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D. Media for freshwater green microalgae
WCWC (Guillard's Freshwater medium)
Freshwater analogue of f/2
- Application
- Freshwater diatoms, green, golden and cryptophyte algae; the CCMP collection
- pH
- 7.2–7.6
- Source
- Guillard & Lorenzen, 1972
Composition
| Component | Amount per 1 L |
|---|---|
| NaNO₃ | 85.01 mg |
| CaCl₂·2H₂O | 36.76 mg |
| MgSO₄·7H₂O | 36.97 mg |
| NaHCO₃ | 12.60 mg |
| K₂HPO₄ | 8.71 mg |
| Na₂SiO₃·9H₂O | 28.42 mg |
| Tris | 500 mg (or HEPES 2.38 g) |
| f/2 trace element solution | 1.0 mL |
| Vitamin solution | 0.5 mL |
| Distilled water | to 1000 mL |
f/2 trace element solution (Guillard's) — working 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 |
|---|---|
| Na₂EDTA·2H₂O | 436 mg |
| FeCl₃·6H₂O | 315 mg |
| MnCl₂·4H₂O | 18.0 mg (or 1 mL of primary stock solution No. 1) |
| Primary stock solution No. 2 (ZnSO₄) | 1 mL |
| Primary stock solution No. 3 (CoCl₂) | 1 mL |
| Primary stock solution No. 4 (CuSO₄) | 1 mL |
| Primary stock solution No. 5 (Na₂MoO₄) | 1 mL |
| Distilled water | to 100 mL |
Primary stock solutions of f/2 trace elements (each prepared separately)
each per 100 mL · 1 mL of each is taken per 100 mL of working solution · storage at 4 °C, 12 months
| Component | Weighed amount / volume |
|---|---|
| No. 1 — MnCl₂·4H₂O | 1.80 g per 100 mL |
| No. 2 — ZnSO₄·7H₂O | 0.220 g per 100 mL |
| No. 3 — CoCl₂·6H₂O | 0.100 g per 100 mL |
| No. 4 — CuSO₄·5H₂O | 0.098 g per 100 mL |
| No. 5 — Na₂MoO₄·2H₂O | 0.063 g per 100 mL |
Vitamin solution (standard f/2 formulation) — working solution
per 100 mL of solution · usage rate 0.5 mL per 1 L of medium · storage at −20 °C in 1–2 mL aliquots, 6 months
| Component | Weighed amount / volume |
|---|---|
| Thiamine·HCl (vitamin B₁) | 20 mg |
| Primary biotin stock solution | 1 mL |
| Primary B₁₂ stock solution | 1 mL |
| Distilled water | to 100 mL |
Primary vitamin stock solutions (prepared separately)
each per 100 mL · 1 mL of each is taken per 100 mL of working solution · storage at −20 °C, 6 months
| Component | Weighed amount / volume |
|---|---|
| Biotin (vitamin H) | 10 mg per 100 mL |
| Cyanocobalamin (vitamin B₁₂) | 10 mg per 100 mL |
Preparation
Dissolve NaNO₃ → MgSO₄·7H₂O → K₂HPO₄ → NaHCO₃ → Tris → 1 mL of the trace element solution → CaCl₂·2H₂O → adjust the pH to 7.2–7.6 → add Na₂SiO₃·9H₂O last → make up to 1000 mL → autoclave → add 0.5 mL of the vitamin solution aseptically to the cooled medium.
Notes
- The silicate is added last and only to a medium whose pH has already been set — otherwise amorphous SiO₂ precipitates. For non-diatom cultures Na₂SiO₃ is omitted.
- The vitamins are added after autoclaving and cooling, by sterile filtration.
- The Tris-free variant is used for cultivation with a CO₂ supply, where a buffer is unnecessary and even interferes with control of the carbonate system.
- Order: dissolve Na₂EDTA first, then FeCl₃ (molar ratio ≈ 1:1, the solution turns straw-yellow), then add 1 mL of each primary stock solution. The trace elements must be added from the primary stock solutions — 0.6–2 mg cannot be weighed out accurately.
- The five solutions are prepared and stored in separate, clearly labelled bottles. They must not be mixed with one another before being added to the working solution.
- Vitamins are NOT autoclaved — sterile filtration through a 0.22 µm membrane only. Thiamine loses 40–70 % of its activity at 121 °C and degrades at pH above 8 even without heating, so vitamins are added to the cooled medium immediately before inoculation. Refreezing of aliquots is not permitted.
- Biotin is poorly soluble in cold water — dissolve it in 1 mL of 50 % ethanol or in warm water at pH 8, then make up the volume. The B₁₂ solution has a characteristic pink-red colour; loss of colour means the vitamin has decomposed.