710 .
THERMOANAEROBACTER (KoKo) MEDIUM
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Equipment needed:
Autoclave
Filter
Gassing station
Hungate tubes
Final gas composition:
100 % N2
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Final pH:
7.0
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Tryptone
(BD Bacto) |
1.00 | g | ||
|
|
Peptone
(meat) |
1.00 | g | ||
|
|
Yeast extract
(BD Bacto) |
1.00 | g | ||
|
|
K2HPO4 | 1.60 | g | ||
|
|
NaH2PO4 x 2 H2O | 1.00 | g | ||
|
|
NH4Cl | 0.50 | g | ||
|
|
MgSO4 x 6 H2O | 0.16 | g | ||
|
|
Trace element solution SL-11 | 1.00 | ml | ||
|
|
Sodium resazurin
(0.1% w/v) |
0.50 | ml | ||
|
|
CaCl2 x 2 H2O | 0.06 | g | ||
|
|
NaHCO3 | 1.00 | g | ||
|
|
D-Glucose | 5.00 | g | ||
|
|
Wolin's vitamin solution (10x) | 1.00 | ml | ||
|
|
L-Cysteine HCl x H2O | 0.30 | g | ||
|
|
Na2S x 9 H2O | 0.30 | g | ||
|
|
Distilled water | 1000.00 | ml | ||
| 1 Dissolve ingredients (except calcium chloride, bicarbonate, glucose, vitamins, cysteine and sulfide), adjust pH to 7.0 and sparge medium with 100% N2 gas for 30 - 45 min to make it anoxic. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. After sterilization add calcium chloride, glucose, cysteine and sulfide from sterile anoxic stock solutions autoclaved under 100% N2 gas atmosphere. Add bicarbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Vitamins are prepared under 100% N2 gas and sterilized by filtration. The pH of the complete medium should be at 7.0. | |||||
| 2 Note: Supplementation of medium with 10.00 g/l MOPS buffer (pH 6.9 - 7.0; 10% (w/v) anoxic stock solution) may enhance the buffer capacity of the medium. | |||||
Trace element solution SL-11 #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Na2-EDTA x 2 H2O | 5.2 | g | ||
|
|
FeCl2 x 4 H2O | 1.5 | g | ||
|
|
ZnCl2 | 70.0 | mg | ||
|
|
MnCl2 x 4 H2O | 100.0 | mg | ||
|
|
H3BO3 | 6.0 | mg | ||
|
|
CoCl2 x 6 H2O | 190.0 | mg | ||
|
|
CuCl2 x 2 H2O | 2.0 | mg | ||
|
|
NiCl2 x 6 H2O | 24.0 | mg | ||
|
|
Na2MoO4 x 2 H2O | 36.0 | mg | ||
|
|
Distilled water | 1000.0 | ml | ||
| 1 Dissolve EDTA in 800 ml distilled water, adjust pH to 7 using 2 N NaOH and add ferrous chloride. After ferrous chloride has dissolved add other compounds. Finally adjust pH to 6.0 and bring volume to 1000 ml. | |||||
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 |