problem preparing Hutners trace elements...

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problem preparing Hutners trace elements...

mahmoud.abdullah.jaber
Hello Marcel

 

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.

 

Best regards

Mahmoud Jaber

Chemist student

 

 

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
Mo7O24(NH4)6·4H2O
(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 filter paper
Store in a brown bottle at 4°.

 

 

 

 

 

Dear netters,
 
I hope some among you have experience in preparing Hutner trace elements
solution. I followed the procedure described on page 579 in the Chlamydomonas
Sourcebook.
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
problem.
 
Does anybody know what could be the cause of this behaviour,
 
thank you very much for your time,
 
sincerely,
Marcel Janssen
Food and Bioprocess Engineering Group
Wageningen Agricultural Universtity
The Netherlands

 

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Re: problem preparing Hutners trace elements...

Elizabeth Harris
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.


>Hello Marcel
>
>
>
>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.
>
>
>
>Best regards
>
>Mahmoud Jaber
>
>Chemist student
>
>
>
>
>
>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
>Mo7O24(NH4)6·4H2O
>(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
>filter paper
>Store in a brown bottle at 4°.
>
>
>
>
>
>
>
>
>
>
>
>Dear netters,
>
>I hope some among you have experience in preparing Hutner trace elements
>solution. I followed the procedure described on page 579 in the Chlamydomonas
>Sourcebook.
>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
>problem.
>
>Does anybody know what could be the cause of this behaviour,
>
>thank you very much for your time,
>
>sincerely,
>Marcel Janssen
>Food and Bioprocess Engineering Group
>Wageningen Agricultural Universtity
>The Netherlands
>
>
>
>_______________________________________________
>Chlamy mailing list
>[hidden email]
>http://www.bio.net/biomail/listinfo/chlamy


--

Elizabeth H. Harris

Chlamydomonas Center
http://www.chlamy.org/


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problem preparing Hutners trace elements...

Adriana Garibay
In reply to this post by mahmoud.abdullah.jaber
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
MEXICO
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Re: problem preparing Hutners trace elements...

René Matagne
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
colour.
Good luck!

René Matagne


----- Original Message -----
From: "Adriana Garibay" <[hidden email]>
To: <[hidden email]>
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
> MEXICO
> _______________________________________________
> Chlamy mailing list
> [hidden email]
> http://www.bio.net/biomail/listinfo/chlamy 

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