Bold's Basal Medium with 3N concentrate (BBM+3N)

*This formulation has been modified by J. Acreman (CPCC) from the original.

Reference: Stein, J.R. (Ed.). (1973). Handbook of phycological methods: Culture methods and growth measurements. Cambridge University Press, Cambridge, 448 pp.

This concentrate is highly enriched and is used for some cyanobacteria.

Stock Substance Primary stock (g/L) Volume used to prepare 200 mL concentrate Concentrated stock (g/L) Final concentration in medium (mg/L) (adding 20 mL/L)
  Purified H2O – 83 mL – –
7. Na2EDTA•2H2O 10 10 mL 0.5 10
KOH 6.2 0.31
8. FeSO4•7H2O 4.98 10 mL 0.249 4.98
H2SO4 (conc.) 1 0.05
1. KH2PO4 175 10 mL 8.75 175
2. CaCl2•2H2O 25 10 mL 1.25 25
3. MgSO4•7H2O 75 10 mL 3.75 75
4. NaNO3 250 30 mL 37.5 250
5. K2HPO4 a 75 –a – a – a
6. NaCl 25 10 mL 1.25 25
9. Trace metal solution See below for concentrations 10 mL See below for concentrations See below for concentrations
10. H3BO3 11.5 7 mL 0.4025 8.05
11. f/2 vitamin solution See below for concentrations 10 mL See below for concentrations See below for concentrations

aNOTE: The K2HPO4 stock solution (BBM Stock 5) that is normally added to BBM medium is removed from the concentrate as it precipitates out. This stock can be added separately during preparation of the medium.

Trace metal solution

Substock Substance Primary stock (g/L) Concentration (g/L) Final concentration in medium (mg/L)
1. H3BO3 2.86 0.143 2.86
2. MnCl2•4H2O 1.81 0.0905 1.81
3. ZnSO4•7H2O 0.222 0.0111 0.222
4. Na2MoO4•2H2O 0.390 0.0195 0.390
5. CuSO4•5H2O 0.079 0.00395 0.079
6. Co(NO3)2•6H2O 0.049 0.00245 0.049

f/2 vitamin solution

Substock Substance Primary stock (g/L) Concentration (g/L) Final concentration in medium (mg/L)
1. Vitamin B12 (Cyanocobalamin) 0.001 0.00005 0.001
2. Biotin 0.001 0.00005 0.001
3. Thiamine 0.200 0.0100 0.200

To prepare concentrate:

To make 200 mL of concentrate, combine stocks solutions, in the order listed above, to make 200 mL total volume. Concentrate may be stored in the fridge in the dark until needed.

To prepare medium:

The final concentrations shown above are to prepare a 100% 3N BBM medium by adding 1 mL of K2HPO4 stock solution (BBM Stock 5) plus 20 mL of the concentrate to 979 mL of purified water (e.g., distilled, Nanopure, Milli-Q). Stir, pH adjust to 6.8 if necessary with 1N HCl or 1N NaOH. If sterile medium is needed, then the final medium may be filter-sterilized through a 0.22 um filter and dispensed aseptically into sterile vessels or autoclaved directly in the vessels. Prepared medium may be stored in the fridge in the dark until needed.