BG-11₀ (nitrogen-free BG-11)

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

The procedure is the same as for BG-11, except that NaNO₃ is omitted and ferric ammonium citrate is replaced by ferric citrate. CaCl₂·2H₂O is added last.

• Key point: ferric ammonium citrate MUST NOT be left in a nitrogen-free medium — it contains NH₄⁺ and the medium is then no longer nitrogen-free. It must be replaced by ferric citrate or Fe-EDTA.
• Before inoculation the inoculum is washed twice in BG-11₀ by centrifugation (3000 g, 5 min) to remove residual nitrate — otherwise heterocysts do not form and the experimental result is unreliable.
• The variant BG-11₀ + 2–5 mM NaHCO₃ (0.17–0.42 g/L) is used for cultivation without CO₂ sparging.
• The first signs of the switch to nitrogen fixation are the appearance of heterocysts after 18–36 h and a transient yellowing of the culture caused by degradation of the phycobilisomes.
• 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.