The recipe for Hutners trace elements quoted in
the previous message (and repeated below) is not
precisely the same as the one that is given in
the Chlamydomonas Sourcebook.
In particular, the Sourcebook recipe specifies
EDTA as the disodium salt, so if the acid were
used instead, this would probably affect the
final pH of the solution.
Also, the recipe in the Sourcebook specifies
making separate solutions of each salt, then
mixing the dissolved salts together, adding the
EDTA solution last, after the mixture is heated
to 70 degrees.
I now recommend that when the color changes don't
occur as expected, that the pH should be checked
again and adjusted with KOH or HCl to bring it
back to near neutrality.
>>>>I have the same problem as you described below.
>Did you descover the reason. I could uspect pH,
>but i am not sure. Hope you can help.
>>I have prepared the mixture as shawn below.
>>>>>>A) Dissolve 50g of acid free EDTA in 250 ml of ddH2O. Heat to dissolve.
>>B) Dissolve the following, one by one, in order,
>heating to approximately 100° in 550 ml ddH2O.
>>BO3H3 11.40 g
>ZnSO4·7H2O 22.00 g
>MnCl2·4H2O 5.06 g
>FeSO4·7H2O 4.99 g
>CoCl2·6H2O 1.61 g
>CuSO4·5H2O 1.57 g
>(Ammonium Molybdate) 1.1 g
>>Mix the two solutions (A & B) together. The
>resulting solution should be blue-green.
>Heat to 100°C. Cool slightly, but don't let the
>temperature drop below 80°-90°.
>Adjust pH to 6.5 to 6.8 with 20% KOH
>(approximately 83 ml). Don't let the temperature
>drop below 70 ° until after the pH is adjusted.
>Make up to 1L and let stand in a 2L Erlenmeyer
>flask, stoppered loosly. The color should change
>from green to purple over a period of days.
>Remove the rust-coloured precipitate by
>filtering, with suction, through 3 layers of
>Whatman #1 filter paper in a Buchner funnel.
>Repeat until no more precipitate is seen on the
>Store in a brown bottle at 4°.
>>I hope some among you have experience in preparing Hutner trace elements
>solution. I followed the procedure described on page 579 in the Chlamydomonas
>The problem occurs during the final step, when I adjust to pH 6.7 by adding
>20% w/v KOH. The solution changes from clear dark green to a suspension of a
>green solution and rust-brown particles. According to the procedure it should
>be clear green initially and after one or two weeks the color changes to
>purple leaving this rust-brown precipitate. However, when I let the solution
>stand for 2 weeks nothing changes and after two weeks I still have a
>suspension of a green solution and a rust-brown precipitate...
>I already tried this procedure for three times and always I have the same
>>Does anybody know what could be the cause of this behaviour,
>>thank you very much for your time,
>Food and Bioprocess Engineering Group
>Wageningen Agricultural Universtity
>Chlamy mailing list
>Chlamy At net.bio.net>http://www.bio.net/biomail/listinfo/chlamy
Elizabeth H. Harris