683 .
TISSIERELLA (AMO.1) MEDIUM
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Equipment needed:
Autoclave
Filter
Gassing station
Hungate tubes
Final pH:
8.3
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
NaCl | 5.80 | g | ||
|
|
MgCl2 x 6 H2O | 0.40 | g | ||
|
|
KCl | 0.30 | g | ||
|
|
CaCl2 x 2 H2O | 0.15 | g | ||
|
|
NH4Cl | 0.27 | g | ||
|
|
KH2PO4 | 0.20 | g | ||
|
|
Trace element solution SL-10 | 1.00 | ml | ||
|
|
Selenite-tungstate solution | 1.00 | ml | ||
|
|
Yeast extract
(OXOID) |
10.00 | g | ||
|
|
Trypticase peptone
(BD BBL) |
0.50 | g | ||
|
|
L-Serine | 2.00 | g | ||
|
|
L-Threonine | 2.00 | g | ||
|
|
N-methylhydantoin
(SIGMA) |
5.64 | g | ||
|
|
Sodium resazurin
(0.1% w/v) |
0.50 | ml | ||
|
|
NaHCO3 | 4.50 | g | ||
|
|
Wolin's vitamin solution (10x) | 1.00 | ml | ||
|
|
L-Cysteine HCl x H2O | 0.50 | g | ||
|
|
Distilled water | 1000.00 | ml | ||
| 1 Dissolve ingredients (except bicarbonate, cysteine and vitamins) and sparge medium with 100% N2 gas for 30 - 45 min to make it anoxic. Adjust pH to 8.3 with 10 N NaOH, add bicarbonate and cysteine and dissolve. Switch gas phase to 80% N2 and 20% CO2 gas mixture and flush only the head space of the medium vessel. Dispense medium under 80% N2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave 15 min at 121°C. Add vitamins to the autoclaved medium from an anoxic stock solution prepared under 100% N2 gas atmosphere and sterilized by filtration. Adjust pH of complete medium to 8.3, if necessary. | |||||
| 2 Note: If creatinine is used as the substrate replacing N-methylhydantoin, prepare separately an anoxic, filter sterilized stock solution of creatinine under 100% N2 gas atmosphere. | |||||
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. | |||||
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 |
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 |