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A. Media for freshwater cyanobacteria
CASTENHOLZ-DCastenholz D medium
For thermophilic cyanobacteria
- Application
- Thermosynechococcus, Synechococcus lividus, Chloroflexus, Mastigocladus laminosus — hot springs (45–70 °C)
- pH
- 8.2 (variant DH — 7.6–8.0)
- Source
- Castenholz, 1969, 1988
Composition
| Component | Amount per 1 L |
|---|---|
| Nitrilotriacetic acid (NTA) | 100 mg |
| CaSO₄·2H₂O | 60 mg |
| MgSO₄·7H₂O | 100 mg |
| NaCl | 8 mg |
| KNO₃ | 103 mg |
| NaNO₃ | 689 mg |
| Na₂HPO₄ | 111 mg |
| Iron solution (Castenholz) | 1.0 mL |
| Nitsch trace element solution | 1.0 mL |
| Distilled water | to 1000 mL |
Iron solution for Castenholz medium
per 100 mL · usage rate 1 mL per 1 L of medium · storage at 4 °C, 6 months
| Component | Weighed amount / volume |
|---|---|
| FeCl₃·6H₂O | 29 mg |
| Distilled water | to 100 mL |
Nitsch trace element solution
per 100 mL of solution · usage rate 1 mL per 1 L of medium · storage at 4 °C, 12 months
| Component | Weighed amount / volume |
|---|---|
| H₃BO₃ | 50 mg |
| MnSO₄·H₂O | 50 mg |
| ZnSO₄·7H₂O | 5.0 mg |
| CuSO₄·5H₂O | 2.5 mg (1 mL of a primary stock solution at 2.5 g/L) |
| Na₂MoO₄·2H₂O | 2.5 mg (1 mL of a primary stock solution at 2.5 g/L) |
| CoCl₂·6H₂O | 2.5 mg (1 mL of a primary stock solution at 2.5 g/L) |
| Distilled water | to 100 mL |
Preparation
Strictly: dissolve NTA first → CaSO₄·2H₂O (dissolves slowly, stir for 10–15 min) → MgSO₄·7H₂O → NaCl → KNO₃ → NaNO₃ → Na₂HPO₄ → 1 mL of the iron solution → 1 mL of the Nitsch solution → make up to 1000 mL → pH 8.2.
Notes
- Departing from this order of addition leads to precipitation of CaSO₄ and calcium phosphate: the NTA must already be in solution BEFORE the calcium, otherwise chelation does not work.
- Cultivation at 45–55 °C is carried out in closed vessels fitted with a water seal — otherwise evaporation raises the salinity of the medium by 20–30 % within a week.
- The DH variant (Castenholz D with HEPES, or with a lowered pH) is used for strains sensitive to high alkalinity.
- EDTA is not added to this solution: chelation is provided by the nitrilotriacetic acid (NTA) that is a component of the medium itself.
- The three primary stock solutions are prepared at 250 mg per 100 mL each and 1 mL is taken from each. The finished solution is acidified with 1 drop of conc. H₂SO₄ — this prevents hydroxides from precipitating during storage.