318 .
METHANOSARCINA THERMOPHILA (BCYT) MEDIUM
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Equipment needed:
Autoclave
Filter
Gassing station
Hungate tubes
Final pH:
6.8
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
KH2PO4 | 0.30 | g | ||
|
|
NaCl | 0.60 | g | ||
|
|
MgCl2 x 6 H2O | 0.10 | g | ||
|
|
CaCl2 x 2 H2O | 0.08 | g | ||
|
|
Trace element solution | 10.00 | ml | ||
|
|
NH4Cl | 1.00 | g | ||
|
|
Yeast extract
(OXOID) |
0.50 | g | ||
|
|
Trypticase peptone
(BD BBL) |
0.50 | g | ||
|
|
Sodium resazurin
(0.1% w/v) |
0.50 | ml | ||
|
|
KHCO3 | 2.00 | g | ||
|
|
Methanol
(50% v/v) |
10.00 | ml | ||
|
|
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 bicarbonate, vitamins, methanol, cysteine and sulfide, then sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense medium under the same gas atmosphere into anoxic Hungate-type tubes or serum vials to 30% of their volume and autoclave. Add vitamins (sterilized by filtration), methanol (50% v/v solution), cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas and bicarbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Prior to use adjust pH of complete medium to 6.8, if necessary. | |||||
Trace element solution #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Nitrilotriacetic acid
(NTA) |
12.80 | g | ||
|
|
FeCl2 x 4 H2O | 1.00 | g | ||
|
|
MnCl2 x 4 H2O | 0.10 | g | ||
|
|
CoCl2 x 6 H2O | 0.03 | g | ||
|
|
CaCl2 x 2 H2O | 0.10 | g | ||
|
|
ZnCl2 | 0.10 | g | ||
|
|
CuCl2 | 0.02 | g | ||
|
|
H3BO3 | 0.01 | g | ||
|
|
Na2MoO4 x 2 H2O | 0.03 | g | ||
|
|
NiCl2 x 6 H2O | 0.10 | g | ||
|
|
NaCl | 1.00 | g | ||
|
|
Na2SeO3 x 5 H2O | 0.03 | g | ||
|
|
Na2WO4 x 2 H2O | 0.04 | g | ||
|
|
Distilled water | 1000.00 | ml | ||
| 1 First dissolve NTA in 200 ml of distilled water and adjust pH to 6.5 with KOH, then dissolve mineral salts. Finally adjust pH to 6.5 with KOH and make up to 1000.00 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 |