Brain heart infusion 9.260 g Trypticase soy broth 7.500 g Yeast extract 2.500 g NaNO3 0.426 g K2HPO4 1.250 g 1. Adjust pH to 7.0. 2. Bring to boil, cool down under N2CO2. Cysteine HCl 0.250 g 3. Fill under N2CO2 in hungate tubes (10 ml) 4. Autoclave at 121 °C for 15 min. Haemin solution 0.050 ml Vitamin K3 solution 0.050 ml Fetal bovine serum 0.500 ml 5. pressurize with 1 bar H2 Haemin solution (from medium 78) Haemin 50.00 mg NaOH (1 N) 1.00 ml Distilled water 100.00 ml Dissolve 50 mg haemin in 1 ml 1 N NaOH; make up to 100 ml with distilled water and filter sterilize. Store refrigerated. Vitamin K3 solution Vitamin K3 5.00 mg Ethanol (95%) 1.00 ml Water 100.00 ml Dissolve 5 mg vitamin K3 in 1 ml 95% ethanol, dilute to 0.05 mg/ml in water and filter sterilize. Store refrigerated in a brown bottle.
