Bold's Basal Medium with vitamins concentrate (BBM+vit)

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

Reference: Stein, J. (ED.) Handbook of Phycological methods. Culture methods and growth measurements. Cambridge University Press. 448 pp.

This concentrate is highly enriched and is used for many freshwater green algae.

Stock Substance Primary stock (g/L) Volume used to prepare 100 mL concentrate Concentrated stock (g/L) Final concentration in medium (mg/L) (adding 10 mL/L)
  Purified H2O – 3 mL – –
7. Na2EDTA•2H2O 10 10 mL 1 10
KOH 6.2 0.62
8. FeSO4•7H2O 4.98 10 mL 0.498 4.98
H2SO4 (conc.) 1 0.1
1. KH2PO4 a 175 10 mL 17.5 175
2. CaCl2•2H2O 25 10 mL 2.5 25
3. MgSO4•7H2O 75 10 mL 7.5 75
4. NaNO3 250 10 mL 25 250
5. K2HPO4 b 75 – b – b – b
6. NaCl 25 10 mL 2.5 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 0.805 8.05
11. f/2 vitamin solution See below for concentrations 10 mL See below for concentrations See below for concentrations

aNOTE: Store the KH2PO4 a primary stock solution (Stock 1) at room temperature (precipitates out in fridge).

bNOTE: The K2HPO4 stock solution that is normally added to BBM medium as Stock 5 is not added to the concentrate as it precipitates out. This should not greatly affect the growth of weedy, green algae; however, if desired, you may add this stock separately during preparation of the medium at a rate of 1 mL/L.

Trace metal solution

Substock Substance Primary stock (g/L) Concentration (g/L) Final concentration in medium (mg/L)
1. H3BO3 2.86 0.286 2.86
2. MnCl2•4H2O 1.81 0.181 1.81
3. ZnSO4•7H2O 0.222 0.022 0.222
4. Na2MoO4•2H2O 0.390 0.039 0.390
5. CuSO4•5H2O 0.079 0.0079 0.079
6. Co(NO3)2•6H2O 0.049 0.0049 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 0.0001 0.001
2. Biotin 0.001 0.0001 0.001
3. Thiamine 0.200 0.0200 0.200

To prepare concentrate:

To make 100 mL of concentrate, combine stocks solutions, in the order listed above, to make 100 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% BBM medium by adding 10 mL of the concentrate to 990 mL of purified water (e.g., distilled, Nanopure, Milli-Q). However, if you are adding Stock 5, then add 10 mL of the concentrate plus 1 mL of Stock 5 to 989 mL of purified water instead. This is a very rich medium. If you wish to prepare a dilute growth medium (i.e., 10% BBM), add 1 mL of the concentrate to 999 mL of purified water. 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 should be stored in the fridge in the dark until needed.