Compound | Amount | Unit | |||
---|---|---|---|---|---|
|
Solution A | 950 | ml | ||
|
Solution B | 30 | ml | ||
|
Solution C | 1 | ml | ||
|
Solution D | 10 | ml | ||
|
Solution E | 10 | ml | ||
1 Sparge solution A with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic, then dispense under same gas atmosphere into anoxic serum vials to 10% volume (e.g., 10 ml medium in 100 ml bottle) and autoclave. Solution B is autoclaved separately after sparging with 80% N2 and 20% CO2 gas mixture. Solution C and D are prepared under 100% N2 gas atmosphere and sterilized by filtration. Solution E is autoclaved under 100% N2 gas atmosphere. To complete the medium add appropriate amounts of solutions B to E to the sterile solution A in the sequence as indicated. | |||||
2 Adjust pH of complete medium to 7.2, if necessary. | |||||
3 Note: Cultures are very sensitive to the nitrite produced during growth. Thus, incubate this strain ony a few hours at one time (8-12 h) and thereafter check growth by microscopy. Immediately stop the incubation at 85°C, when the medium begins to show some turbidity. |
Compound | Amount | Unit | |
---|---|---|---|
|
NaCl | 18.00 | g |
|
MgCl2 x 6 H2O | 4.30 | g |
|
CaCl2 x 2 H2O | 0.14 | g |
|
KCl | 0.34 | g |
|
K2HPO4 | 0.14 | g |
|
NH4Cl | 0.24 | g |
|
KNO3 | 1.00 | g |
|
Modified Wolin's mineral solution | 10.00 | ml |
|
Distilled water | 940.00 | ml |
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. |
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 |