Reference: Olaveson, Mary M. and Pamela M. Stokes. 1989. Responses of the acidophilic alga Euglena mutabilis (Euglenophyceae) to carbon enrichment at pH 3. Journal of Phycology. 25: 529-539.
| Stock | Substance | Concentration | Amount added to medium (mL/L) |
|---|---|---|---|
| 1. | (NH4)2SO4 | 50 g/L | 10 mL |
| 2. | CaCl2•2H2O | 1 g/L | 10 mL |
| 3. | MgSO4•7H2O | 50 g/L | 10 mL |
| 4. | KH2PO4 | 30 g/L | 10 mL |
| 5. | NaCl | 3.0 g/L | 10 mL |
| 6. | Na2EDTA•2H2O | 20 g/L | 0.5 mL |
| 7. | Trace metal solution | See below | 1 mL |
| 8. | FeSO4•7H2O | 4.98 g/L | 1 mL |
| H2SO4 (concentrated) | 1 mL/L | ||
| 9. | f/2 vitamin solution | See below | 1 mL |
If adding agar, make medium double strength and adjust the pH to 3.5. Autoclave separately from agar. Add the agar to equivalent volume of purified water (for most purposes use 12 g/L) and autoclave. When cooled slightly, combine the two. This will prevent autolyzing of the agar at low pH (e.g. < 4.0).
Trace metal solution
| Substance | Concentration (g/L) |
|---|---|
| 1. H3BO3 | 2.86 g |
| 2. MnCl2•4H2O | 1.81 g |
| 3. ZnSO4•7H2O | 0.222 g |
| 4. Na2MoO4•2H2O | 0.390 g |
| 5. CuSO4•5H2O | 0.079 g |
| 6. Co(NO3)2•6H2O | 0.0494 g |
Dissolve each of the above substances separately prior to adding the next on the list.
f/2 vitamin solution
Substock solutions:
First prepare separate substock solutions of cyanocobalamin (Vitamin B12) and biotin in purified water (e.g., Milli-Q, Nanopure, distilled water).
| Substock | Substance | Concentration |
|---|---|---|
| 1. | Vitamin B12 (Cyanocobalamin) | 10 mg/10 mL |
| 2. | Biotin | 5 mg/50 mL |
Working stock solution:
Then prepare the final working solution by combining the following amounts of the two substock solutions plus thiamine HCl to 100 mL of purified water.
| Substance | Concentration (per 100 mL) |
|---|---|
| 1. Vitamin B12 (Cyanocobalamin) | 0.1 mL |
| 2. Biotin | 1.0 mL |
| 3. Thiamine HCl | 20 mg |
Dispense the working solution as 1 mL aliquots in cryovials for convenient use when preparing the medium. Store the remainder of the primary stocks and working solution in polyethylene bottles. Wrap vials/bottles with Parafilm to avoid moisture loss and store all solutions frozen.
References: Guillard and Ryther 1962, Guillard 1975
Modified MAM for metal uptake experiments
Make as in the regular formulation but reduce levels of FeSO4•7H2O + H2SO4, Na2EDTA•2H2O, and trace metal solution to 10% of the above levels.