BBM — Bold's Basal Medium

EDTA-KOH solution for BBM

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

Iron solution (FeSO₄-H₂SO₄) for BBM

per 100 mL · usage rate 1 mL per 1 L of medium · storage at 4 °C in dark glass, 2–3 months

Boric acid solution for BBM

per 100 mL · usage rate 1 mL per 1 L of medium · storage at 4 °C, 12 months

BBM trace element solution (Bold's)

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

In 900 mL of water dissolve NaNO₃ → MgSO₄·7H₂O → NaCl → K₂HPO₄ → KH₂PO₄ → add 1 mL each of the EDTA-KOH solution, the iron solution, the boric acid solution and the trace element solution → last of all, with vigorous stirring, add CaCl₂·2H₂O → make up to 1000 mL → pH 6.6–6.8.

• The four auxiliary solutions are prepared and stored SEPARATELY — this is a fundamental requirement of Bold's formulation: mixing the EDTA-KOH and FeSO₄-H₂SO₄ concentrates produces an immediate precipitate.
• The phosphates and CaCl₂ are added from different stock solutions and always into a dilute solution; otherwise Ca₃(PO₄)₂ precipitates as a characteristic white turbidity.
• The K₂HPO₄ + KH₂PO₄ pair forms a phosphate buffer that maintains a slightly acidic pH; no additional buffer is needed.
• Molar equivalent of nitrogen: NaNO₃ — 2.94 mM, six times lower than in BG-11; for batch cultures 3N-BBM is used.
• The alkali is added first: in neutral water EDTA dissolves extremely slowly. Store this solution separately from the iron solution — mixing the concentrates produces a precipitate.
• The sulfuric acid acts as a preservative: without it Fe²⁺ is rapidly oxidised to Fe³⁺ and precipitates as the hydroxide. The appearance of rusty turbidity indicates that the solution is no longer usable; renew it every 2–3 months.
• In Bold's formulation boron is taken out of the general trace element solution and given its own solution because of its high concentration. It dissolves slowly — warm to 40 °C.
• Molybdenum trioxide MoO₃ is practically insoluble in water — dissolve it beforehand in 0.1 mL of 1 M NaOH. With an equimolar substitution by Na₂MoO₄·2H₂O this step is unnecessary.