Bold's Basal Medium without vitamins concentrate (BBM w/o vit) | Canadian Phycological Culture Centre

*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	–	13 mL	–	–
7.	Na₂EDTA•2H₂O	10	10 mL	1	10
7.	KOH	6.2	10 mL	0.62	10
8.	FeSO₄•7H₂O	4.98	10 mL	0.498	4.98
8.	H₂SO₄ (conc.)	1	10 mL	0.1	4.98
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

^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

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.