1094 .
METHANOSPHAERULA (PEAT) MEDIUM
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Equipment needed:
Autoclave
Filter
Gassing station
Hungate tubes
Final pH:
5.7
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Solution A | 943 | ml | ||
|
|
Solution B | 13 | ml | ||
|
|
Solution C | 20 | ml | ||
|
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Solution D | 1 | ml | ||
|
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Solution E | 10 | ml | ||
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Solution F | 10 | ml | ||
|
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Solution G | 10 | ml | ||
| 1 Sparge solution A with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make anoxic. Dispense under same gas atmosphere into anoxic Hungate-type tubes or serum vials to 30% of their volume and autoclave. Solutions B, C, D and G are prepared under 100% N2 gas atmosphere and sterilized by filtration. Solutions E and F are prepared under 100% N2 gas atmosphere and autoclaved. To complete the medium add appropriate amounts of solutions B to G to the sterile solution A in the sequence indicated and let medium equilibrate overnight. Adjust pH of complete medium to 5.7, if necessary. | |||||
| 2 After inoculation pressurize vials to 1 bar overpressure with sterile 80% H2 and 20% CO2 gas mixture. | |||||
| 3 Note: The complete medium is not stable and can be stored only for a short time. | |||||
Solution A #
943
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
KCl
(0.1% w/v) |
1.5 | ml |
|
|
KH2PO4
(0.1% w/v) |
13.6 | ml |
|
|
NH4Cl
(0.1% w/v) |
26.8 | ml |
|
|
Trace element solution | 1.0 | ml |
|
|
Distilled water | 900.0 | ml |
Solution B #
13
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
Tris-HCl buffer
(1.0 M solution, pH 8.0) |
7.20 | ml |
|
|
Na3-NTA x H2O
(0.5 M solution) |
4.80 | ml |
|
|
TiCl3
(15% w/v solution in HCl; Riedel-de Haen) |
0.55 | ml |
Solution C #
20
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
2-(N-Morpholino)ethanesulfonic acid
(MES) |
1.95 | g | ||
|
|
NaOH | 0.20 | g | ||
|
|
Distilled water | 20.00 | ml | ||
| 1 Adjust to pH 7.5 with NaOH. | |||||
Solution E #
10
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
2-Mercaptoethanesulfonic acid
(Coenzyme M) |
0.08 | g |
|
|
Distilled water | 10.00 | ml |
Trace element solution #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Na2-EDTA | 37.23 | g | ||
|
|
FeCl2 x 4 H2O | 1.34 | g | ||
|
|
AlK(SO4)2 x 12 H2O | 3.45 | g | ||
|
|
CaCl2 x 2 H2O | 2.34 | g | ||
|
|
MgSO4 x 7 H2O | 1.56 | g | ||
|
|
CoCl2 x 6 H2O | 24.00 | mg | ||
|
|
ZnCl2 | 75.00 | mg | ||
|
|
H3BO3 | 19.00 | mg | ||
|
|
NiCl2 x 6 H2O | 24.00 | mg | ||
|
|
Na2Mo4 x 2 H2O | 24.00 | mg | ||
|
|
MnSO4 x 4 H2O | 26.00 | mg | ||
|
|
CuSO4 x 5 H2O | 9.00 | mg | ||
|
|
Distilled water | 1000.00 | ml | ||
| 1 Dissolve EDTA in distilled water, adjust pH to 7 using 2 N NaOH and add ferrous chloride. After ferrous chloride has dissolved add other compounds. | |||||
Wolin's vitamin solution (10x) #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
Biotin | 20 | mg |
|
|
Folic acid | 20 | mg |
|
|
Pyridoxine hydrochloride | 100 | mg |
|
|
Thiamine HCl | 50 | mg |
|
|
Riboflavin | 50 | mg |
|
|
Nicotinic acid | 50 | mg |
|
|
Calcium D-(+)-pantothenate | 50 | mg |
|
|
Vitamin B12 | 1 | mg |
|
|
p-Aminobenzoic acid | 50 | mg |
|
|
(DL)-alpha-Lipoic acid | 50 | mg |
|
|
Distilled water | 1000 | ml |