f/2 (Guillard's medium)

The principal medium worldwide for marine phytoplankton

Application: Nannochloropsis, Isochrysis, Tetraselmis, Pavlova, marine diatoms and flagellates; aquaculture
pH: 8.0–8.2 (the natural pH of seawater)
Source: Guillard & Ryther, 1962; Guillard, 1975

Composition

Component	Amount per 1 L
Seawater (filtered, 0.22 µm)	1000 mL
NaNO₃	75 mg (1 mL of a solution of 7.5 g per 100 mL)
NaH₂PO₄·H₂O	5 mg (1 mL of a solution of 0.5 g per 100 mL)
Na₂SiO₃·9H₂O	30 mg (f/2 + Si only, for diatoms)
f/2 trace element solution	1.0 mL
Vitamin solution	0.5 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

Filter the seawater and autoclave it (or pasteurise it), then cool. Prepare concentrates in 100 mL: NaNO₃ 7.5 g and NaH₂PO₄·H₂O 0.5 g — take 1 mL of each per litre. To the cooled sterile seawater add aseptically 1 mL of the nitrate, 1 mL of the phosphate, 1 mL of the trace element solution and 0.5 mL of the vitamin solution; the silicate is added as a separate sterile solution (3 g per 100 mL), 1 mL per litre.

Notes

The name "f/2" denotes a twofold dilution of the original "f" medium. f/4 and f/10 (for oligotrophic species) and "f" itself (double strength, for intensive growth) are also used.
The seawater is sterilised SEPARATELY and the nutrients are added after cooling: autoclaving seawater together with phosphate and silicate yields colloidal SiO₂ and a carbonate precipitate.
Pasteurisation (80 °C for 30 min, three times at 24 h intervals) is a gentler alternative to autoclaving that preserves the carbonate equilibrium of the seawater.
Molar equivalents: NaNO₃ — 882 µM; NaH₂PO₄·H₂O — 36.2 µM (an N:P ratio of ≈ 24:1).
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.