377a .
PALAEOCOCCUS MEDIUM
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Equipment needed:
Autoclave
Filter
Gassing station
Hungate tubes
Final gas composition:
100 % N2
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Final pH:
6.5
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
NaCl | 30.00 | g | ||
|
|
MgSO4 x 7 H2O | 3.50 | g | ||
|
|
MgCl2 x 6 H2O | 2.75 | g | ||
|
|
KCl | 0.33 | g | ||
|
|
NaBr | 0.05 | g | ||
|
|
H3BO3 | 15.00 | mg | ||
|
|
SrCl2 x 6 H2O
(0.1% w/v) |
7.00 | ml | ||
|
|
(NH4)2SO4 | 10.00 | mg | ||
|
|
Citric acid
(0.1% w/v) |
5.00 | ml | ||
|
|
KI
(0.01% w/v) |
0.50 | ml | ||
|
|
CaCl2 x 2 H2O | 0.75 | g | ||
|
|
KH2PO4 | 0.50 | g | ||
|
|
NiCl2 x 6 H2O
(0.1% w/v) |
2.00 | ml | ||
|
|
Modified Wolin's mineral solution | 10.00 | ml | ||
|
|
Sodium resazurin
(0.1% w/v) |
0.50 | ml | ||
|
|
Sulfur
(powdered) |
30.00 | g | ||
|
|
Peptone
(BD Bacto) |
5.00 | g | ||
|
|
Yeast extract
(OXOID) |
1.00 | g | ||
|
|
Wolin's vitamin solution (10x) | 1.00 | ml | ||
|
|
Neutralized sulfide solution 3% (w/v) | 15.00 | ml | ||
|
|
Distilled water | 960.00 | ml | ||
| 1 Dissolve ingredients except sulfur, peptone, yeast extract, vitamins, and sulfide, adjust the pH to 6.5 and sparge medium with 100% N2 gas for 30 - 45 min to make it anoxic. Distribute medium under the same gas atmosphere into anoxic Hungate-type tubes or serum vials that already contain the appropriate amount of sulfur. Sterilize the medium by autoclaving at 110°C for 20 min. After sterilization add peptone, yeast extract, and vitamins from sterile stock solutions prepared under 100% N2 gas atmosphere and sulfide from a neutralized stock solution. Vitamins should be sterilized by filtration. | |||||
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 |
Neutralized sulfide solution 3% (w/v) #
100
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Na2S x 9 H2O | 3 | g | ||
|
|
Distilled water | 100 | ml | ||
| 1 The sulfide solution is prepared in a 250 ml screw-capped bottle with a butyl rubber septum and a magnetic stirrer. The solution is bubbled with nitrogen gas, closed and autoclaved for 15 min. at 121°C. After cooling to room temperature the pH is adjusted to about 7.0 by adding of sterile 2 M H2SO4 drop-wise with a syringe without opening the bottle. | |||||