944 .
MW MEDIUM FOR METHANOSARCINA
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Equipment needed:
Anaerobic workbench
Autoclave
Filter
Final pH:
6.5
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
NaCl | 1.00 | g | ||
|
|
KCl | 0.50 | g | ||
|
|
MgCl2 x 6 H2O | 0.40 | g | ||
|
|
NH4Cl | 0.25 | g | ||
|
|
CaCl2 x 2 H2O | 0.15 | g | ||
|
|
Trace elements solution | 10.00 | ml | ||
|
|
Resazurin | 1.00 | mg | ||
|
|
Distilled water | 946.00 | ml | ||
| 1 Mix components thoroughly, and autoclave under a N2-CO2 (80:20, v/v) gas atmosphere. After cooling, aseptically and anaerobically add the following solutions (autoclaved or *filter-sterilized): | |||||
|
|
KH2PO4
(10%) |
2.00 | ml | ||
|
|
NaHCO3
(*, 8%) |
32.00 | ml | ||
|
|
Vitamin solution
(*) |
10.00 | ml | ||
| 2 Aseptically distribute the medium into culture vessels under a H2-CO2 (80:20, v/v) gas mixture and seal with butyl rubber stoppers (leave >80% gas phase by volume). Finally, add per liter the following solutions from anaerobic stocks (autoclaved and stored under a N2 atmosphere): | |||||
|
|
L-Cysteine HCl x H2O
(5%) |
6.00 | ml | ||
|
|
Na2S x 9 H2O
(5%) |
6.00 | ml | ||
| 3 Pressurize the inoculated vessels to 200 kPa H2-CO2 (80:20, v/v). | |||||
Trace elements solution #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Nitrilotriacetic acid | 1.50 | g | ||
|
|
Fe(NH4)2(SO4)2 x 6 H2O | 0.20 | g | ||
|
|
Na2SeO3 x 5 H2O | 0.20 | g | ||
|
|
MnCl2 x 4 H2O | 0.11 | g | ||
|
|
CoCl2 x 6 H2O | 0.10 | g | ||
|
|
Na2MoO4 x 2 H2O | 0.10 | g | ||
|
|
Na2WO4 x 2 H2O | 0.10 | g | ||
|
|
NiCl2 x 6 H2O | 0.25 | g | ||
|
|
ZnSO4 x 7 H2O | 0.20 | g | ||
|
|
H3BO3 | 0.01 | g | ||
|
|
CuSO4 x 5 H2O | 2.00 | mg | ||
|
|
Distilled water | 1000.00 | ml | ||
| 1 Dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH. Then proceed to add minerals. Adjust final pH to 7.0. | |||||
Vitamin solution #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
p-Aminobenzoic acid | 10 | mg |
|
|
Nicotinic acid | 10 | mg |
|
|
DL-Calcium pantothenate | 10 | mg |
|
|
Pyridoxine hydrochloride | 10 | mg |
|
|
Riboflavin | 10 | mg |
|
|
Thiamine HCl | 10 | mg |
|
|
Biotin | 5 | mg |
|
|
Folic acid | 5 | mg |
|
|
Lipoic acid | 5 | mg |
|
|
Vitamin B12 | 5 | mg |
|
|
Distilled water | 1000 | ml |