858 .
ANAEROBIC CHOLESTEROL MEDIUM
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Equipment needed:
Anaerobic workbench
Autoclave
Filter
Final pH:
6.8 - 7.2
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Solution A | 9.000 | ml | ||
|
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Solution B | 0.500 | ml | ||
|
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Solution C | 0.025 | ml | ||
|
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Solution D | 0.050 | ml | ||
|
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Solution E | 0.025 | ml | ||
|
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Solution F | 0.500 | ml | ||
| 1 Prepare solution A, distribute approbiate amounts of cholesterol (e.g. 5 mg for 10 ml medium) and solution A in anaerobic tubes, and autoclave anaerobically under 80% N2 + 20% CO2. Solutions B and F are also autoclaved under these conditions. Solution C, D and E may be prepared aerobically. After cooling, add to 9 ml solution A: 500 µl sol. B, 500 µl sol. F, 25 µl (one drop) sol. C and E, each, and 50 µl sol. D under anaerobic conditions. Adjust pH to 7.0±0.2. | |||||
Solution A #
200
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
MgSO4 x 7 H2O | 0.10 | g |
|
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NH4Cl | 0.10 | g |
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CaCl2 | 0.02 | g |
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NaNO3 | 0.16 | g |
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Distilled water | 200.00 | ml |
Solution C #
1
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Trace element solution SL-10 | 1 | ml | ||
| 1 Trace elements SL-10 (see medium 320) | |||||
Vitamin solution #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Vitamin B12 | 50 | mg | ||
|
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Pantothenic acid | 50 | mg | ||
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Riboflavin | 50 | mg | ||
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Pyridoxamine hydrochloride | 10 | mg | ||
|
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Biotin | 20 | mg | ||
|
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Folic acid | 20 | mg | ||
|
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Nicotinic acid | 25 | mg | ||
|
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Nicotine amide | 25 | mg | ||
|
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alpha-lipoic acid | 50 | mg | ||
|
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p-Aminobenzoic acid | 50 | mg | ||
|
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Thiamine-HCl x 2 H2O | 50 | mg | ||
|
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Distilled water | 1000 | ml | ||
| 1 Stir for some hours, filter sterilize the solution. | |||||
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 | ||
|
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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 | ||
|
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Na2MoO4 x 2 H2O | 36.0 | mg | ||
|
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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. | |||||
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 |
|
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Distilled water | 1000.0 | ml |