# 330: RUMEN BACTERIA MEDIUM ## Main sol. 330 38 ml **Mineral solution** 0.3 g K2HPO4 2 g Trypticase peptone (BD BBL) 0.5 g Yeast extract (OXOID) 3.1 ml **Volatile fatty acid mixture** 2 ml **Haemin solution** (0.05% w/v) 0.5 g Glycerol 0.5 ml Sodium resazurin (0.1% w/v) 4 g Na2CO3 0.5 g D-Glucose 0.5 g Maltose 0.5 g Cellobiose 0.5 g Starch, soluble 0.25 g L-Cysteine HCl x H2O 0.25 g Na2S x 9 H2O 960 ml Distilled water 1. Dissolve ingredients (except carbonate, glucose, maltose, cellobiose, soluble starch, cysteine and sulfide), then sparge medium with 100% CO2 gas for 30 - 45 min to make it anoxic. Add the carbonate and equilibrate the medium with the CO2 gas to pH 6.8. Distribute under 100% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Thereafter, add glucose, maltose, cellobiose, soluble starch, cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas atmosphere. Adjust pH of complete medium to 6.7 - 6.8, if necessary. ## Mineral solution 6 g KH2PO4 12 g NaCl 6 g (NH4)2SO4 1.6 g CaCl2 x 2 H2O 2.5 g MgSO4 x 7 H2O 1000 ml Distilled water ## Volatile fatty acid mixture 548.5 ml Acetic acid 193.5 ml Propionic acid 129 ml Butyric acid 32.25 ml n-Valeric acid 32.25 ml iso-Butyric acid 32.25 ml DL-2-Methylbutyric acid 32.25 ml iso-Valeric acid ## Haemin solution 50 mg Haemin 1 ml NaOH (1 N) 100 ml Distilled water 1. Dissolve 50 mg haemin in 1 ml 1 N NaOH; make up to 100 ml with distilled water and filter sterilize. Store refrigerated.