Rumin fluid (see below) 30.0 ml
Glucose 0.05 g
Maltose 0.05 g
Soluble Starch 0.05 g
Peptone 0.05 g
Yeast Extract 0.05 g
Cellobiose 0.1 g
(NH4)2SO4 0.04 g
Resazurin Soln. (see below) 1.0 mg
Salts Solution (see below) 50.0 ml
L-Cystein x H2O 0.05 g
DI Water 20.0 ml
Rumin Fluid:
Filter rumen contents, obtained from a cow fed on an alfalfa-hay concentrate ration, through two layers of cheesecloth to remove large particles. Store under CO2 in quart milk bottles in the refrigerator. Much of the particulate matter settles.
Use only the supernatant. Resazurin Solution: Dissolve one resazurin tablet in 44 ml of distilled water.
Salts Solution:
CaCl2 (anhydrous) 0.2 g
MgSO4 0.2 g
K2HPO4 1.0 g
KH2PO4 1.0 g
NaHCO3 10.0 g
NaCl 2.0 g
Mix CaCl2 and MgSO4 in 300 ml
of distilled water until dissolved. Add 500 ml water and add the remaining salts while swirling slowly.
Add 200 ml of distilled water, mix and store at 4 C.
Mix all of the ingredients in an Erlenmyer flask.
Flask should have a small heat space to minimize air volume that must be purged during cooling.
Fit a removable chimney to the boiling flask to prevent media from boiling over.
Boil (10-20 minutes) until medium changes from pink to yellowish.
Cool in ice water bath under O2-Free CO2. The flow of CO2 should cause gentle bubbling (sufficient to exclude air).
Remove from ice bathe and add 0.05 g L-cysteine x H2O. Adjust pH to 7.0 with 8N NaOH or 5N HCl.
Stopper with #1 butyl rubber stoppers (or black rubber stoppers).
The nitrogen prevents a pH change during storage.
Place the rack of tubes in a press and sterilize for 12-15 minutes at 121 C.