1630a .
NITROSOPUMILUS MEDIUM
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Equipment needed:
Autoclave
Filter
Final pH:
7.2
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
NaCl | 26.00 | g | ||
|
|
MgSO4 x 7 H2O | 5.00 | g | ||
|
|
MgCl2 x 6 H2O | 5.00 | g | ||
|
|
CaCl2 x 2 H2O | 1.50 | g | ||
|
|
KBr | 0.10 | g | ||
|
|
KH2PO4
(0.1% w/v) |
2.00 | ml | ||
|
|
HEPES buffer solution | 10.00 | ml | ||
|
|
NH4Cl | 0.05 | g | ||
|
|
Trace element solution SL-11 | 1.00 | ml | ||
|
|
Na-pyruvate | 0.05 | g | ||
|
|
NaHCO3 | 0.25 | g | ||
|
|
Wolin's vitamin solution | 10.00 | ml | ||
|
|
Distilled water | 1000.00 | ml | ||
| 1 Dissolve ingredients (except trace elements, HEPES buffer, ammonium chloride, pyruvate, bicarbonate and vitamins), dispense medium into Erlenmeyr flasks with screw caps (e.g., 20 ml medium per 100 ml flask) and autoclave. Add HEPES buffer and ammonium chloride from stock solutions sterilized by autoclaving. Trace elements, pyruvate, bicarbonate and vitamins are added from stock solutions sterilized by filtration. Adjust pH of the complete medium to 7.2 using sterile solutions of bicarbonate (5% w/v) or 1 N HCl. | |||||
| 2 Note: Use at least 5% (v/v) inoculum and incubate in the dark without shaking. | |||||
HEPES buffer solution #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
NaOH
(pellets) |
24.0 | g | ||
|
|
HEPES
(free acid) |
238.4 | g | ||
|
|
Distilled water | 1000.0 | ml | ||
| 1 Fist dissolve NaOH in ca 600 ml water, then add HEPES, adjust pH to 7.5 and fill up to 1000 ml volume. | |||||
Trace element solution SL-11 #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Na2-EDTA x 2 H2O | 5.2 | g | ||
|
|
FeCl2 x 4 H2O | 1.5 | g | ||
|
|
ZnCl2 | 70.0 | mg | ||
|
|
MnCl2 x 4 H2O | 100.0 | mg | ||
|
|
H3BO3 | 6.0 | mg | ||
|
|
CoCl2 x 6 H2O | 190.0 | mg | ||
|
|
CuCl2 x 2 H2O | 2.0 | mg | ||
|
|
NiCl2 x 6 H2O | 24.0 | mg | ||
|
|
Na2MoO4 x 2 H2O | 36.0 | mg | ||
|
|
Distilled water | 1000.0 | ml | ||
| 1 Dissolve EDTA in 800 ml distilled water, adjust pH to 7 using 2 N NaOH and add ferrous chloride. After ferrous chloride has dissolved add other compounds. Finally adjust pH to 6.0 and bring volume to 1000 ml. | |||||
Wolin's vitamin solution #
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 D-(+)-pantothenate | 5.0 | mg |
|
|
Vitamin B12 | 0.1 | mg |
|
|
p-Aminobenzoic acid | 5.0 | mg |
|
|
(DL)-alpha-Lipoic acid | 5.0 | mg |
|
|
Distilled water | 1000.0 | ml |