334 .
METHANOTHRIX MEDIUM
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Equipment needed:
Autoclave
Filter
Gassing station
Serum bottle
Final pH:
7.2
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Solution A | 951 | ml | ||
|
|
Solution B | 25 | ml | ||
|
|
Solution C | 10 | ml | ||
|
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Solution D | 10 | ml | ||
|
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Solution E | 1 | ml | ||
|
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Solution F | 10 | ml | ||
| 1 Sparge solution A with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic, dispense under same gas atmosphere into anoxic serum vials (e.g., 20 ml medium in 50 ml bottles) and autoclave. Prior to inoculation complete the medium by adding appropriate amounts of sterile solutions C, D, E and F prepared under 100% N2 gas and solution B prepared under 80% N2 and 20% CO2 gas atmosphere. Vitamins are sterilized by filtration. Adjust the pH of the complete medium to 7.2. | |||||
| 2 Note: Use 20% (v/v) inoculum. | |||||
Solution A #
951
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
KH2PO4 | 0.22 | g |
|
|
Na2HPO4 x 2 H2O | 0.86 | g |
|
|
Na-acetate | 6.80 | g |
|
|
Na2-EDTA
(0.1% w/v) |
1.00 | ml |
|
|
Trace elements solution | 10.00 | ml |
|
|
Sodium resazurin
(0.1% w/v) |
0.50 | ml |
|
|
Distilled water | 940.00 | ml |
Solution C #
10
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
NaCl | 0.30 | g |
|
|
CaCl2 x 2 H2O | 0.11 | g |
|
|
MgCl2 x 6 H2O | 0.10 | g |
|
|
Distilled water | 10.00 | ml |
Trace elements solution #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Nitrilotriacetic acid
(NTA) |
12.80 | g | ||
|
|
FeCl3 x 6 H2O | 1.35 | g | ||
|
|
MnCl2 x 4 H2O | 0.10 | g | ||
|
|
CoCl2 x 6 H2O | 0.03 | g | ||
|
|
CaCl2 x 2 H2O | 0.10 | g | ||
|
|
ZnCl2 | 0.10 | g | ||
|
|
CuCl2 | 0.03 | g | ||
|
|
H3BO3 | 0.01 | g | ||
|
|
Na2MoO4 x 2 H2O | 0.03 | g | ||
|
|
NiCl2 x 6 H2O | 0.12 | g | ||
|
|
NaCl | 1.00 | g | ||
|
|
Na2SeO3 x 5 H2O | 0.03 | g | ||
|
|
Distilled water | 1000.00 | ml | ||
| 1 First dissolve NTA in 200 ml of distilled water and adjust pH to 6.5 with KOH, then dissolve mineral salts. Finally adjust pH to 6.5 with KOH and make up to 1000.00 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 |