1526e .
LIMIHALOGLOBUS MEDIUM
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Equipment needed:
Autoclave
Filter
Gassing station
Hungate tubes
Final pH:
7.3 - 7.5
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
NaCl | 60.0 | g | ||
|
|
MgCl2 x 6 H2O | 6.0 | g | ||
|
|
KCl | 1.5 | g | ||
|
|
Na2SO4 | 1.0 | g | ||
|
|
NH4Cl | 1.0 | g | ||
|
|
CaCl2 x 2 H2O | 0.4 | g | ||
|
|
K2HPO4 | 0.4 | g | ||
|
|
Modified Wolin's mineral solution | 10.0 | ml | ||
|
|
Sodium resazurin
(0.1% w/v) |
0.5 | ml | ||
|
|
Sulfur
(powder) |
10.0 | g | ||
|
|
Na2CO3 | 1.5 | g | ||
|
|
D-Glucose | 1.0 | g | ||
|
|
Yeast extract | 1.0 | g | ||
|
|
Wolin's vitamin solution (10x) | 1.0 | ml | ||
|
|
L-Cysteine HCl x H2O | 0.3 | g | ||
|
|
DL-Dithiothreitol
(DTT) |
0.3 | g | ||
|
|
Distilled water | 1000.0 | ml | ||
| 1 Dissolve ingredients except sulfur, carbonate, glucose, yeast extract, vitamins, cysteine and DTT, then sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense medium under the same gas atmosphere into anoxic Hungate-type tubes or serum vials containing already the appropriate amount of sulfur and autoclave at 110°C for 20 min. Add glucose, yeast extract, vitamins, cysteine and DTT from sterile anoxic stock solutions prepared under 100% N2 gas (vitamins and DTT are sterilized by filtration) and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Prior to use adjust pH of complete medium to 7.3 - 7.5, 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 |