# 711a: EUBACTERIUM AGGREGANS MEDIUM ## Main sol. 711a 1 g NH4Cl 0.3 g K2HPO4 0.3 g KH2PO4 0.2 g MgCl2 x 6 H2O 0.1 g CaCl2 x 2 H2O 0.1 g KCl 0.6 g NaCl 1 g Yeast extract 5 g Fructose 1.5 ml **Trace element solution SL-10** 0.5 ml Sodium resazurin (0.1% w/v) 1.5 g Na2CO3 10 ml **Wolin's vitamin solution** 1.19 g 3,4,5-Trimethoxycinnamic acid (TMC) 0.3 g L-Cysteine HCl x H2O 0.3 g Na2S x 9 H2O 1000 ml Distilled water 1. Dissolve ingredients (except carbonate, vitamins, TMC and reducing agents) 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 and autoclave. After autoclaving add vitamins, TMC, 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. The stock solution of TMC (6% w/v) should be neutralized with NaOH prior to sterilization by filtration. Adjust pH of complete medium to 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 2 mg Biotin 2 mg Folic acid 10 mg Pyridoxine hydrochloride 5 mg Thiamine HCl 5 mg Riboflavin 5 mg Nicotinic acid 5 mg Calcium D-(+)-pantothenate 0.1 mg Vitamin B12 5 mg p-Aminobenzoic acid 5 mg (DL)-alpha-Lipoic acid 1000 ml Distilled water