My 1st. TC experience

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Highlander

 
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Re: My 1st. TC experience

by Highlander » Fri Nov 29, 2013 10:52 am

Hi rellenburg,

I'm sorry but my english is bad :(
I put into autoclave all tubes and caps but the medium was into a autoclavable bottle(2 x 500ml).

Thanks for the info..I really have to change some products concentration and the time to expose the tissue.

So I'll disolve my H2O2 to get 3%.
I'll write the news soon.

Best regards

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Highlander

 
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Re: My 1st. TC experience

by Highlander » Mon Dec 02, 2013 5:54 pm

Hey friends,

Following the sterilization methods:

I just bought a soap with triclosan but how can I do a solution with it?
Only dissolve it in sterilized water?

Many thanks

Ricardo

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rellenburg

 
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Re: My 1st. TC experience

by rellenburg » Mon Dec 02, 2013 11:20 pm

I use it as a first clean step, so I just use tap water. I fill a 4oz baby food jar with tap water and 1 drop of soap. Shake for 2 min. Pour explants into a tea ball. Rinse under running tap water for 3 min. Then procede to the chemicals. I'm sure others do it differently.

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coline

 
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Re: My 1st. TC experience

by coline » Tue Dec 03, 2013 2:43 am

Since that soap normally has that hormonal disruptor in it, and it is in a bottle with a "applicator" I press it 2 times (about 3ml I think) for a 150ml jar with tap water, shake and rinse in almost sterile distilled water.

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rellenburg

 
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Re: My 1st. TC experience

by rellenburg » Tue Dec 03, 2013 3:56 am

Haha. I knew someone would have a different method :-) I've never used it that strong.

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Highlander

 
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Re: My 1st. TC experience

by Highlander » Tue Dec 03, 2013 11:03 am

Hello,

Ok I'll mix 3ml of my liquid soap in 150ml distiled water hehehehe.

I want to try with Drosera capensis(8cm leaf), Drosera burkeana(2cm leaf) and vft wacky trap(1-2cm leaf)

How long time I have to expose this explants in yours opinion?

Rellenburg / Coline >>> Thanks a lot!

Regards
Ricardo

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coline

 
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Re: My 1st. TC experience

by coline » Tue Dec 03, 2013 11:28 am

The soap is not very harmful since what I have seen, so any time between 5-30min

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Highlander

 
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Re: My 1st. TC experience

by Highlander » Tue Dec 03, 2013 11:46 am

Ok I'll do!
Thanks :D

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rellenburg

 
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Re: My 1st. TC experience

by rellenburg » Tue Dec 03, 2013 1:41 pm

Let us know how it goes for you. I have not experimented with the length of time with the soap, so I really can't give any other advice than what I've posted already. The majority of my failures result in dead tissue, not contamination, so I would be reluctant to go longer in any step. Unless of course I went longer in the soap and backed off some time in the bleach step. Hmmmm, may ahve to try that. Good luck!

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Highlander

 
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Re: My 1st. TC experience

by Highlander » Tue Dec 03, 2013 9:53 pm

Can I use a magnetic stirrer to make the sterilization?
I can do it easily....but I don´t know if this method would be good, maybe reduce the time to expose....

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Re: My 1st. TC experience

by BigBella » Tue Dec 03, 2013 11:23 pm

Highlander wrote:Can I use a magnetic stirrer to make the sterilization?
I can do it easily....but I don´t know if this method would be good, maybe reduce the time to expose....


Yes, I successfully use a magnetic stirrer in the cleaning of explants or seed. Generally, I use the tiniest ones, about the size of a pencil's eraser, which will fit within small 50 ml beakers or even flat-bottomed test tubes . . .

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Highlander

 
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Re: My 1st. TC experience

by Highlander » Tue Dec 03, 2013 11:28 pm

I have baby food jars(30-40ml) and beckers(50ml) :D

I just read that my liquid soap has 1% triclosan so I have to add 1 or 2 drops into 50ml dest. water? is ok?

Thanks
Regards

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BigBella

 
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Re: My 1st. TC experience

by BigBella » Wed Dec 04, 2013 2:34 am

Highlander wrote:I have baby food jars(30-40ml) and beckers(50ml) :D

I just read that my liquid soap has 1% triclosan so I have to add 1 or 2 drops into 50ml dest. water? is ok?

Thanks
Regards


Yes, that soap will work; but definitely wash the explant under running water first, for five minutes at the very least, before soaking . . .

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Highlander

 
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Re: My 1st. TC experience

by Highlander » Wed Dec 04, 2013 10:39 pm

I did a step by step to make my sterilization method as described bellow:

I read the FAQ and the answers on this topic, so I made (at home) 4 main chemical solutions to try here.

1) Triclosan solution( a bottle with 1 liter of distilled water + 1full cap of liquid soap 1% troclosan);
2)H2O2 3% solution ( 3 flasks with 50ml bought in a drugstore);
3)NAClO 10% solution + tween20 ( a bottle with 900ml of distilled water + 100ml of bleach + 40 drops of tween20);
4)PPM solution (a bottle with 1 liter >>> ppm 1ml/L);

I want to do this method with 4 or 5 leafs from my vft , each about 2-3cm lenght.
All steps will be made with a magnetic stirrer and jars about 40-50ml(except running water) to clean the explants.

1) wash 10 min. in running water
2) wash 10 min in triclosan solution
3) wash 5 min in distilled water
4) wash 2 min in H2O2 3% solution
5) wash 5 min in distilled water
6) wash 5 min in NAClO 10% + tween20 solution
7) wash 5 min in distilled water
8) wash 10 min in PPM solution
9) wash 5 min in distilled water
10) wash 5 min in distilled water

I'm waiting for suggestions because I think that will make this process until the next weekend.

Thanks in advance

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coline

 
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Re: My 1st. TC experience

by coline » Thu Dec 05, 2013 3:54 am

You are killing any kind of effect that PPM gives to the culture using Tween, the manufacturer says that
For everything else it is a good procedure

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