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
MEXICO