1097 .
SEAWATER PYRUVATE THIOSULFATE MEDIUM
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Equipment needed:
Anaerobic workbench
Autoclave
Filter
Final pH:
n.d.
Select a strain (optional):
ml
| 1 Mix components and autoclave under a N2-CO2 (4:1, v/v) atmosphere. After cooling, add the following solutions (autoclaved or *filter-sterilized): | |||||
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
NaCl | 20.00 | g | ||
|
|
MgCl2 x 6 H2O | 3.00 | g | ||
|
|
CaCl2 x 2 H2O | 0.15 | g | ||
|
|
NH4Cl | 0.25 | g | ||
|
|
KH2PO4 | 0.20 | g | ||
|
|
KCl | 0.50 | g | ||
|
|
RST trace elements | 10.00 | ml | ||
|
|
Resazurin | 1.00 | mg | ||
|
|
Distilled water | 922.50 | ml | ||
|
|
NaHCO3
(*, 8%) |
37.50 | ml | ||
|
|
RST vitamin solution
(*) |
10.00 | ml | ||
|
|
Sodium pyruvate
(1 M) |
20.00 | ml | ||
|
|
Yeast extract
(1%) |
10.00 | ml | ||
| 2 Anaerobically and aseptically distribute the medium into appropriate culture vessels (e.g., 20 ml medium in 60 ml serum bottles) under a N2-CO2 (4:1, v/v) gas stream and seal with butyl rubber stoppers. Finally, add per liter the following solution: | |||||
|
|
Reducing agent solution | 10.00 | ml | ||
RST vitamin solution #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
Coenzyme M | 5 | mg |
|
|
Biotin | 2 | mg |
|
|
Folic acid | 2 | mg |
|
|
Pyridoxine hydrochloride | 10 | mg |
|
|
Thiamine HCl | 5 | mg |
|
|
Riboflavin | 5 | mg |
|
|
Nicotinic acid | 5 | mg |
|
|
DL-Calcium pantothenate | 5 | mg |
|
|
p-Aminobenzoic acid | 5 | mg |
|
|
Lipoic acid | 5 | mg |
|
|
Distilled water | 1000 | ml |
RST trace elements #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Nitrilotriacetic acid | 2.00 | g | ||
|
|
MnSO4 x n H2O | 1.00 | g | ||
|
|
Fe(NH4)2(SO4)2 x 6 H2O | 0.80 | g | ||
|
|
CoCl2 x 6 H2O | 0.20 | g | ||
|
|
ZnSO4 x 7 H2O | 0.20 | g | ||
|
|
CuCl2 x 2 H2O | 0.02 | g | ||
|
|
NiCl2 x 6 H2O | 0.02 | g | ||
|
|
Na2MoO4 x 2 H2O | 0.02 | g | ||
|
|
Na2SeO4 | 0.02 | g | ||
|
|
Na2WO4 x 2 H2O | 0.02 | g | ||
|
|
Distilled water | 1000.00 | ml | ||
| 1 Dissolve nitrilotriacetic acid and adjust pH to 6.0 with KOH. Then proceed to add minerals. | |||||
Reducing agent solution #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Na2S x 9 H2O | 12.5 | g | ||
|
|
L-Cysteine HCl x H2O | 12.5 | g | ||
|
|
Distilled water | 1000.0 | ml | ||
| 1 Autoclave and store under a N2 atmosphere. | |||||
| 2 Comment: For strain JCM 13588, inoculate with a 20% inoculum, and incubate the vessels in inverted position. | |||||