991 .
CALDISPHAERA MEDIUM
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Equipment needed:
Autoclave
Filter
Gassing station
Hungate tubes
Serum bottle
Water bath
Final pH:
4.0 - 4.5
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
(NH4)2SO4 | 1.30 | g | ||
|
|
KH2PO4 | 0.28 | g | ||
|
|
MgSO4 x 7 H2O | 0.25 | g | ||
|
|
CaCl2 x 2 H2O | 0.07 | g | ||
|
|
FeCl3 x 6 H2O | 0.02 | g | ||
|
|
Allen's trace element solution | 10.00 | ml | ||
|
|
Trisodium citrate x 2 H2O | 2.94 | g | ||
|
|
Sodium resazurin
(0.1% w/v) |
0.50 | ml | ||
|
|
Sulfur
(powdered) |
10.00 | g | ||
|
|
Yeast extract
(OXOID) |
0.50 | g | ||
|
|
Wolin's vitamin solution (10x) | 1.00 | ml | ||
|
|
Na2S x 9 H2O | 0.50 | g | ||
|
|
Distilled water | 1000.00 | ml | ||
| 1 Dissolve ingredients (except sulfur, yeast extract, vitamins and sulfide), adjust pH to 3.5 with 10 N H2SO4 and sparge medium with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials (e.g., 20 ml medium in 100 ml bottles) containing already the appropriate amount of sulfur. Sterilize medium by heating cultivation vessels in a water bath to 90 - 100°C for 1 - 2 hours on each of 3 successive days. Add yeast extract, vitamins (sterilized by filtration) and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas. Adjust pH of complete medium to 4.0 - 4.5, if necessary. | |||||
| 2 After inoculation pressurize vials to 1 bar overpressure with sterile 80% H2 and 20% CO2 gas mixture. | |||||
Allen's trace element solution #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
MnCl2 x 4 H2O | 180 | mg | ||
|
|
Na2B4O7 x 10 H2O | 450 | mg | ||
|
|
ZnSO4 x 7 H2O | 22 | mg | ||
|
|
CuCl2 x 2 H2O | 5 | mg | ||
|
|
Na2MoO4 x 2 H2O | 3 | mg | ||
|
|
VOSO4 x 2 H2O | 3 | mg | ||
|
|
CoSO4 x 7 H2O | 1 | mg | ||
|
|
Distilled water | 1000 | ml | ||
| 1 Adjust pH of final solution to 2 with 1 N HCl. | |||||
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 |