1277b .
CALDIMICROBIUM THIODISMUTANS MEDIUM
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* Medium for Dissulfurispira thermophila DSM 110365
Replace pyrosulfite with 2.5 g/l Na2S2O3 x 5 H2O added from an anoxic stock solution sterilized by filtration.
Growth conditions: 7-14 days
anaerobic
55 °C
Equipment needed:
Autoclave
Filter
Gassing station
Hungate tubes
Final pH:
7.5 - 8.0
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
MgCl2 x 6 H2O | 0.2 | g | ||
|
|
CaCl2 x 2 H2O | 0.1 | g | ||
|
|
(NH4)2SO4 | 0.1 | g | ||
|
|
KH2PO4 | 0.1 | g | ||
|
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KCl | 0.1 | g | ||
|
|
Trace element solution SL-10 | 1.0 | ml | ||
|
|
Selenite-tungstate solution | 1.0 | ml | ||
|
|
Amorphous Fe(OH)3 | 45.0 | ml | ||
|
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Na2CO3 | 2.5 | g | ||
| Na2S2O5* | 0.9 | g | |||
|
|
Seven vitamins solution | 1.0 | ml | ||
|
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Na2S2O3 x 5 H2O | 2.5 | g/l | ||
|
|
Distilled water | 960.0 | ml | ||
| 1 Dissolve ingredients (except carbonate, pyrosulfite and vitamins), then sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense medium under same gas atmosphere into Hungate-type tubes or serum vials and autoclave. Add pyrosulfite from a 10% (w/v) stock solution adjusted to pH 7.5-8.0 with NaOH, made anoxic with 100% N2 gas and sterilized by filtration. Vitamins are added from a stock solution prepared under 100% N2 gas atmosphere and sterilized by filtration and carbonate is added from a sterile stock solution prepared under 80% N2 and 20% CO2 gas atmosphere. Adjust pH of complete medium to 7.5 - 8.0. Incubate without shaking. | |||||
Trace element solution SL-10 #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
HCl
(25%) |
10.0 | ml | ||
|
|
FeCl2 x 4 H2O | 1.5 | g | ||
|
|
ZnCl2 | 70.0 | mg | ||
|
|
MnCl2 x 4 H2O | 100.0 | mg | ||
|
|
H3BO3 | 6.0 | mg | ||
|
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CoCl2 x 6 H2O | 190.0 | mg | ||
|
|
CuCl2 x 2 H2O | 2.0 | mg | ||
|
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NiCl2 x 6 H2O | 24.0 | mg | ||
|
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Na2MoO4 x 2 H2O | 36.0 | mg | ||
|
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Distilled water | 990.0 | ml | ||
| 1 First dissolve FeCl2 in the HCl, then dilute in water, add and dissolve the other salts. Finally make up to 1000.00 ml. | |||||
Selenite-tungstate solution #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
NaOH | 0.5 | g |
|
|
Na2SeO3 x 5 H2O | 3.0 | mg |
|
|
Na2WO4 x 2 H2O | 4.0 | mg |
|
|
Distilled water | 1000.0 | ml |
Amorphous Fe(OH)3 #
400
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
FeCl3 x 6 H2O
(60.00 g/l) |
320 | ml | ||
|
|
NaOH
, adjust if required
(10% w/v) |
80 | ml | ||
| 1 Slowly titrate 320 ml of a FeCl3 x 6 H2O stock solution (60.00 g/l) with 10% (w/v) NaOH to pH 8.0-8.5 under agitation (use magnetic stirrer). Total amount of added NaOH approx. 80 - 100 ml. The precipitated Fe(OH)3 should be stored at room temperature overnight with surface covered with water. Thereafter, centrifuge at 2000 rpm for 5 min and discard the supernatant. Wash several times with distilled water. Resuspend the pellet in medium as described above. For storage autoclave under 100% N2 atmosphere. | |||||
Seven vitamins solution #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
Vitamin B12 | 100 | mg |
|
|
p-Aminobenzoic acid | 80 | mg |
|
|
D-(+)-biotin | 20 | mg |
|
|
Nicotinic acid | 200 | mg |
|
|
Calcium pantothenate | 100 | mg |
|
|
Pyridoxine hydrochloride | 300 | mg |
|
|
Thiamine-HCl x 2 H2O | 200 | mg |
|
|
Distilled water | 1000 | ml |