High Salt Medium (HSM) with AAP Trace Metal Solution | Canadian Phycological Culture Centre

*This formulation has been modified from the original.

Reference: Sueoka, N., Chiang, K.S. and Kates, J.R. (1967) Deoxyribonucleic acid replication in meiosis of Chlamydomonas reinhardtii. Isotopic transfer experiments with a strain producing eight zoospores. J. Mol. Biol. 25: 44-67.

This medium is used for many freshwater Chlamydomonas spp., as well as other species.

Stock	Substance	Concentration	Amount added to medium (mL/L)
1.	Beijerinck's #2:		50 mL
	NH₄Cl	5 g/500 mL
	MgSO₄•7H₂O	0.2 g/500 mL
	CaCl₂•2H₂O	0.1 g/500 mL
2.	Phosphate #2:		25 mL
	KH₂PO₄	14.8 g/500 mL
	K₂HPO₄	28.8 g/500 mL
3.	AAP trace metal solution	See below for concentrations	1 mL

Final pH of medium is 6.8 and if necessary can be adjusted using 1N HCl and 1N NaOH.

Autoclave for 20 minutes or longer if using larger quantities than 1 Litre. Allow medium to stand overnight before use.

AAP Trace metal solution

Substance	Concentration (mg/500 mL)
1. H₃BO₃	92.76 mg
2. MnCl₂•4H₂O	207.69 mg
3. ZnCl₂	1.64 mg ^a
4. FeCl₃•6H₂O	79.89 mg
5. Na₂EDTA•2H₂O	150.00 mg
6. CoCl₂•6H₂O	1.30 mg ^b
7. Na₂MoO₄•2H₂O	3.63 mg ^c
8. CuCl₂•2H₂O	0.006 mg ^d

^aNOTE: Weigh out 0.1635 g of ZnCl₂ and dilute to 100 mL. Add 1 mL of this solution to Stock Solution 3.

^bNOTE: Weigh out 0.714 g of CoCl₂●6H₂0 and dilute to 100 mL. Add 1 mL of this solution to Stock Solution 3.

^cNOTE: Weigh out 0.363 g of Na₂MoO₄●2H₂O and dilute to 100 mL. Add 1 mL of this solution to Stock Solution 3.

^dNOTE: Weigh out 0.060 g of CuCl₂●2H₂O and dilute to 100 mL (= Stock 3d(1)). Dilute 1 mL of this solution to 100 mL (= Stock 3d(2)). Add 1 mL of this second solution to Stock Solution 3.

Additional notes for stock 3:

Substocks: First prepare 3d(1). Then prepare 3a, 3b, 3c. Then prepare 3d(2).

First add Na₂EDTA, then FeCl₃, then add the rest of the stock chemicals and substock solutions (3a, 3b, 3c, 3d(2)).