# 1526b: KIRITIMATIELLA (TYG) MEDIUM ## Main sol. 1526b 60 g Sea Salt (SIGMA) 1 ml **Wolfe's mineral elixir** 1 g Trypticase peptone (BD BBL) 1 g Yeast extract (OXOID) 1 g Glucose 0.5 ml Sodium resazurin (0.1% w/v) 1 g Na2CO3 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 carbonate, 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 and autoclave. Add glucose, vitamins, cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas (vitamins are sterilized by filtration) 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 7.3 - 7.5, if necessary. ## Wolfe's mineral elixir 30 g MgSO4 x 7 H2O 5 g MnSO4 x H2O 10 g NaCl 1 g FeSO4 x 7 H2O 1.8 g CoCl2 x 6 H2O 1 g CaCl2 x 2 H2O 1.8 g ZnSO4 x 7 H2O 0.1 g CuSO4 x 5 H2O 0.18 g AlK(SO4)2 x 12 H2O 0.1 g H3BO3 0.1 g Na2MoO4 x 2 H2O 2.8 g (NH4)2Ni(SO4)2 x 6 H2O 0.1 g Na2WO4 x 2 H2O 0.1 g Na2SeO4 1000 ml Distilled water 1. First adjust pH to 1.0 with diluted H2SO4, then add and dissolve the salts. ## 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