1665 .
DESULFAMPLUS MEDIUM
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Equipment needed:
Autoclave
Filter
Gassing station
Hungate tubes
Final gas composition:
100 % N2
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Final pH:
7.2 - 7.5
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
NaCl | 20.0 | g | ||
|
|
MgCl2 x 6 H2O | 6.0 | g | ||
|
|
Na2SO4 | 3.0 | g | ||
|
|
CaCl2 x 2 H2O | 0.2 | g | ||
|
|
KCl | 0.5 | g | ||
|
|
NH4Cl | 0.3 | g | ||
|
|
Wolfe's mineral elixir | 2.0 | ml | ||
|
|
HEPES
(SIGMA) |
2.4 | g | ||
|
|
Na2-fumarate | 1.0 | g | ||
|
|
Yeast extract
(OXOID) |
0.2 | g | ||
|
|
Sodium resazurin
(0.1% w/v) |
0.5 | ml | ||
|
|
K-phosphate buffer
(0.5 M, pH 7.0) |
2.0 | ml | ||
|
|
NaHCO3 | 1.0 | g | ||
|
|
FeCl2 x 4 H2O
(0.2% w/v in 0.02 N HCl) |
10.0 | ml | ||
|
|
Wolin's vitamin solution (10x) | 1.0 | ml | ||
|
|
L-Cysteine HCl x H2O | 0.5 | g | ||
|
|
Distilled water | 1000.0 | ml | ||
| 1 Dissolve ingredients (except phosphate buffer, bicarbonate, ferrous chloride, vitamins and cysteine), adjust pH to 7.2 with NaOH and sparge medium with 100% N2 gas for 30 - 45 min to make it anoxic. Dispense medium under the same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. After autoclaving add phosphate buffer, ferrous chloride, vitamins and cysteine from sterile anoxic stock solutions prepared under 100% N2 gas atmosphere and bicarbonate from a sterile stock solution prepared under 80% N2 and 20% CO2 gas mixture. Vitamins should be sterilized by filtration. Adjust pH of the complete medium to 7.2 - 7.5 , if necessary. | |||||
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 |