3N-BBM + V

BBM with triple nitrogen and vitamins

Application: Fastidious strains of green algae and vitamin auxotrophs; the CCAP collection standard
pH: 6.6
Source: CCAP; modification of Bischoff & Bold

Composition

Component	Amount per 1 L
NaNO₃	0.75 g (×3 that of BBM)
CaCl₂·2H₂O	0.025 g
MgSO₄·7H₂O	0.075 g
K₂HPO₄	0.075 g
KH₂PO₄	0.175 g
NaCl	0.025 g
EDTA-KOH solution	1.0 mL
Iron solution (FeSO₄-H₂SO₄)	1.0 mL
Boric acid solution	1.0 mL
BBM trace element solution	1.0 mL
Thiamine (B₁) solution	1.0 mL
Cyanocobalamin (B₁₂) solution	1.0 mL
Distilled water	to 1000 mL

EDTA-KOH solution for BBM

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

Component	Weighed amount / volume
Na₂EDTA	5.0 g
KOH	3.1 g
Distilled water	to 100 mL

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

Component	Weighed amount / volume
FeSO₄·7H₂O	498 mg
H₂SO₄ (conc.)	0.1 mL (2 drops)
Distilled water	to 100 mL

Boric acid solution for BBM

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

Component	Weighed amount / volume
H₃BO₃	1.142 g
Distilled water	to 100 mL

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

Component	Weighed amount / volume
ZnSO₄·7H₂O	882 mg
CuSO₄·5H₂O	157 mg
MnCl₂·4H₂O	144 mg
MoO₃	71 mg (or Na₂MoO₄·2H₂O 119 mg)
Co(NO₃)₂·6H₂O	49 mg
Distilled water	to 100 mL

Thiamine (B₁) solution for 3N-BBM + V

per 100 mL · usage rate 1 mL per 1 L of medium · storage at −20 °C, 6 months

Component	Weighed amount / volume
Thiamine·HCl	1.2 mg (or 1 mL of a primary stock solution at 0.12 g/L)
Distilled water	to 100 mL

Cyanocobalamin (B₁₂) solution for 3N-BBM + V

per 100 mL · usage rate 1 mL per 1 L of medium · storage at −20 °C, 6 months

Component	Weighed amount / volume
Cyanocobalamin (B₁₂)	1.0 mg (or 1 mL of a primary stock solution at 0.1 g/L)
Distilled water	to 100 mL

Preparation

Follow the BBM procedure exactly (calcium last), then autoclave the medium, cool it to room temperature and only then add 1 mL of each sterile-filtered vitamin solution.

Notes

Vitamins are NEVER autoclaved together with the medium: thiamine loses 40–70 % of its activity at 121 °C, which makes the medium useless for precisely those auxotrophs for which it is prepared.
Final vitamin concentrations in the medium: thiamine·HCl — 0.012 mg/L, cyanocobalamin — 0.01 mg/L.
The vitamin solutions are stored at −20 °C in 1–2 mL aliquots; the shelf life is 6 months and refreezing is not permitted.
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.
Primary thiamine stock solution: 12 mg per 100 mL; take 1 mL of it. The final concentration in the medium is 0.012 mg/L. Sterilise by filtration only (0.22 µm).
Primary B₁₂ stock solution: 10 mg per 100 mL; take 1 mL of it. The final concentration in the medium is 0.01 mg/L. The two vitamin solutions are prepared and stored separately.