462 .
SALTWATER MEDIUM WITH LACTATE
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Equipment needed:
Autoclave
Filter
Hungate tubes
Final pH:
7.0
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
NaCl | 20.00 | g | ||
|
|
Na2SO4 | 4.00 | g | ||
|
|
MgCl2 x 6 H2O | 3.00 | g | ||
|
|
CaCl2 x 2 H2O | 0.15 | g | ||
|
|
NH4Cl | 0.75 | g | ||
|
|
KH2PO4 | 0.60 | g | ||
|
|
KBr | 0.09 | g | ||
|
|
KCl | 0.50 | g | ||
|
|
NaHCO3 | 2.50 | g | ||
|
|
Trace element solution | 1.00 | ml | ||
|
|
Vitamin solution | 1.00 | ml | ||
|
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Thiamine solution | 1.00 | ml | ||
|
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Vitamin B12 solution 0.05 g/L | 1.00 | ml | ||
|
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Riboflavin solution 0.025 g/L | 1.00 | ml | ||
|
|
Selenite-tungstate solution | 1.00 | ml | ||
|
|
Sodium lactate | 2.25 | g | ||
|
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Na2S x 9 H2O | 0.30 | g | ||
|
|
Resazurin | 1.00 | mg | ||
| 1 Dissolve components except NaHCO3, Vitamin solution, Thiamine solution, Vitamin B12 solution, sodium lactate and Na2S x 9H2O in 900 ml distilled water and adjust pH to 7.0. Bring to a boil and cool down under a N2-CO2 (4:1, v/v) gas stream. Distribute the medium into culture vessels (e.g., 9 ml of the medium in Hungate tubes) under a N2-CO2 (4:1, v/v) gas mixture, seal with butyl rubber stoppers and autoclave. Separately autoclave 0.2 M sodium lactate under a N2 atmosphere. After cooling, add 1/9 volume of the sodium lactate solution, and filter-sterilized 8% NaHCO3 solution, Vitamin solution, Thiamine solution and Vitamin B12 solution to the medium. Readjust pH to 7.0, if necessary. Prior to use, reduce the medium with Na2S x 9H2O (3% solution, autoclaved and stocked under N2). | |||||
Trace element solution #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
HCl
(25%) |
12.5 | ml |
|
|
FeSO4 x 7 H2O | 2.1 | g |
|
|
H3BO3 | 30.0 | mg |
|
|
MnCl2 x 4 H2O | 100.0 | mg |
|
|
CoCl2 x 6 H2O | 190.0 | mg |
|
|
NiCl2 x 6 H2O | 24.0 | mg |
|
|
CuCl2 x 2 H2O | 2.0 | mg |
|
|
ZnSO4 x 7 H2O | 144.0 | mg |
|
|
Na2MoO4 x 2 H2O | 36.0 | mg |
|
|
Distilled water | 987.0 | ml |
Vitamin solution #
100
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
p-Aminobenzoic acid | 4 | mg |
|
|
Biotin | 1 | mg |
|
|
Nicotinic acid | 10 | mg |
|
|
DL-Calcium pantothenate | 5 | mg |
|
|
Pyridoxine hydrochloride | 10 | mg |
|
|
Sodium phosphate buffer
(10 mM, pH 7.1) |
100 | ml |
Thiamine solution #
100
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
Thiamine HCl | 10 | mg |
|
|
Sodium phosphate buffer
(25 mM, pH 3.4) |
100 | ml |
Selenite-tungstate solution #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
NaOH | 0.4 | g |
|
|
Na2SeO3 x 5 H2O | 6.0 | mg |
|
|
Na2WO4 x 2 H2O | 8.0 | mg |
|
|
Distilled water | 1000.0 | ml |