ATCC medium: 1471 Sludge medium for Methanobacteria
KH2PO4 .........................................0.5 g
MgSO4 . 7H2O ...................................0.4 g
NaCl...........................................0.4 g
NH4Cl ..........................................0.4 g
CaCl2 . 2H2O ...................................0.05 g
FeSO4 . 7H2O ...................................2.0 mg
Trace Elements Solution SL-6 (see below).......1.0 ml
Yeast extract..................................1.0 g
Sodium acetate.................................1.0 g
Sodium formate.................................2.0 g
Sludge Fluid (see below)......................50.0 ml
NaHCO3 .........................................4.0 g
Fatty Acid Mixture (see below)................20.0 ml
Resazurin......................................1.0 mg
L-Cysteine HCl.................................0.5 g
Na2S . 9H2O ....................................0.5 g
Distilled water..............................940.0 ml
Adjust pH to 6.7-7.0. Prepare and dispense medium anaerobically under
80% N2, 20% CO2 into aluminum seal-type culture tubes. Autoclave at 121C
for 15 minutes under fast exhaust.
Trace Elements Solution SL-6:
ZnSO4 . 7H2O .......................0.10 g
MnCl2 . 4H2O .......................0.03 g
H3BO3 ..............................0.3 g
CoCl2 . 6H2O .......................0.2 g
CuCl2 . 2H2O .......................0.01 g
NiCl2 . 6H2O .......................0.02 g
Na2MoO4 . 2H2O......................0.03 g
Distilled water....................1.0 L
Adjust final pH of Trace Elements Solution SL-6 to 3.4.
Sludge Fluid:
To sludge from an anaerobic digester, add yeast extract to a
concentration of 0.4% and after gassing with nitrogen gas for a few
minutes incubate at 37C for 24 hours. Centrifuge at 13000 g and remove
the supernate. Autoclave the supernate in screw-capped bottles under
nitrogen gas. The sludge fluid can be stored at room temperature in the
dark.
Fatty Acid Mixture:
Valeric acid.................0.5 g
Isovaleric acid..............0.5 g
alpha-Methylbutyric acid.....0.5 g
Isobutyric acid..............0.5 g
Distilled water.............20.0 ml
Adjust pH to 7.5 with concentrated NaOH.