Guillard, R.R.L. and Ryther, J.H. 1962. Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea Cleve. Can. J. Microbiol. 8: 229-239
Guillard, R.R.L. 1975. Culture of phytoplankton for feeding marine invertebrates. pp 26-60. In Smith, W.L. and Chanley, M.H. (Eds) Culture of Marine Invertebrate Animals, Plenum Press, New York.
Prepare stocks using purified water. To 950 mL filtered seawater or artificial seawater (see recipe below) add stocks:
| Stock | Substance | Concentration | Amount added to medium (mL/L) | Molar concentration in final medium |
|---|---|---|---|---|
| 1. | NaNO3 | 75 g/L dH2O | 1 mL | 8.83 x 10-4 M |
| 2. | NaH2PO4•H2O | 5 g/L dH2O | 1 mL | 3.63 x 10-5 M |
| 3. | Na2SiO3•9H2O* | 30 g/L dH2O | 1 mL | 1.07 x 10-4 M |
| 4. | f/2 trace metal solution | See below | 1 mL | |
| 5. | f/2 vitamin solution | See below | 0.5 mL |
*Note: Autoclaved f/2 medium may produce extensive silica precipitate. If this is the case, you may choose to delete the silicate when it is not required by the alga.
Make final volume up to 1 L with filtered seawater (or artificial seawater) and adjust to pH 8 using 1N HCl or 1N NaOH. Autoclave. If seawater is not available, use artificial seawater as in ESAW. Combine equal parts of Salt Solution I and Salt Solution II to make the seawater base.
| Salt solution I | Concentration (g/L dH2O) |
|---|---|
| 1. NaCl | 20.758 g/L |
| 2. Na2SO4 | 3.477 g/L |
| 3. KCl | 0.587 g/L |
| 4. NaHCO3 | 0.170 g/L |
| 5. NaBr | 0.0746 g/L |
| 6. H3BO3 | 0.0225 g/L |
| 7. NaF | 0.0027 g/L |
| Salt solution II | Concentration (g/L dH2O) |
|---|---|
| 1. MgCl2•6H2O | 9.395 g/L |
| 2. CaCl2•2H2O | 1.316 g/L |
| 3. SrCl2•6H2O | 0.0214 g/L |
If you experience excessive precipitation when using artificial seawater, you may choose to add the following 2 ingredients after adjusting the pH to 8.0 and before autoclaving:
| Substance | Concentration |
|---|---|
| NaHCO3 | 0.12 g/L |
| HCl (1N) | 1.44 mL/L |
f/2 trace metal solution
To 950 mL dH2O add:
| Substance | Concentration | Amount added to stock | Molar concentration in final medium |
|---|---|---|---|
| 1. FeCl3•6H2O | – | 3.15 g | 1 x 10-5 M |
| 2. Na2EDTA•2H2O | – | 4.36 g | 1 x 10-5 M |
| 3. CuSO4•5H2O | 9.8 g/L dH2O | 1 mL | 4 x 10-8 M |
| 4. Na2MoO4•2H2O | 6.3 g/L dH2O | 1 mL | 3 x 10-8 M |
| 5. ZnSO4•7H2O | 22.0 g/L dH2O | 1 mL | 8 x 10-8 M |
| 6. CoCl2•6H2O | 10.0 g/L dH2O | 1 mL | 5 x 10-8 M |
| 7. MnCl2•4H2O | 180.0 g/L dH2O | 1 mL | 9 x 10-7 M |
Make final volume up to 1 L with dH2O.
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