318b .
ANAEROTIGNUM NEOPROPIONICUM MEDIUM
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Equipment needed:
Autoclave
Filter
Gassing station
Hungate tubes
Final pH:
7.0 - 7.2
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
KH2PO4 | 0.30 | g | ||
|
|
NaCl | 0.60 | g | ||
|
|
MgCl2 x 6 H2O | 0.10 | g | ||
|
|
CaCl2 x 2 H2O | 0.08 | g | ||
|
|
Trace element solution | 10.00 | ml | ||
|
|
NH4Cl | 1.00 | g | ||
|
|
Yeast extract
(OXOID) |
0.50 | g | ||
|
|
Trypticase peptone
(BD BBL) |
0.50 | g | ||
|
|
Sodium resazurin
(0.1% w/v) |
0.50 | ml | ||
|
|
KHCO3 | 4.00 | g | ||
|
|
Ethanol | 1.30 | ml | ||
|
|
Wolin's vitamin solution (10x) | 1.00 | ml | ||
|
|
L-Cysteine HCl x H2O | 0.30 | g | ||
|
|
Na2S x 9 H2O | 0.30 | g | ||
|
|
Distilled water | 1000.00 | ml | ||
| 1 Dissolve ingredients (except bicarbonate, ethanol, vitamins, cysteine and sulfide), then sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Add and dissolve bicarbonate and adjust pH to 7.0, then dispense under 80% N2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. After sterilization add ethanol, cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas. Vitamins are prepared under 100% N2 gas and sterilized by filtration. Adjust pH of complete medium to 7.0 - 7.2, if necessary. | |||||
Trace element solution #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Nitrilotriacetic acid
(NTA) |
12.80 | g | ||
|
|
FeCl2 x 4 H2O | 1.00 | g | ||
|
|
MnCl2 x 4 H2O | 0.10 | g | ||
|
|
CoCl2 x 6 H2O | 0.03 | g | ||
|
|
CaCl2 x 2 H2O | 0.10 | g | ||
|
|
ZnCl2 | 0.10 | g | ||
|
|
CuCl2 | 0.02 | g | ||
|
|
H3BO3 | 0.01 | g | ||
|
|
Na2MoO4 x 2 H2O | 0.03 | g | ||
|
|
NiCl2 x 6 H2O | 0.10 | g | ||
|
|
NaCl | 1.00 | g | ||
|
|
Na2SeO3 x 5 H2O | 0.03 | g | ||
|
|
Na2WO4 x 2 H2O | 0.04 | g | ||
|
|
Distilled water | 1000.00 | ml | ||
| 1 First dissolve NTA in 200 ml of distilled water and adjust pH to 6.5 with KOH, then dissolve mineral salts. Finally adjust pH to 6.5 with KOH and 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 |
|
|
Vitamin B12 | 1 | mg |
|
|
p-Aminobenzoic acid | 50 | mg |
|
|
(DL)-alpha-Lipoic acid | 50 | mg |
|
|
Distilled water | 1000 | ml |