Compound | Amount | Unit | |||
---|---|---|---|---|---|
|
NH4Cl | 1.0 | g | ||
|
MgCl2 x 6 H2O | 1.0 | g | ||
|
CaCl2 x 2 H2O | 0.4 | g | ||
|
KH2PO4 | 0.4 | g | ||
|
Wolfe's mineral elixir | 1.0 | ml | ||
|
Yeast extract | 2.0 | g | ||
|
Trypticase peptone
(BD BBL) |
2.0 | g | ||
|
Sodium resazurin
(0.1% w/v) |
0.5 | ml | ||
|
Na2CO3 | 1.0 | g | ||
|
Na-crotonate solution (1 M) | 20.0 | ml | ||
|
2-Mercaptoethanesulfonic acid
(coenzyme M) |
0.5 | g | ||
|
Wolin's vitamin solution (10x) | 1.0 | ml | ||
|
Na2S x 9 H2O | 0.3 | g | ||
|
Distilled water | 1000.0 | ml | ||
1 Dissolve ingredients (except carbonate, crotonate, coenzyme M, vitamins and sulfide), 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 (e.g., 5 ml medium in Balch-type tubes) and autoclave. Add crotonate, coenzyme M, vitamins and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Stock solutions of crotonate, coenzyme M and vitamins should be sterilized by filtration. Adjust pH of the complete medium to 7.0 - 7.2. | |||||
2 Note: Use 10% (v/v) as inoculum. |
Compound | Amount | Unit | |||
---|---|---|---|---|---|
|
MgSO4 x 7 H2O | 30.00 | g | ||
|
MnSO4 x H2O | 5.00 | g | ||
|
NaCl | 10.00 | g | ||
|
FeSO4 x 7 H2O | 1.00 | g | ||
|
CoCl2 x 6 H2O | 1.80 | g | ||
|
CaCl2 x 2 H2O | 1.00 | g | ||
|
ZnSO4 x 7 H2O | 1.80 | g | ||
|
CuSO4 x 5 H2O | 0.10 | g | ||
|
AlK(SO4)2 x 12 H2O | 0.18 | g | ||
|
H3BO3 | 0.10 | g | ||
|
Na2MoO4 x 2 H2O | 0.10 | g | ||
|
(NH4)2Ni(SO4)2 x 6 H2O | 2.80 | g | ||
|
Na2WO4 x 2 H2O | 0.10 | g | ||
|
Na2SeO4 | 0.10 | g | ||
|
Distilled water | 1000.00 | ml | ||
1 First adjust pH to 1.0 with diluted H2SO4, then add and dissolve the salts. |
Compound | Amount | Unit | |||
---|---|---|---|---|---|
|
Crotonic acid
(ALDRICH 113018) |
86 | g | ||
|
NaOH
(10 N) |
100 | ml | ||
|
Distilled water | 900 | ml | ||
1 Dissolve crotonic acid in 800 ml distilled water, add around 100 ml of 10 N NaOH and adjust pH to around 7. Then add water to reach a volume of 1000 ml. Sterilize by filtration under 100% N2 gas atmosphere. |
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 |