Compound | Amount | Unit | |||
---|---|---|---|---|---|
|
Solution A | 500 | ml | ||
|
Solution B | 500 | ml | ||
|
Solution C | 1 | ml | ||
|
Solution D | 1 | ml | ||
|
Solution E | 5 | ml | ||
|
Solution F | 10 | ml | ||
|
Solution G | 5 | ml | ||
|
Solution H | 10 | ml | ||
|
Solution I | 10 | ml | ||
1 Sterilize solutions A and B in closed thick-walled screw-top bottles (e.g., SCHOTT) for 20 min at 120°C. The pH of solution A after sterilization should be 10. There is some precipitate forming that settles at the bottom after 3 - 4 days. It is best to remove precipitates by decantation before using solution A for medium preparation. | |||||
2 Combine solution A with solution B and sparge medium with 100% N2 gas for at least 30 - 45 min to make it anoxic, then dispense under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Solutions C to I are sterilized separately under 100% N2 gas. Vitamins and pyruvate should be sterilized by filtration. To complete the medium appropriate amounts of solutions C to I are added to the combined sterile solutions A and B in the sequence as indicated. Final pH of the medium should be 9.5. | |||||
3 Note: Addition of 10 - 20 mg sodium dithionite per liter (e.g. from 5% (w/v) solution, freshly prepared under N2 and filter-sterilized) may stimulate growth at the beginning. For transfers use 5 - 10% inoculum. |
Compound | Amount | Unit | |
---|---|---|---|
|
Na2CO3 | 92.5 | g |
|
NaHCO3 | 17.5 | g |
|
NaCl | 8.0 | g |
|
K2HPO4 | 0.5 | g |
|
Distilled water | 500.0 | ml |
Compound | Amount | Unit | |
---|---|---|---|
|
NaCl | 120.00 | g |
|
K2HPO4 | 1.25 | g |
|
KCl | 2.50 | g |
|
Distilled water | 500.00 | ml |
Compound | Amount | Unit | |||
---|---|---|---|---|---|
|
EDTA | 5.00 | g | ||
|
FeSO4 x 7 H2O | 2.20 | g | ||
|
ZnSO4 x 7 H2O | 0.10 | g | ||
|
MnCl2 x 4 H2O | 0.03 | g | ||
|
H3BO3 | 0.03 | g | ||
|
CoCl2 x 6 H2O | 0.20 | g | ||
|
CuCl2 x 2 H2O | 0.03 | g | ||
|
NiCl2 x 6 H2O | 0.03 | g | ||
|
Na2MoO4 x 2 H2O | 0.03 | g | ||
|
Distilled water | 1000.00 | ml | ||
1 pH 3.0-4.0 |
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 |
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 |