Modified Zarrouk medium (production-scale)

A5 + Co trace element solution

per 100 mL of solution · usage rate 1 mL per 1 L of medium (sufficient for 100 L) · storage at 4 °C in dark glass, 12 months

Allow the water to settle and filter it → dissolve NaNO₃ → K₂SO₄ → NaCl → MgSO₄·7H₂O → add the FeSO₄ + EDTA complex → 1 mL of A5 → CaCl₂ (or omit it if the water is hard) → prepare a separate solution of NaHCO₃ + K₂HPO₄ and pour it in last, with stirring.

• Reducing NaHCO₃ to 8 g/L is permissible only when CO₂ is supplied to the culture: the bicarbonate serves both as a carbon source and as a means of maintaining the alkalinity that protects against contamination.
• When urea is used, the concentration of free NH₃ rises sharply at pH 10 and becomes toxic. The safe limit is no more than 3–5 mM total ammonium nitrogen per addition; urea is supplied in small, divided doses.
• The hardness of the technical-grade water is taken into account by reducing the CaCl₂ dose, up to omitting it entirely: at a hardness above 4 meq/L calcium is already present in excess.
• In production the A5 solution is often replaced by technical-grade iron sulfate and micronutrient fertilisers; in that case monitoring of boron and molybdenum is mandatory — it is their deficiency that limits growth first.
• Dissolve the salts one at a time, in the order given; H₃BO₃ dissolves slowly — warming to 40 °C is permissible. Cobalt is added from a primary stock solution: a weighed amount of 4.94 mg cannot be measured on an ordinary balance. Primary Co stock solution: 494 mg Co(NO₃)₂·6H₂O in 100 mL of water, take 1 mL. Without cobalt the solution is simply called "A5". The appearance of a precipitate or turbidity indicates that the solution is no longer usable.