ASN-III

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

Dissolve NaCl → MgCl₂·6H₂O → MgSO₄·7H₂O → KCl → NaNO₃ → K₂HPO₄ → citric acid → ferric ammonium citrate → Na₂EDTA → Na₂CO₃ → 1 mL of A5 + Co → CaCl₂·2H₂O last, with stirring → make up to 1000 mL → pH 7.5–8.0.

• Nitrogen-free variant ASN-III₀: omit NaNO₃ and replace ferric ammonium citrate with ferric citrate.
• For halotolerant strains NaCl is varied from 10 to 40 g/L, with MgCl₂·6H₂O and MgSO₄·7H₂O changed proportionally so as to preserve the ionic ratios of seawater.
• The Mg:Ca ratio in ASN-III is substantially higher than in freshwater media — when adapting a freshwater strain to ASN-III, halve the concentration of all salts (½ ASN-III) and raise it gradually.
• 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.