Rumen fluid, clarified...........400.00 ml
K2HPO4 .............................0.45 g
KH2PO4 .............................0.45 g
NaCl...............................0.90 g
(NH4)2SO4...........................0.90 g
CaCl2 ..............................0.09 g
MgSO4 . 7H2O .......................0.18 g
NaHCO3 .............................6.37 g
Cellobiose.........................5.0 g
Casitone...........................5.0 g
Resazurin..........................1.0 mg
Agar, if needed...................15.0 g
Distilled water..................575.0 ml
Bring above mixture to boil under 98% CO2, 2% H2. Cool slightly and add the following freshly prepared, alkaline solution:
L-Cysteine . HCl...................0.5 g
Na2S . 9H2O ........................0.25 g
Distilled water...................25.00 ml
Dispense medium using anaerobic techniques and the same gas phase. Autoclave medium at 121C for 15 minutes. Adjust final pH to 6.8, +/- 0.2.