1221 .
MODIFIED NITRATE MINERAL SALTS MEDIUM-2
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Equipment needed:
Autoclave
Filter
Final pH:
6.8
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
MgSO4 x 7 H2O | 1.00 | g | ||
|
|
CaCl2 x 2 H2O | 0.20 | g | ||
|
|
KNO3 | 0.25 | g | ||
|
|
FeCl2 solution | 1.00 | ml | ||
|
|
Trace element solution | 1.00 | ml | ||
|
|
HEPES
(pH 7.0, 1 M) |
2.00 | ml | ||
|
|
Ammonium ferric citrate | 4.00 | mg | ||
|
|
Distilled water | 1000.00 | ml | ||
| 1 Mix components thoroughly and autoclave. After cooling, add the following solutions (autoclaved) to the medium: | |||||
|
|
Phosphate buffer solution | 2.00 | ml | ||
|
|
Vitamin solution | 10.00 | ml | ||
| 2 Methane: Aseptically distribute the medium into sterile serum bottles (leave &; 80% gas phase by volume) and seal with rubber stoppers. Replace the gas phase with an air-methane gas mixture (75:25, v/v). Cultivate with shaking. | |||||
| 3 Adjust pH to 6.8, if necessary. Cultivate with methane or methanol as a substrate as described below: | |||||
| 4 Methanol: Aseptically distribute the medium into culture vessels (e.g., 20 ml in 60 ml serum bottles), supplement with 4.0 ml per liter filter-sterilized methanol and seal with rubber stoppers. | |||||
Phosphate buffer solution #
100
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
KH2PO4 | 1.4 | g |
|
|
Na2HPO4 x 2 H2O | 3.6 | g |
|
|
Distilled water | 100.0 | ml |
Vitamin solution #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
Trace vitamins | 1000 | ml |
|
|
Vitamin B12 | 20 | mg |
|
|
L-Ascorbic acid | 5 | mg |
|
|
Nicotinamide | 5 | mg |
FeCl2 solution #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
HCl
(7.7 M, 25%) |
10.0 | ml |
|
|
FeCl2 x 4 H2O | 1.5 | g |
|
|
Distilled water | 990.0 | ml |
Trace element solution #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
ZnCl2 | 70 | mg |
|
|
MnCl2 x 4 H2O | 100 | mg |
|
|
H3BO3 | 6 | mg |
|
|
CoCl2 x 6 H2O | 190 | mg |
|
|
CuCl2 x 2 H2O | 2 | mg |
|
|
NiCl2 x 6 H2O | 24 | mg |
|
|
Na2MoO4 x 2 H2O | 36 | mg |
|
|
Distilled water | 1000 | ml |
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 |