ATCC medium: 983 E medium for anaerobes with 0.2% rutin
ATCC Medium 982 (see below) with 1% phloroglucinol replaced by 0.2% rutin.
ATCC Medium 982:....................... 30.0 ml
Rumen Fluid (see below) (NH4)2SO4....................................... 0.5 g
Resazurin Solution (see below)................. 0.4 ml
Salt Solution (see below)..................... 50.0 ml
L-Cysteine . HCl............................... 0.05 g
Sterile 1% phloroglucinol solution............ 30.0 ml
Mix all the ingredients in an Erlenmeyer flask. Flask should have a small head space to minimize air volume that must be purged during cooling. Fit a removable chimney to the boiling flask to prevent medium from boiling over. Boil (10-20 min) until medium changes from pink to yellow. Replace chimney with stopper fitted with glass tubing which bubbles O2-free gas, composed of N 2 and H2 through the medium to maintain in reduced state (97% N2, 3% H2 is employed at the ATCC) and place in an ice bath medium into tubes under O2-free N2 and H2 . Stopper with butyl rubber stoppers. Place rack of tubes in press so that stoppers do not dislodge, and autoclave at 121C for 12-15 minutes on fast exhaust. Rumen Fluid: Filter rumen contents, obtained from a cow fed an alfalfa-hay concentrated ration, through two layers of cheesecloth to remove large particles. Store bottles in the freezer. Much of the particulate matter settles.
Use only the supernatant.
Resazurin Solution:
Resazurin................... 25.0 mg
Distilled water............ 100.0 ml
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
Dissolve CaCl2 and MgSO4 in 300 ml of distilled water. Add 500 ml water and add the remaining salts while swirling slowly. Add 200 ml of distilled water, mix, and store at 4C.
Phloroglucinol: Filter-sterilize freshly prepared phloroglucinol solution. Wrap glassware in aluminum foil to exclude light.