448 .
KA22 MEDIUM
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Equipment needed:
Autoclave
Filter
Final pH:
6.5
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Sea Salt
(Sigma) |
30.00 | g | ||
|
|
Mg(NO3)2 x 6 H2O | 2.54 | g | ||
|
|
KH2PO4 | 0.30 | g | ||
|
|
NaHCO3 | 1.00 | g | ||
|
|
MES | 1.95 | g | ||
|
|
Sulfur
(powder) |
12.00 | g | ||
|
|
Trace minerals | 10.00 | ml | ||
|
|
Trace vitamins | 0.50 | ml | ||
|
|
Na2S x 9 H2O | 0.50 | g | ||
|
|
Resazurin | 1.00 | mg | ||
| 1 Mix ingredients except sulfur, NaHCO3 and Na2S x 9H2O, adjust pH to 6.5 and autoclave. Filter-sterilize 8% NaHCO3 solution and add to the medium. Steam sulfur for 3 hr on each of 3 successive days. Separately autoclave 5% Na2S x 9H2O solution under a N2 atmosphere. Aseptically distribute the medium into culture vessels containing appropriate amount of sulfur (e.g., 20 ml culture medium in 120 ml serum bottle containing 0.24 g sulfur) under a H2-CO2 (4:1, v/v) gas mixture and seal with butyl rubber stoppers. Prior to inoculation, add the sterile Na2S x 9H2O solution and readjust pH to 6.5, if necessary. Pressurize the inoculated bottles to 200 kPa H2-CO2 (4:1, v/v). | |||||
Trace minerals #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Nitrilotriacetic acid | 1.50 | g | ||
|
|
MgSO4 x 7 H2O | 3.00 | g | ||
|
|
MnSO4 x n H2O | 0.50 | g | ||
|
|
NaCl | 1.00 | g | ||
|
|
FeSO4 x 7 H2O | 0.10 | g | ||
|
|
CoSO4 x 7 H2O | 0.10 | g | ||
|
|
CaCl2 x 2 H2O | 0.10 | g | ||
|
|
ZnSO4 x 7 H2O | 0.10 | g | ||
|
|
CuSO4 x 5 H2O | 0.01 | g | ||
|
|
AlK(SO4)2 | 0.01 | g | ||
|
|
H3BO3 | 0.01 | g | ||
|
|
Na2MoO4 x 2 H2O | 0.01 | g | ||
|
|
Distilled water | 1000.00 | ml | ||
| 1 Dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH solution. Then proceed to add minerals. Adjust final pH to 7.0. | |||||
Trace vitamins #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
Biotin | 2.0 | mg |
|
|
Folic acid | 2.0 | mg |
|
|
Pyridoxine hydrochloride | 10.0 | mg |
|
|
Thiamine HCl | 5.0 | mg |
|
|
Riboflavin | 5.0 | mg |
|
|
Nicotinic acid | 5.0 | mg |
|
|
Calcium pantothenate | 5.0 | mg |
|
|
Vitamin B12 | 0.1 | mg |
|
|
p-Aminobenzoic acid | 5.0 | mg |
|
|
Lipoic acid | 5.0 | mg |
|
|
Distilled water | 1000.0 | ml |