135b .
ACETOBACTERIUM DEHALOGENANS MEDIUM
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Equipment needed:
Autoclave
Filter
Gassing station
Hungate tubes
Final pH:
7.4
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
NH4Cl | 1.00 | g | ||
|
|
KH2PO4 | 0.33 | g | ||
|
|
K2HPO4 | 0.45 | g | ||
|
|
MgSO4 x 7 H2O | 0.10 | g | ||
|
|
Modified Wolin's mineral solution | 20.00 | ml | ||
|
|
Yeast extract | 2.00 | g | ||
|
|
Sodium resazurin
(0.1% w/v) |
0.50 | ml | ||
|
|
Na2CO3 | 1.50 | g | ||
|
|
Na-syringate solution (0.3 M) | 15.00 | ml | ||
|
|
Wolin's vitamin solution (10x) | 1.00 | ml | ||
|
|
L-Cysteine HCl x H2O | 0.50 | g | ||
|
|
Distilled water | 970.00 | ml | ||
| 1 Dissolve ingredients except for carbonate, Na-syringate solution, vitamins, and cysteine, bring to the boil and cool to room temperature under 80% N2 and 20% CO2 gas atmosphere. Thereafter, dispense under the same gas atmosphere in anoxic Hungate-type tubes or serum vials and autoclave. Add Na-syringate solution, vitamins, and cysteine from sterile anoxic stock solutions prepared under 100% N2 gas and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas atmosphere. Vitamins should be sterilized by filtration. The pH of the completed medium should be at 7.4. | |||||
| 2 Note: After growth has been started add an additional amount of Na-syringate to reach a final concentration of 2.00 g/l. | |||||
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 |
Na-syringate solution (0.3 M) #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Syringic acid
(SIGMA S6881) |
60 | g | ||
|
|
NaOH
(5 N) |
50 | ml | ||
|
|
Distilled water | 950 | ml | ||
| 1 Add syringic acid to 700 ml distilled water, add about 50 ml of 5 N NaOH and adjust the pH to about 7.5 to dissolve it. Then make up to a volume of 1000 ml with water. Make the solution anoxic by sparging with 100% N2 gas, distribute in anoxic serum vials and autoclave at 121°C. | |||||