Provasoli ES (enriched seawater)

Medium for fastidious marine algae

Application: Marine flagellates, haptophytes, red and brown microalgae, macrophyte zoospores
pH: 7.8–8.2
Source: Provasoli, 1968

Composition

Component	Amount per 1 L
Seawater	980 mL
ES stock solution	20 mL

ES stock solution (Provasoli's)

per 1 L · usage rate 20 mL per 1 L of medium · storage at 4 °C in dark glass, 6 months

Component	Weighed amount / volume
NaNO₃	3.5 g
Na₂ glycerophosphate	0.5 g
Tris	0.5 g
P II metals solution	25 mL
Fe-EDTA (1:1) solution	2.5 mL
Thiamine solution (1 g/L)	0.5 mL
Biotin solution (10 mg/L)	0.5 mL
B₁₂ solution (10 mg/L)	1.0 mL
Distilled water	to 1000 mL

P II metals solution (Provasoli's) — working solution

per 100 mL · usage rate 25 mL per 1 L of ES stock solution · storage at 4 °C, 12 months

Component	Weighed amount / volume
H₃BO₃	114 mg
Na₂EDTA	100 mg
MnSO₄·4H₂O	16.4 mg
Primary FeCl₃ stock solution	1 mL
Primary ZnSO₄ stock solution	1 mL
Primary CoSO₄ stock solution	1 mL
Distilled water	to 100 mL

Primary stock solutions for P II (prepared separately)

each per 100 mL · 1 mL of each is taken · storage at 4 °C, 12 months

Component	Weighed amount / volume
FeCl₃·6H₂O	0.490 g per 100 mL
ZnSO₄·7H₂O	0.220 g per 100 mL
CoSO₄·7H₂O	0.048 g per 100 mL

Fe-EDTA (1:1) solution for Provasoli ES

per 100 mL · usage rate 2.5 mL per 1 L of ES stock solution · storage at 4 °C, 6 months

Component	Weighed amount / volume
Fe(NH₄)₂(SO₄)₂·6H₂O (Mohr's salt)	70.2 mg
Na₂EDTA	60.0 mg
Distilled water	to 100 mL

Preparation

Filter and sterilise the seawater separately. Sterilise the ES stock solution by filtration (it contains vitamins). Add 20 mL of the stock solution aseptically to the cooled seawater.

Notes

Na₂ glycerophosphate is an organic phosphorus source with slow release. It produces practically no calcium phosphate precipitate on autoclaving, which is the medium's chief advantage for marine cultures.
The ES/2 and ES/4 variants (10 and 5 mL of stock solution per litre) are used for oligotrophic species that tolerate a rich medium poorly.
The Tris in the stock solution stabilises the pH after the concentrate is added to the seawater.
Prepared per litre because of the high usage rate (20 mL/L): one litre is enough for 50 L of medium. It contains vitamins and therefore is NOT autoclaved — sterilise by filtration through 0.22 µm. Vitamin solutions: thiamine 100 mg per 100 mL, biotin 1 mg per 100 mL, B₁₂ 1 mg per 100 mL; the final concentrations in the stock solution are 0.5 mg/L thiamine, 5 µg/L biotin and 10 µg/L B₁₂.
Na₂EDTA is dissolved first and the iron salts last.
The FeCl₃ solution is acidified with 1 drop of conc. HCl; otherwise the iron hydrolyses during storage.
Both salts are dissolved together and stirred for 30 min until the complex forms — a stable yellow colour develops with no precipitate. The molar ratio of Fe:EDTA is 1:1 (1.79 mmol/L of each).