1223 .
WIDDEL FRESHWATER MEDIUM WITH LACTATE
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Equipment needed:
Anaerobic workbench
Autoclave
Filter
Hungate tubes
Final pH:
7.5
Select a strain (optional):
ml
| 1 Mix components, adjust pH to 7.5 and autoclave under a N2 gas atmosphere. After cooling, add the following solutions from sterile anaerobic stocks (autoclaved or *filter-sterilized): | |||||
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
NaCl | 1.00 | g | ||
|
|
MgCl2 x 6 H2O | 0.40 | g | ||
|
|
CaCl2 x 2 H2O | 0.10 | g | ||
|
|
NH4Cl | 0.25 | g | ||
|
|
KH2PO4 | 0.20 | g | ||
|
|
KCl | 0.50 | g | ||
|
|
Na2SO4 | 4.00 | g | ||
|
|
Resazurin | 1.00 | mg | ||
|
|
Distilled water | 988.00 | ml | ||
|
|
Trace element solution | 1.00 | ml | ||
|
|
Vitamin solution
(*) |
1.00 | ml | ||
|
|
Thiamine solution
(*) |
1.00 | ml | ||
|
|
Vitamin B12 solution 0.05 g/L
(*) |
1.00 | ml | ||
|
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Selenite-tungstate solution | 1.00 | ml | ||
|
|
L-Sodium lactate
(1.0 M) |
7.30 | ml | ||
| 2 Readjust pH to 7.5, if necessary. Separately autoclave and dry iron metal (grain). Aseptically distribute the medium and iron metal into culture vessels (e.g., 10 ml medium and 100 mg iron in Hungate tubes) under a N2 gas stream and seal with butyl rubber stoppers. Prior to use, add per liter the following solution: | |||||
|
|
Na2S x 9 H2O
(5%) |
2.00 | ml | ||
Trace element solution #
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Nitrilotriacetic acid | 12.80 | g | ||
|
|
FeSO4 x 7 H2O | 0.42 | g | ||
|
|
H3BO3 | 0.01 | g | ||
|
|
MnCl2 x 4 H2O | 0.10 | g | ||
|
|
CoCl2 x 6 H2O | 0.17 | g | ||
|
|
CuCl2 x 2 H2O | 0.02 | g | ||
|
|
ZnSO4 x 7 H2O | 0.21 | g | ||
|
|
Na2MoO4 x 2 H2O | 0.01 | g | ||
| 1 Dissolve nitrilotriacetic acid in 800 ml distilled water and adjust pH to 6.5 with NaOH. Then proceed to add the minerals and bring volume to 1.0 L. | |||||
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 |