1159 .
MOLLICUTES H-RF3 MEDIUM
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Equipment needed:
Anaerobic workbench
Autoclave
Filter
Final pH:
7.0
Select a strain (optional):
ml
| 1 Mix components and adjust pH to 7.5. Bring to a boil and cool down under a N2-CO2 (4:1, v/v) gas stream. Distribute the medium into culture vessels (e.g., 20 ml medium in 60 ml serum bottles) under the same gas mixture, seal with butyl rubber stoppers and autoclave. After cooling, aseptically and anaerobically add per liter the following solutions (autoclaved or *filter-sterilized): | |||||
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
KH2PO4 | 0.14 | g | ||
|
|
MgCl2 x 6 H2O | 0.20 | g | ||
|
|
CaCl2 x 2 H2O | 0.15 | g | ||
|
|
NH4Cl | 0.54 | g | ||
|
|
Casamino acids
(BD-Difco) |
1.00 | g | ||
|
|
Yeast extract | 1.00 | g | ||
|
|
Trace minerals | 1.00 | ml | ||
|
|
Resazurin | 0.50 | mg | ||
|
|
Distilled water | 1000.00 | ml | ||
|
|
NaHCO3
(*, 8%) |
30.00 | ml | ||
|
|
Trace vitamins
(*) |
10.00 | ml | ||
|
|
L-Cysteine HCl x H2O
(5%) |
6.00 | ml | ||
|
|
Na2S x 9 H2O
(5%) |
6.00 | ml | ||
| 2 Readjust pH to 7.0, if necessary. | |||||
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 |