854 .
HIPPEA MEDIUM
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Equipment needed:
Autoclave
Filter
Gassing station
Hungate tubes
Final pH:
6.0 - 6.2
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
NH4Cl | 0.33 | g | ||
|
|
CaCl2 x 2 H2O | 0.33 | g | ||
|
|
MgCl2 x 6 H2O | 0.33 | g | ||
|
|
KCl | 0.33 | g | ||
|
|
KH2PO4 | 0.33 | g | ||
|
|
NaCl | 25.00 | g | ||
|
|
MOPS buffer
(SIGMA) |
3.00 | g | ||
|
|
Modified Wolin's mineral solution | 10.00 | ml | ||
|
|
Yeast extract | 0.10 | g | ||
|
|
Na-acetate | 5.00 | g | ||
|
|
Sodium resazurin
(0.1% w/v) |
0.50 | ml | ||
|
|
Sulfur
(powdered) |
10.00 | g | ||
|
|
Wolin's vitamin solution (10x) | 1.00 | ml | ||
|
|
Na2S x 9 H2O | 0.50 | g | ||
|
|
Distilled water | 1000.00 | ml | ||
| 1 Dissolve ingredients (except sulfur, vitamins, and sulfide), adjust pH to 5.5 - 6.0, and sparge the medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense the medium under the same gas atmosphere into anoxic Hungate-type tubes or serum vials that contain already an appropriate amount of sulfur powder. Autoclave 20 min at 110°C. Prior to use add vitamins and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas. Vitamins are sterilized by filtration. Adjust the pH of the complete medium to 6.0 - 6.2, if necessary. | |||||
Modified Wolin's mineral solution #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Nitrilotriacetic acid | 1.50 | g | ||
|
|
MgSO4 x 7 H2O | 3.00 | g | ||
|
|
MnSO4 x H2O | 0.50 | g | ||
|
|
NaCl | 1.00 | g | ||
|
|
FeSO4 x 7 H2O | 0.10 | g | ||
|
|
CoSO4 x 7 H2O | 0.18 | g | ||
|
|
CaCl2 x 2 H2O | 0.10 | g | ||
|
|
ZnSO4 x 7 H2O | 0.18 | g | ||
|
|
CuSO4 x 5 H2O | 0.01 | g | ||
|
|
AlK(SO4)2 x 12 H2O | 0.02 | g | ||
|
|
H3BO3 | 0.01 | g | ||
|
|
Na2MoO4 x 2 H2O | 0.01 | g | ||
|
|
NiCl2 x 6 H2O | 0.03 | g | ||
|
|
Na2SeO3 x 5 H2O | 0.30 | mg | ||
|
|
Na2WO4 x 2 H2O | 0.40 | mg | ||
|
|
Distilled water | 1000.00 | ml | ||
| 1 First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Adjust final to pH 7.0 with KOH. | |||||
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 |