355 .
METHANOSPHAERA MEDIUM
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Equipment needed:
Anaerobic workbench
Autoclave
Final pH:
7.0
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Rumen fluid
, clarified
(see below) |
100.000 | ml | ||
|
|
Yeast extract
(BD-Difco) |
2.000 | g | ||
|
|
Trypticase peptone
(BD-BBL) |
2.000 | g | ||
|
|
Sodium acetate | 0.500 | g | ||
|
|
Sodium formate | 0.500 | g | ||
|
|
Trace minerals | 10.000 | ml | ||
|
|
Trace vitamins | 1.000 | ml | ||
|
|
Na2SeO4 | 0.190 | mg | ||
|
|
NiCl2 x 6 H2O | 0.700 | mg | ||
|
|
FeSO4 x 7 H2O | 3.000 | mg | ||
|
|
KH2PO4 | 2.800 | g | ||
|
|
K2HPO4 | 0.600 | g | ||
|
|
(NH4)2SO4 | 0.300 | g | ||
|
|
NH4Cl | 1.000 | g | ||
|
|
NaCl | 0.600 | g | ||
|
|
MgSO4 x 7 H2O | 0.150 | g | ||
|
|
CaCl2 x 2 H2O | 0.076 | g | ||
|
|
Methanol | 5.000 | ml | ||
|
|
Resazurin | 1.000 | mg | ||
|
|
L-Cysteine HCl x H2O | 0.875 | g | ||
|
|
Na2S x 9 H2O | 0.375 | g | ||
|
|
Distilled water | 900.000 | ml | ||
| 1 Mix ingredients, except methanol, L-cysteine x HCl x H2O and Na2S x 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 x HCl x H2O and Na2S x 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 x HCl x H2O and Na2S x 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. | |||||
Trace minerals #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Nitrilotriacetic acid | 1.50 | g | ||
|
|
MgSO4 x 7 H2O | 3.00 | g | ||
|
|
MnSO4 x n H2O | 0.50 | g | ||
|
|
NaCl | 1.00 | g | ||
|
|
FeSO4 x 7 H2O | 0.10 | g | ||
|
|
CoSO4 x 7 H2O | 0.10 | g | ||
|
|
CaCl2 x 2 H2O | 0.10 | g | ||
|
|
ZnSO4 x 7 H2O | 0.10 | g | ||
|
|
CuSO4 x 5 H2O | 0.01 | g | ||
|
|
AlK(SO4)2 | 0.01 | g | ||
|
|
H3BO3 | 0.01 | g | ||
|
|
Na2MoO4 x 2 H2O | 0.01 | g | ||
|
|
Distilled water | 1000.00 | ml | ||
| 1 Dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH solution. Then proceed to add minerals. Adjust final pH to 7.0. | |||||
Trace vitamins #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
Biotin | 2.0 | mg |
|
|
Folic acid | 2.0 | mg |
|
|
Pyridoxine hydrochloride | 10.0 | mg |
|
|
Thiamine HCl | 5.0 | mg |
|
|
Riboflavin | 5.0 | mg |
|
|
Nicotinic acid | 5.0 | mg |
|
|
Calcium pantothenate | 5.0 | mg |
|
|
Vitamin B12 | 0.1 | mg |
|
|
p-Aminobenzoic acid | 5.0 | mg |
|
|
Lipoic acid | 5.0 | mg |
|
|
Distilled water | 1000.0 | ml |