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
----- 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
>> 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.
>> Adriana Garibay
> Cellular Engineering & Biocatalysis Dpt.
> Instituto de Biotecnologia-UNAM
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