Medium data

Search for medium no. = [266].
Unless otherwise stated, sterilize media by autoclaving at 121°C for 15 min.

266   METHANOMICROBIUM MOBILE MEDIUM

Rumen fluid, clarified (see below)  300.0     ml
KH2PO4  0.3     g
K2HPO4  0.3     g
(NH4)2SO4  0.3     g
NaCl  0.6     g
MgSO4·7H2O  0.13    g
CaCl2·2H2O  8.0     mg
Trace minerals (see Medium No. 151)  10.0     ml
Trace vitamins (see Medium No. 197)  10.0     ml
FeSO4·7H2O  2.0     mg
NaHCO3  2.0     g
Yeast extract (BD-Difco)  1.0     g
Trypticase peptone (BD-BBL)  1.0     g
Fatty acid mixture (see below)  20.0     ml
Resazurin  1.0     mg
Na2S·9H2O  0.5     g
L-Cysteine·HCl·H2O  0.5     g
Distilled water  660.0     ml
Mix ingredients, except NaHCO3, L-cysteine·HCl·H2O and Na2S·9H2O, and adjust pH to 6.5. Bring to a boil for 5-10 sec, then, cool down under a stream of H2-CO2 (80:20, v/v) and add NaHCO3 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 H2-CO2 (80:20, v/v) 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 L-cysteine·HCl·H2O and Na2S·9H2O solutions. Pressurize the inoculated bottles to 200 kPa H2-CO2 (80:20, v/v).
To prepare the clarified rumen fluid, preheat rumen content at 120C for 15 min. Use the supernatant after centrifuging at 25,000 x g for 15 min.
Fatty acid mixture:
Valeric acid  0.5     g
Isovaleric acid  0.5     g
α-Methylbutyric acid  0.5     g
Isobutyric acid  0.5     g
Distilled water  20.0     ml
Adjust pH to 7.5 with concentrated NaOH solution.