195d .
KNH MEDIUM
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Equipment needed:
Autoclave
Filter
Gassing station
Hungate tubes
Final pH:
7.1 - 7.4
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Solution A | 952 | ml | ||
|
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Solution B | 30 | ml | ||
|
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Solution C | 10 | ml | ||
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Solution D | 1 | ml | ||
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Solution E | 13 | ml | ||
| 1 Solution A is sparged with 80% H2 and 20% CO2 gas mixture to reach a pH below 6 (at least 30 min), then distributed under the same gas atmosphere in anoxic Hungate-type tubes or serum vials to 30% of their volume and autoclaved. Solution B is autoclaved separately under 80% N2 and 20% CO2 gas atmosphere. Solution C is autoclaved under 100% N2 gas. Solutions D and E are prepared under 100% N2 gas atmosphere and sterilized by filtration. To complete the medium solutions B to E are added to the sterile solution A in the sequence as indicated. Final pH of the medium should be 7.1 - 7.4. | |||||
| 2 After inoculation add 1 bar overpressure of sterile 80% H2 and 20% CO2 gas mixture. | |||||
Solution A #
952
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
Na2SO4 | 3.00 | g |
|
|
KH2PO4 | 0.20 | g |
|
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NH4Cl | 0.30 | g |
|
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NaCl | 21.00 | g |
|
|
MgCl2 x 6 H2O | 3.00 | g |
|
|
KCl | 0.50 | g |
|
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CaCl2 x 2 H2O | 0.15 | g |
|
|
Yeast extract | 0.50 | g |
|
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Ascorbic acid | 0.10 | g |
|
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Trace element solution SL-10 | 1.00 | ml |
|
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Selenite-tungstate solution | 1.00 | ml |
|
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Sodium resazurin
(0.1% w/v) |
0.50 | ml |
|
|
Distilled water | 950.00 | ml |
Solution E #
13
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Tris-HCl buffer
(1.0 M solution, pH 8.0) |
7.20 | ml | ||
|
|
Na3-NTA x H2O
(0.5 M solution) |
4.80 | ml | ||
|
|
TiCl3
(15% w/v solution in HCl; Riedel-de Haen) |
0.55 | ml | ||
| 1 Sterilize by filtration. | |||||
Selenite-tungstate solution #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
NaOH | 0.5 | g |
|
|
Na2SeO3 x 5 H2O | 3.0 | mg |
|
|
Na2WO4 x 2 H2O | 4.0 | mg |
|
|
Distilled water | 1000.0 | ml |
Trace element solution SL-10 #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
HCl
(25%) |
10.0 | ml | ||
|
|
FeCl2 x 4 H2O | 1.5 | g | ||
|
|
ZnCl2 | 70.0 | mg | ||
|
|
MnCl2 x 4 H2O | 100.0 | mg | ||
|
|
H3BO3 | 6.0 | mg | ||
|
|
CoCl2 x 6 H2O | 190.0 | mg | ||
|
|
CuCl2 x 2 H2O | 2.0 | mg | ||
|
|
NiCl2 x 6 H2O | 24.0 | mg | ||
|
|
Na2MoO4 x 2 H2O | 36.0 | mg | ||
|
|
Distilled water | 990.0 | ml | ||
| 1 First dissolve FeCl2 in the HCl, then dilute in water, add and dissolve the other salts. Finally 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 |
|
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Vitamin B12 | 1 | mg |
|
|
p-Aminobenzoic acid | 50 | mg |
|
|
(DL)-alpha-Lipoic acid | 50 | mg |
|
|
Distilled water | 1000 | ml |