1116 .
STEROIDOBACTER MEDIUM
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Equipment needed:
Anaerobic workbench
Autoclave
Filter
Final pH:
7.2
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
NaCl | 1.00 | g | ||
|
|
MgCl2 x 6 H2O | 0.40 | g | ||
|
|
KH2PO4 | 0.20 | g | ||
|
|
KCl | 0.50 | g | ||
|
|
NH4Cl | 0.25 | g | ||
|
|
CaCl2 x 2 H2O | 0.15 | g | ||
|
|
Na2SO4 | 0.07 | g | ||
|
|
NaNO3 | 0.42 | g | ||
|
|
Distilled water | 1000.00 | ml | ||
| 1 For growth with testosterone, dissolve testosterone in acetone (20 mg/ml), dispense portions of the solution, e.g. 0.1 ml portions for 10 ml medium, to anaerobic culture tubes. Let the solvent evaporate to dryness. Dispense 10 ml portions of medium and stream with N2/CO2 (80:20). Close the vessels and autoclave. Treat the vessels in an ultrasonic bath to detach and suspend the testosterone. | |||||
| 2 For growth with heptanoate, omit testosterone. Sterilize the gassed medium in anaerobic vessels by autoclaving. After cooling, add 325 mg/l heptanoic acid from a 20fold, pH adjusted, filter sterilized anaerobic stock solution. | |||||
| 3 After autoclaving, add (per 10 ml) 0.3 ml NaHCO3-solution (84 g/l, autoclaved under CO2-atmosphere), 1 µl trace element solution SL 10 (see below), 1 µl selenite tungstate solution (see below), and 1 µl vitamin solution (see below). Adjust pH to 7.2. Incubate the cultures in the dark for up to 4 weeks. Briefly shake the cultures once per day. | |||||
Trace element solution SL-10 #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
HCl
(25%; 7.7 M) |
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 |
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 |
Vitamin solution #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Vitamin B12 | 50 | mg | ||
|
|
Pantothenic acid | 50 | mg | ||
|
|
Riboflavin | 50 | mg | ||
|
|
Pyridoxamine hydrochloride | 10 | mg | ||
|
|
Biotin | 20 | mg | ||
|
|
Folic acid | 20 | mg | ||
|
|
Nicotinic acid | 25 | mg | ||
|
|
Nicotine amide | 25 | mg | ||
|
|
alpha-lipoic acid | 50 | mg | ||
|
|
p-Aminobenzoic acid | 50 | mg | ||
|
|
Thiamine-HCl x 2 H2O | 50 | mg | ||
|
|
Distilled water | 1000 | ml | ||
| 1 Stir for some hours, filter sterilize the solution. | |||||