Johnson medium for Dunaliella

Hypersaline medium

Application: Dunaliella salina, D. bardawil, D. viridis — accumulation of β-carotene and glycerol
pH: 7.5 (7.0–7.5 for carotenogenesis)
Source: Johnson et al., 1968; Borowitzka & Borowitzka, 1988

Composition

Component	Amount per 1 L
NaCl	87.7 g (1.5 M); range 29–234 g (0.5–4 M)
MgCl₂·6H₂O	1.5 g
MgSO₄·7H₂O	0.5 g
KCl	0.2 g
CaCl₂·2H₂O	0.2 g
KNO₃	1.0 g (or NaNO₃ 0.84 g)
NaHCO₃	0.043 g
KH₂PO₄	0.035 g
FeCl₃·6H₂O	1.45 mg
Na₂EDTA	189 mg
Johnson trace element solution	1.0 mL
Distilled water	to 1000 mL

Trace element solution for Johnson medium

per 100 mL · usage rate 1 mL per 1 L of medium · storage at 4 °C, 6 months

Component	Weighed amount / volume
H₃BO₃	61.0 mg
MnCl₂·4H₂O	41.0 mg
Primary ZnCl₂ stock solution	1 mL
Primary CuCl₂ stock solution	1 mL
Primary molybdate stock solution	1 mL
Primary CoCl₂ stock solution	1 mL
Distilled water	to 100 mL

Primary stock solutions for Johnson medium (prepared separately)

each per 100 mL · 1 mL of each is taken · storage at 4 °C, 6 months

Component	Weighed amount / volume
ZnCl₂	0.410 g per 100 mL
CuCl₂·2H₂O	0.410 g per 100 mL
(NH₄)₆Mo₇O₂₄·4H₂O	0.380 g per 100 mL
CoCl₂·6H₂O	0.150 g per 100 mL

Preparation

Dissolve NaCl in 700 mL of water → MgCl₂·6H₂O → MgSO₄·7H₂O → KCl → KNO₃ → KH₂PO₄ → prepare the FeCl₃ + Na₂EDTA complex separately and add it → 1 mL of the trace element solution → CaCl₂·2H₂O → NaHCO₃ last of all → make up to 1000 mL → pH 7.5 → sterilise by filtration.

Notes

At high salinity the medium is sterilised by FILTRATION: autoclaving causes CaSO₄ and Mg(OH)₂ to precipitate and produces a sharp pH shift.
Dunaliella has no cell wall. On dilution of the medium with distilled water the cells lyse instantly — all manipulations (washing, dilution for counting, subculturing) are carried out with iso-osmotic solutions.
Induction of carotenogenesis: raise NaCl to 3–4 M, reduce nitrogen 5- to 10-fold and increase irradiance to 300–500 µmol photons·m⁻²·s⁻¹. The culture then changes colour from green to orange-red.
The ratio of Na₂EDTA to iron here is unusually high (189 mg against 1.45 mg of FeCl₃·6H₂O): the excess chelator also binds heavy metals, which are toxic at high ionic strength.
In the original formulation the solution is ten times more dilute and is added at 10 mL per litre of medium. Here it has been rescaled to the usage rate of 1 mL/L used throughout this reference — the final trace element concentrations in the medium are identical to the original.
The four solutions are stored in separate bottles. ZnCl₂ is hygroscopic — weigh the amount quickly and close the jar immediately.