1280 .
METHANOREGULA (PEAT) MEDIUM
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Equipment needed:
Autoclave
Filter
Gassing station
Serum bottle
Final pH:
5.1
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Solution A | 954 | ml | ||
|
|
Solution B | 10 | ml | ||
|
|
Solution C | 13 | ml | ||
|
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Solution D | 10 | ml | ||
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Solution E | 10 | ml | ||
|
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Solution F | 10 | ml | ||
| 1 Dissolve ingredients of solution A, adjust pH to 5.0 with 0.1 N HCl and sparge with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense solution A under same gas atmosphere into anoxic Hungate-type tubes or serum vials (e.g., 20 ml medium in 50 ml bottles) and autoclave. Solutions B - F are prepared under 100% N2 gas atmosphere and sterilized by filtration. Prior to inoculation complete the medium by adding appropriate amounts of solutions B to F to the sterile solution A in the sequence as indicated. | |||||
| 2 Adjust pH of the complete medium to 5.1 with a sterile anoxic solution of 1 N HCl. After inoculation pressurize vials to 1 bar overpressure with sterile 80% H2 and 20% CO2 gas mixture. | |||||
| 3 Note: Use 10-20% (v/v) as inoculum. | |||||
Solution A #
954
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
NH4Cl | 27.0 | mg |
|
|
KH2PO4 | 14.0 | mg |
|
|
KCl
(0.1% w/v) |
2.0 | ml |
|
|
Na-acetate | 17.0 | mg |
|
|
Trace elements solution | 1.0 | ml |
|
|
Sodium resazurin
(0.1% w/v) |
0.5 | ml |
|
|
Distilled water | 950.0 | ml |
Solution B #
10
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Homo-PIPES
(SIGMA) |
1.58 | g | ||
|
|
NaOH
(pellets) |
0.18 | g | ||
|
|
Distilled water | 10.00 | ml | ||
| 1 Adjust pH of buffer to 5.5. | |||||
Solution C #
13
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
NaOH
(0.38 M) |
7.20 | ml |
|
|
Nitrilotriacetate
(0.5 M, neutralized with 10 N NaOH) |
4.80 | ml |
|
|
TiCl3
(15% w/v in HCl, RIEDEL-de HAEN) |
0.55 | ml |
Solution E #
10
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
2-Mercaptoethanesulfonate
(coenzyme M) |
0.07 | g |
|
|
Distilled water | 10.00 | ml |
Trace elements solution #
1010
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
HCl
(25%) |
10.00 | ml | ||
|
|
FeCl2 x 4 H2O | 1.34 | g | ||
|
|
CoCl2 x 6 H2O | 24.00 | mg | ||
|
|
ZnCl2 | 75.00 | mg | ||
|
|
H3BO3 | 19.00 | mg | ||
|
|
NiCl2 x 6 H2O | 24.00 | mg | ||
|
|
Na2MoO4 x 2 H2O | 24.00 | mg | ||
|
|
MnSO4 x 4 H2O | 26.00 | mg | ||
|
|
MgSO4 x 7 H2O | 1.56 | g | ||
|
|
CaCl2 x 2 H2O | 2.34 | g | ||
|
|
CuSO4 x 5 H2O | 9.00 | mg | ||
|
|
AlK(SO4)2 x 12 H2O | 3.45 | g | ||
|
|
Distilled water | 1000.00 | ml | ||
| 1 First dissolve ferrous chloride in the HCl, then dilute in water and dissolve the other salts. Finally make up to 1000 ml. | |||||
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 |