827 .
DEHALOCOCCOIDES MEDIUM
What is this page?
Equipment needed:
Anaerobic workbench
Autoclave
Filter
Final pH:
9.0
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
NaCl | 1.000 | g | ||
|
|
MgCl2 x 6 H2O | 0.410 | g | ||
|
|
NH4Cl | 0.270 | g | ||
|
|
KCl | 0.520 | g | ||
|
|
CaCl2 x 2 H2O | 0.015 | g | ||
|
|
KH2PO4 | 0.200 | g | ||
|
|
FeCl2 solution | 1.000 | ml | ||
|
|
Trace element solution | 1.000 | ml | ||
|
|
Selenite-tungstate solution | 1.000 | ml | ||
|
|
Sodium acetate | 0.410 | g | ||
|
|
Resazurin | 0.250 | mg | ||
|
|
Distilled water | 1000.000 | ml | ||
| 1 Mix components thoroughly, bring to a boil and cool down under a N2 atmosphere. Add the following solutions from anaerobic stocks, then dispense the medium into culture vessels leaving 40 to 50% volume of a gas phase (e.g., 30 ml of the medium in a 50 ml serum bottle) and autoclave under a N2 atmosphere: | |||||
|
|
L-Cysteine HCl x H2O
(5%) |
1.000 | ml | ||
|
|
Na2S x 9 H2O
(5%) |
1.000 | ml | ||
| 2 After cooling, replace the gas phase with a N2-CO2-H2 (8:1:1, v/v) gas mixture. Add per liter the following filter-sterilized solutions from anaerobic stocks: | |||||
|
|
NaHCO3
(8%) |
10.000 | ml | ||
|
|
MOPS
(pH 7.4, 1.0 M) |
20.000 | ml | ||
|
|
25 mM Ti(III) NTA solution
(25 mM) |
20.000 | ml | ||
|
|
Vitamin solution | 1.000 | ml | ||
| 3 To complete the medium, add per liter the following filter-sterilized chlorosolvent: | |||||
|
|
Trichloroethylene
(10 mg/ml in methanol) |
0.700 | ml | ||
| 4 Check final pH to be about 7.2. | |||||
Vitamin solution #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
Biotin | 10 | mg |
|
|
Folic acid | 2 | mg |
|
|
Pyridoxine hydrochloride | 250 | mg |
|
|
Thiamine HCl | 20 | mg |
|
|
Riboflavin | 5 | mg |
|
|
Nicotinic acid | 100 | mg |
|
|
DL-Calcium pantothenate | 10 | mg |
|
|
Vitamin B12 | 25 | mg |
|
|
p-Aminobenzoic acid | 50 | mg |
|
|
Lipoic acid | 5 | mg |
|
|
Distilled water | 1000 | ml |
25 mM Ti(III) NTA solution #
300
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Nitrilotriacetic acid | 9.6 | g | ||
|
|
Water
(anaerobic ) |
300.0 | ml | ||
| 1 To prepare anaerobic water, boil 1.0 L distilled water and cool down under a stream of oxygen-free N2 gas. Mix components under N2 gas. Adjust pH to 9.0 with NaOH. Slowly add 9.6 ml of 20% TiCl3 to the chelator solution. Keep the pH above 2.0 by the addition of anaerobic saturated Na2CO3 solution. Adjust pH to 7.0 with Na2CO3 solution, adjust pH to 7.0 and bring the final volume to 500 ml with the anaerobic water. Filter-sterilize and keep the solution under a N2 gas atmosphere. | |||||
FeCl2 solution #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
HCl
(7.7 M, 25%) |
10.0 | ml |
|
|
FeCl2 x 4 H2O | 1.5 | g |
|
|
Distilled water | 990.0 | ml |
Trace element solution #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
ZnCl2 | 70 | mg |
|
|
MnCl2 x 4 H2O | 100 | mg |
|
|
H3BO3 | 6 | mg |
|
|
CoCl2 x 6 H2O | 190 | mg |
|
|
CuCl2 x 2 H2O | 2 | mg |
|
|
NiCl2 x 6 H2O | 24 | mg |
|
|
Na2MoO4 x 2 H2O | 36 | mg |
|
|
Distilled water | 1000 | ml |
Selenite-tungstate solution #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
NaOH | 0.4 | g |
|
|
Na2SeO3 x 5 H2O | 6.0 | mg |
|
|
Na2WO4 x 2 H2O | 8.0 | mg |
|
|
Distilled water | 1000.0 | ml |