 6 H2O 0.08 g CaCl2 x 2 H2O 10 ml **Trace element solution** 1 g NH4Cl 0.5 g Yeast extract (OXOID) 0.5 g Trypticase peptone (BD BBL) 0.5 ml Sodium resazurin (0.1% w/v) 2.5 g KHCO3 1.9 g Na-gallate 1 ml **Wolin's vitamin solution (10x)** 0.3 g L-Cysteine HCl x H2O 0.3 g Na2S x 9 H2O 1000 ml Distilled water 1. Dissolve ingredients except bicarbonate, vitamins, gallate, 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 and autoclave. Add vitamins, gallate, cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas and bicarbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Stock solutions of vitamins and gallate are sterilized by filtration. Prior to use adjust pH of complete medium to 7.3, if necessary. ## Trace element solution 12.8 g Nitrilotriacetic acid (NTA) 1 g FeCl2 x 4 H2O 0.1 g MnCl2 x 4 H2O 0.03 g CoCl2 x 6 H2O 0.1 g CaCl2 x 2 H2O 0.1 g ZnCl2 0.02 g CuCl2 0.01 g H3BO3 0.03 g Na2MoO4 x 2 H2O 0.1 g NiCl2 x 6 H2O 1 g NaCl 0.03 g Na2SeO3 x 5 H2O 0.04 g Na2WO4 x 2 H2O 1000 ml Distilled water 1. First dissolve NTA in 200 ml of distilled water and adjust pH to 6.5 with KOH, then dissolve mineral salts. Finally adjust pH to 6.5 with KOH and 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