1042 .
VULCANIBACILLUS MEDIUM
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Equipment needed:
Autoclave
Filter
Gassing station
Hungate tubes
Final gas composition:
100 % N2
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Final pH:
6.8 - 7.0
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Sea Salt
(SIGMA) |
30.0 | g | ||
|
|
NaNO3 | 1.0 | g | ||
|
|
Wolfe's mineral elixir | 2.0 | ml | ||
|
|
VOSO4 x 5 H2O
(0.01% w/v) |
0.5 | ml | ||
|
|
Sodium resazurin
(0.1% w/v) |
0.5 | ml | ||
|
|
FeSO4 x 7 H2O | 0.1 | g | ||
|
|
NaHCO3 | 1.0 | g | ||
|
|
Yeast extract | 0.5 | g | ||
|
|
Na-pyruvate | 1.0 | g | ||
|
|
Wolin's vitamin solution (10x) | 2.0 | ml | ||
|
|
Distilled water | 1000.0 | ml | ||
| 1 Dissolve ingredients (except ferrous sulfate, bicarbonate, yeast extract, pyruvate and vitamins), adjust pH to 7.0 and sparge medium with 100% N2 gas for 30 - 45 min to make it anoxic. Dispense the medium under same gas atmosphere into Hungate-type tubes or serum vials and autoclave. Add ferrous sulfate, bicarbonate, yeast extract, pyruvate and vitamins from sterile anoxic stock solutions prepared under 100% N2 gas and bicarbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas atmosphere. Prepare the stock solution of ferrous sulfate by dissolving 1% (w/v) FeSO4 x 7 H2O in 0.1 N H2SO4. The stock solutions of pyruvate and vitamins should be sterilized by filtration. Adjust pH of the complete medium to 6.8-7.0. | |||||
| 2 Note: It may be necessary to add 10-20 mg sodium dithionite per liter (e.g. from 5% (w/v) solution, freshly prepared under 100% N2 and filter-sterilized), if the medium is not completely reduced after inoculation. | |||||
Wolfe's mineral elixir #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
MgSO4 x 7 H2O | 30.00 | g | ||
|
|
MnSO4 x H2O | 5.00 | g | ||
|
|
NaCl | 10.00 | g | ||
|
|
FeSO4 x 7 H2O | 1.00 | g | ||
|
|
CoCl2 x 6 H2O | 1.80 | g | ||
|
|
CaCl2 x 2 H2O | 1.00 | g | ||
|
|
ZnSO4 x 7 H2O | 1.80 | g | ||
|
|
CuSO4 x 5 H2O | 0.10 | g | ||
|
|
AlK(SO4)2 x 12 H2O | 0.18 | g | ||
|
|
H3BO3 | 0.10 | g | ||
|
|
Na2MoO4 x 2 H2O | 0.10 | g | ||
|
|
(NH4)2Ni(SO4)2 x 6 H2O | 2.80 | g | ||
|
|
Na2WO4 x 2 H2O | 0.10 | g | ||
|
|
Na2SeO4 | 0.10 | g | ||
|
|
Distilled water | 1000.00 | ml | ||
| 1 First adjust pH to 1.0 with diluted H2SO4, then add and dissolve the salts. | |||||
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 |