236 .
THERMOCLADIUM MEDIUM
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Equipment needed:
Anaerobic workbench
Autoclave
Filter
Final pH:
3.5
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Modified Brock's salt base solution | 1000.00 | ml | ||
|
|
Trisodium citrate x 2 H2O | 2.94 | g | ||
|
|
Yeast extract
(BD-Difco) |
0.50 | g | ||
|
|
Trace vitamins | 10.00 | ml | ||
|
|
Resazurin | 1.00 | mg | ||
|
|
Na2S x 9 H2O | 0.50 | g | ||
|
|
Sulfur
(powder) |
10.00 | g | ||
| 1 Combine Modified Brock's salt base solution, trisodium citrate and resazurin, and adjust pH to 3.5 with 10 N H2SO4. Autoclave under a N2 atmosphere. Separately autoclave yeast extract and Na2S x 9H2O as 10% and 5% solutions, respectively, under a N2 atmosphere. Filter-sterilize Trace vitamins. Steam sulfur for 3 hr on each of 3 successive days. Aseptically mix components except Na2S x 9H2O in suitable culture vessels (e.g., 20 ml of the medium in 120 ml serum bottles) while gassing with H2-CO2 (80:20) gas mixture and seal with butyl rubber stoppers. Prior to inoculation, aseptically and anaerobically add the Na2S x 9H2O solutions and adjust pH to 4.0-4.5, if necessary. When the indicator (resazurin) turns to colorless, inoculate the culture and pressurize the vessels to 100 kPa H2-CO2 (80:20). | |||||
Modified Brock's salt base solution #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
(NH4)2SO4 | 1.30 | g |
|
|
KH2PO4 | 0.28 | g |
|
|
MgSO4 x 7 H2O | 0.25 | g |
|
|
CaCl2 x 2 H2O | 0.07 | g |
|
|
FeCl3 x 6 H2O | 2.00 | mg |
|
|
MnCl2 x 4 H2O | 1.80 | mg |
|
|
Na2B4O7 x 10 H2O | 4.50 | mg |
|
|
ZnSO4 x 7 H2O | 0.22 | mg |
|
|
CuCl2 x 2 H2O | 0.05 | mg |
|
|
Na2MoO4 x 2 H2O | 0.03 | mg |
|
|
VOSO4 x n H2O | 0.03 | mg |
|
|
CoSO4 x 7 H2O | 0.01 | mg |
|
|
Distilled water | 1000.00 | ml |
Trace vitamins #
1000
| 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 pantothenate | 5.0 | mg |
|
|
Vitamin B12 | 0.1 | mg |
|
|
p-Aminobenzoic acid | 5.0 | mg |
|
|
Lipoic acid | 5.0 | mg |
|
|
Distilled water | 1000.0 | ml |