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Main sol. 161

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Internal Solution-ID: S277

Equipment needed: Hungate tubes Autoclave Filter Gassing station Centrifuge
Compound Amount Unit Conc. [g/L] Conc. [mM]
Clarified rumen fluid 300.00 ml - -
K2HPO4 0.30 g 0.3 1.721
KH2PO4 0.30 g 0.3 2.202
(NH4)2SO4 0.30 g 0.3 2.268
NaCl 0.60 g 0.599 10.257
MgSO4 x 7 H2O 0.13 g 0.13 0.527
CaCl2 x 2 H2O
(0.1% w/v) 		8.00 ml 0.008 0.054
Modified Wolin's mineral solution 10.00 ml - -
FeSO4 x 7 H2O
(0.1% w/v in 0.1 N H2SO4) 		2.00 ml 0.002 0.007
Yeast extract
(DIFCO) 		1.00 g 0.999 -
Trypticase
(BBL) 		1.00 g 0.999 -
Fatty acid mixture 20.00 ml - -
Sodium resazurin
(0.1% w/v) 		0.50 ml 5.0e-4 0.002
NaHCO3 2.00 g 1.998 23.784
Wolin's vitamin solution (10x) 1.00 ml - -
L-Cysteine HCl x H2O 0.50 g 0.5 2.844
Na2S x 9 H2O 0.50 g 0.5 2.08
Distilled water 660.00 ml - -
1 Dissolve ingredients (except bicarbonate, vitamins, cysteine and sulfide) and sparge medium for 30 - 45 min with 80% H2 and 20% CO2 gas mixture to make it anoxic. Add and dissolve bicarbonate and adjust pH to 6.8, then distribute under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. After sterilization add cysteine and sulfide from sterile anoxic stock solutions autoclaved under 100% N2 gas atmosphere. Vitamins are prepared under 100% N2 gas atmosphere and sterilized by filtration. Adjust pH of complete medium to 6.5. For incubation use sterile 80% H2 and 20% CO2 gas mixture at two atmospheres of pressure.
161 METHANOFOLLIS MEDIUM