144 .
THERMOANAEROBACTER BROCKII MEDIUM
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* Medium for Halanaerobium acetethylicum DSM 3532
Add 10% NaCl; anaerobic
Growth conditions: 35 °C
Equipment needed:
Autoclave
Filter
Gassing station
Hungate tubes
Final gas composition:
100 % N2
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Final pH:
7.2 - 7.4
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
NH4Cl | 0.90 | g | ||
|
|
NaCl | 0.90 | g | ||
|
|
MgCl2 x 6 H2O | 0.40 | g | ||
|
|
KH2PO4 | 0.75 | g | ||
|
|
K2HPO4 | 1.50 | g | ||
|
|
Trace element solution | 9.00 | ml | ||
|
|
FeSO4 x 7 H2O
(0.1% w/v in 0.1 N H2SO4) |
3.00 | ml | ||
|
|
Yeast extract | 3.00 | g | ||
|
|
Trypticase peptone
(BD BBL) |
10.00 | g | ||
|
|
Sodium resazurin
(0.1% w/v) |
0.50 | ml | ||
|
|
Wolin's vitamin solution | 5.00 | ml | ||
|
|
D-Glucose | 5.00 | g | ||
|
|
Na2S x 9 H2O | 1.00 | g | ||
|
|
NaCl | 100.00 | g/l | ||
|
|
Distilled water | 1000.00 | ml | ||
| 1 Dissolve ingredients (except vitamins, glucose and sulfide), sparge medium with 100% N2 gas for 30 - 45 min to make it anoxic. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Add glucose, vitamins (sterilized by filtration) and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas. Adjust pH of complete medium to 7.2 - 7.4. | |||||
Trace element solution #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Nitrilotriacetic acid
(NTA) |
12.80 | g | ||
|
|
FeCl2 x 4 H2O | 0.20 | g | ||
|
|
MnCl2 x 4 H2O | 0.10 | g | ||
|
|
CoCl2 x 6 H2O | 0.17 | g | ||
|
|
CaCl2 x 2 H2O | 0.10 | g | ||
|
|
ZnCl2 | 0.10 | g | ||
|
|
CuCl2 | 0.02 | g | ||
|
|
H3BO3 | 0.01 | g | ||
|
|
Na2MoO4 x 2 H2O | 0.01 | g | ||
|
|
NiCl2 x 6 H2O | 0.03 | g | ||
|
|
NaCl | 1.00 | g | ||
|
|
Na2SeO3 x 5 H2O | 0.03 | g | ||
|
|
Distilled water | 1000.00 | ml | ||
| 1 First dissolve NTA in 200 ml of distilled water and adjust pH to 6.5 with KOH, then dissolve mineral salts. Finally adjust pH to 6.5 with KOH and make up to 1000.00 ml. | |||||
Wolin's vitamin solution #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
Biotin | 2.0 | mg |
|
|
Folic acid | 2.0 | mg |
|
|
Pyridoxine hydrochloride | 10.0 | mg |
|
|
Thiamine HCl | 5.0 | mg |
|
|
Riboflavin | 5.0 | mg |
|
|
Nicotinic acid | 5.0 | mg |
|
|
Calcium D-(+)-pantothenate | 5.0 | mg |
|
|
Vitamin B12 | 0.1 | mg |
|
|
p-Aminobenzoic acid | 5.0 | mg |
|
|
(DL)-alpha-Lipoic acid | 5.0 | mg |
|
|
Distilled water | 1000.0 | ml |