High salt medium (HSM) with Hutner's Trace Elements Solution

*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.Ìý 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: Ìý

50 mL
NH4Cl 5 g/500 mL
MgSO4•7H2O 0.2 g/500 mL
CaCl2•2H2O 0.1 g/500 mL
2. Phosphate #2: Ìý

25 mL
KH2PO4ÌýÌýÌý 14.8 g/500 mL
K2HPO4 28.8 g/500 mL
3. Hutner's trace elements 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.Ìý

Autoclave for 45 minutes or longer if using larger quantities than 1 Litre.Ìý Allow medium to stand overnight before use.ÌýStore unused medium in the fridge until needed.

Hutner's trace elements solution

Substance Concentration (mL/L)
1.Ìý Na2EDTA 50 g
2.Ìý ZnSO4•7H2O 22 g
3.Ìý H3BO3 11.4 g
4.Ìý MnCl2•4H2O 5.06 g
5.Ìý CoCl2•6H2O 1.61 g
6.Ìý CuSO4•5H2O 1.57 g
7.Ìý (NH4)6Mo7O24•4H2O 1.10 g
8.Ìý FeSO4•7H2OÌý 4.99 g

Modified CPCC instructions:

  1. Prepare a 20% KOH solution by adding 20 g to 100 mL of purified water. Boil until dissolved.
  2. Prepare EDTA solution by adding 50 g to 250 mL of purified water. Boil until dissolved.
  3. Mix the remainder of the trace elements in 550 mL of purified water. Heat until dissolved (minimum 70°C). Then carefully add EDTA. The mixture should turn green.
  4. Cool solution to 70°C.
  5. Add 85 mL/L of the hot 20% KOH.
  6. Cool to room temperature and top up to final 1L volume.
  7. pH should be ~6.7. Add 6N KOH or HCl to adjust.
  8. Use cotton plug on flask to allow air exchange. Swirl once a day for 1-2 weeks.
  9. Solution should be clear green, then a deep reddish-purple and leave a rust-brown precipitate.
  10. Decant through Whatman #1 filter paper without disturbing the precipitate; repeat, if necessary, until the solution is clear.
  11. Store refrigerated or frozen in convenient aliquots.

This can be made in smaller or larger batches. More detailed instructions may be found on (based on Hutner et al., Proc. Am. Philos. Soc. 94: 152-170 (1950)).