[Chlamydomonas] problem preparing Hutners trace elements...

René Matagne via chlamy%40net.bio.net (by rf.matagne from ulg.ac.be)
Fri Oct 26 03:29:24 EST 2012

Hi Adriana,

We have always prepared the Hutner's trace element solution according to the 
described recipe. An important point was to keep the temperature to 70 
degrees C or higher when adjusting the pH to 6.5-6.8 with KOH. We never used 
bubbling air to accelerate the formation of the precipitate and we stored 
the flask stopped with a cotton plug for several weeks (more than 1-2 weeks) 
in the dark at room temperature, until obtaining the nice classical purple 
Good luck!

René Matagne

----- Original Message ----- 
From: "Adriana Garibay" <adriagh from ibt.unam.mx>
To: <chlamy from magpie.bio.indiana.edu>
Sent: Thursday, October 18, 2012 3:43 AM
Subject: [Chlamydomonas] problem preparing Hutners trace elements...

> Hi everybody,
> I've been trying to prepare the Hutner's trace element solution and I've 
> been having some problems, therefore I'm asking for your help.
> I prepared the solution according to the recipe provided at the chlamy.org 
> site which is the following one:
>    For 1 liter final mix, dissolve each compound in the volume of water
>    indicated.
>    The EDTA should be dissolved in boiling water, and the FeSO_4 should
>    be prepared last to avoid oxidation.
>    compound amount water
>    EDTA disodium salt 50 g 250 ml
>    ZnSO_4 . 7 H_2 O 22 g 100 ml
>    H_3 BO_3 11.4 g 200 ml
>    MnCl_2 . 4 H_2 O 5.06 g 50 ml
>    CoCl_2 . 6 H_2 O 1.61 g 50 ml
>    CuSO_4 . 5 H_2 O 1.57 g 50 ml
>    (NH_4 )_6 Mo_7 O_24 . 4 H_2 O 1.10 g 50 ml
>    FeSO_4 . 7 H_2 O 4.99 g 50 ml
>    Mix all solutions except EDTA. Bring to boil, then add EDTA
>    solution. The mixture should turn green. When everything is
>    dissolved, cool to 70 degrees C. Keeping temperature at 70, add 85
>    ml hot 20% KOH solution (20 grams / 100 ml final volume). Do NOT use
>    NaOH to adjust the pH.
>    Bring the final solution to 1 liter total volume. It should be clear
>    green initially. Stopper the flask with a cotton plug and let it
>    stand for 1-2 weeks, shaking it once a day. The solution should
>    eventually turn purple and leave a rust-brown precipitate, which can
>    be removed by filtering through two layers of Whatman#1 filter
>    paper, repeating the filtration if necessary until the solution is
>    clear. Store refrigerated or frozen convenient aliquots. Some people
>    shorten the time for formation of the precipiate by bubbling the
>    solution with filtered air.
>    If no precipitate forms, the solution is still usable. However, you
>    might want to check the pH in this case and adjust it to around 7.0
>    using either KOH or HCl as needed.
> Everything was ok until I bubbled filtered air to the solution for aprox. 
> 6 hrs; the precipitate was clearly formed, nevertheless the filtered 
> solution didn't have a purple color, instead, it had still a 
> brown-greenish color (browner than greener). Accordingly, I checked the pH 
> of the solution and it was of 6.0, so I decided to adjust the pH to 7 with 
> KOH (20% w/v) at room temperature. When I was increasing the pH, the 
> solution immediately began to form a considerable ammount of precipitate 
> (no so rusty as the first one, but still brown) and the pH began to 
> diminish by itself; I continued adjusting the pH until it didn't change 
> too much (it stayed at aprox. 6.8). Then, I filtered the solution 
> remaining a considerable amount of precipitate; the solution was not so 
> green, but it still had a brown-green color that wasn't similar to purple. 
> I left the filtered solution with bubbling air for more time, and it seems 
> that it's forming a little bit more of precipitate.
> My question is: is the procedure that I followed ok? will the solution 
> still work for culturing Chlamydomonas?
> Any help will be kindly appreciated.
> Sincerely,
> Adriana Garibay
> Cellular Engineering & Biocatalysis Dpt.
> Instituto de Biotecnologia-UNAM
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