Medium data

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230   METHANOSARCIA MEDIUM

KH2PO4  0.227   g
K2HPO4  0.348   g
NH4Cl  0.5     g
MgSO4·7H2O  0.5     g
CaCl2·2H2O  0.25    g
NaCl  2.25    g
FeSO4·7H2O  2.0     mg
Yeast extract (Difco)  2.0     g
Casitone (Difco)  2.0     g
Trace vitamins (see Medium No. 197)  10.0     ml
FeCl2 solution (see below)  1.0     ml
Trace element solution (see below)  1.0     ml
NaHCO3  0.85    g
Methanol  10.0     ml
L-Cysteine·HCl·H2O  0.3     g
Na2S·9H2O  0.3     g
Resazurin  1.0     mg
Distilled water  980.0     ml
Mix ingredients, except NaHCO3, methanol, cysteine·HCl and Na2S·9H2O, and bring to a boil for 5 - 10 sec. Cool down under a stream of N2-CO2 (80:20), and add NaHCO3 and methanol to the medium. Dispense the medium into suitable culture vessels (e.g., 20 ml of the medium in 120 ml serum bottles) under a stream of N2-CO2 (80:20) and seal with butyl rubber stoppers. After autoclaving, stand the medium overnight. Separately autoclave cysteine·HCl and Na2S·9H2O as 5% solutions, respectively, under a N2 atmosphere. Prior to inoculation, aseptically and anaerobically add the cysteine·HCl and Na2S·9H2O solutions.
FeCl2 solution:
25% HCl (7.7 M)  10.0     ml
FeCl2·4H2O  1.5     g
Distilled water  990.0     ml
Trace element solution:
ZnCl2  70.0     mg
MnCl2·4H2O  100.0     mg
H3BO3  6.0     mg
CoCl2·6H2O  190.0     mg
CuCl2·2H2O  2.0     mg
NiCl2·6H2O  24.0     mg
Na2MoO4·2H2O  36.0     mg
Distilled water  1.0     L