Medium data

Search for medium no. = [355].
Unless otherwise stated, sterilize media by autoclaving at 121°C for 15 min.

355   METHANOSPHAERA MEDIUM

Rumen fluid, clarified (see below)  100.0     ml
Yeast extract (BD-Difco)  2.0     g
Trypticase peptone (BD-BBL)  2.0     g
Sodium acetate  0.5     g
Sodium formate  0.5     g
Trace minerals (see Medium No. 151)  10.0     ml
Trace vitamins (see Medium No. 197)  1.0     ml
Na2SeO4  0.19    mg
NiCl2·6H2O  0.7     mg
FeSO4·7H2O  3.0     mg
KH2PO4  2.8     g
K2HPO4  0.6     g
(NH4)2SO4  0.3     g
NH4Cl  1.0     g
NaCl  0.6     g
MgSO4·7H2O  0.15    g
CaCl2·2H2O  0.076   g
Methanol  5.0     ml
Resazurin  1.0     mg
L-Cysteine·HCl·H2O  0.875   g
Na2S·9H2O  0.375   g
Distilled water  900.0     ml
Mix ingredients, except methanol, L-cysteine·HCl·H2O and Na2S·9H2O, and adjust pH to 7.0. Bring to a boil for 5-10 sec, then, cool down under a stream of N2 and add methanol to the medium. Dispense the medium into suitable culture vessels (e.g., 20 ml of the medium in a 100-ml serum bottle) under a stream of N2 and seal. After autoclaving, stand the medium overnight. Separately autoclave L-cysteine·HCl·H2O and Na2S·9H2O as 5% solutions, respectively, under a N2 atmosphere. Prior to inoculation, replace the gas phase with H2-CO2 (80:20, v/v), and add the L-cysteine·HCl·H2O and Na2S·9H2O solutions aseptically and anaerobically. 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.