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Moderator: Matt

By rellenburg
Posts:  241
Joined:  Fri Aug 10, 2012 2:57 am
#178022
Also look over the sterilizing explants section provided by this site.

Everyone that is brand new to tc should not overlook the tissue culture basics section on this site. Matt (and others i believe) have done an excellent job putting it together so that you may have some success. I used it my first time. After that I tweaked it some to better fit my needs, but to get started use their suggestions as much as you can. It is there so you have a starting point and can spend your time worrying about sterile technique instead of working out all the details he provided. Good luck!
By Highlander
Posts:  27
Joined:  Mon May 06, 2013 1:20 pm
#178024
rellenburg wrote:As for the autoclaving media section I would...
1. Pour prepared media into a pot (or other container that is safe for a stove top or hotplate)
2. Heat on stove or hotplate almost to a boil while stirring media until everything dissolves.
3. Dispense hot liquid (you can let it cool a little) into vessels and secure lids.
4. Autoclave at 15psi for 20-25 min (depends on how much media you're making)
5. When cooled and gelled it is ready for use.
Thanks rellenburg!

Maybe I´m wrong but in the autoclaving process, the temperature isn't enough to boil the media and dissolve the agar?
I wanna make 1L of media.

Regards
By Highlander
Posts:  27
Joined:  Mon May 06, 2013 1:20 pm
#178025
rellenburg wrote:Also look over the sterilizing explants section provided by this site.

Everyone that is brand new to tc should not overlook the tissue culture basics section on this site. Matt (and others i believe) have done an excellent job putting it together so that you may have some success. I used it my first time. After that I tweaked it some to better fit my needs, but to get started use their suggestions as much as you can. It is there so you have a starting point and can spend your time worrying about sterile technique instead of working out all the details he provided. Good luck!
You're right!
I have to learn more about sterilization method wrote in this forum.
I suggested my step-by-step because I saw a video from youtube about VFT TC.

Thank you one more time

Regards
By coline
Posts:  1230
Joined:  Wed Feb 27, 2013 1:57 pm
#178029
rellenburg wrote:As for the autoclaving media section I would...
1. Pour prepared media into a pot (or other container that is safe for a stove top or hotplate)
2. Heat on stove or hotplate almost to a boil while stirring media until everything dissolves.
3. Dispense hot liquid (you can let it cool a little) into vessels and secure lids.
4. Autoclave at 15psi for 20-25 min (depends on how much media you're making)
5. When cooled and gelled it is ready for use.
I second this procedure as it is the most contamination free method normally used, even in big laboratories. Pouring media is used only with disposable petri dishes.

Also, a minor change in order:

1- Dissolve the MS+vit powder in 900mL of destilled water and stir
2- Add the mix for plant tc
3- Add 30g of suggar and stir
4- Add food coloring and stir
5- Complete with destilled water up to 1L mark
6- Get the ph of the solution and adjust until ph5.6
7- Add 2,5g of gellan gum powder(see picture) and stir
8- get to boil in microwave 8min/L


That because the gel normally damages the electrode in the meter by clogging the tiny pores it has, causing errors in future uses. And the gel need to get dissolved before autoclaving to have best results, that is done even when using the pouring medium procedure after autoclaving.

Also this:

1- wash the explant in current water about 2 minutes (optional With soap in water, antibacterial)
2- wash the explant in destilled water about 2 minutes
Dip into 10% v/v NaClO for 5-7 minutes
wash the explant in destilled water about 2 minutes

3- Put the explant in a esterilized plate and cut off top(trap)/botton(white tissue) parts
4- wash the explant in destilled water+PPM about 4 minutes
4- wash the explant in destilled water about 2 minutes
5- wash the explant in destilled water about 2 minutes

6- Put the explant into the media and cap the tube using a plastic film as a seal.

PPM instructions say that you do not wash the explant after using PPM, you use it and transfer to media, so do not do the Red section.
Also, PPM is just a bacteriostatic agent, it does not kill bacteria, as you are using a plant, that tissue has too much bacteria to handle it with just PPM, it is ok with seed but not tissue. That is why I added the green section.

Matt has written a very good protocol for introducing tissue, you may follow it and have good results.
By Highlander
Posts:  27
Joined:  Mon May 06, 2013 1:20 pm
#178047
Thanks Coline,

I just print your tips.
I have to read about others methods in this forum and tomorrow I'll do my exp.


Pura vida!
Regards
Highlander liked this
By Highlander
Posts:  27
Joined:  Mon May 06, 2013 1:20 pm
#181235
Hello friends,
I just received IBA and NAA I'll updating my supply list.

Waiting for Kinetin, IAA and MS media (micro+macro with vitamins)

Best regards
Ricardo
By Highlander
Posts:  27
Joined:  Mon May 06, 2013 1:20 pm
#181365
Hey friends, I need your help about the kind of media to buy, I have some options here in Brazil:

1)Murashige and Skoog Basal Medium/powder, plant cell culture tested
Formula variant >>> With the macro- and micronutrients, and vitamins as described by Murashige and Skoog (1962).

2)Murashige and Skoog Basal Medium/powder
Formula variant >>> With the macro- and micronutrients as described by Murashige and Skoog (1962) and the vitamins as described by Gamborg, et al. (1968).

What Should I buy?

Thanks
Regards
Ricardo
By Highlander
Posts:  27
Joined:  Mon May 06, 2013 1:20 pm
#181504
hello!

I made my own laminar flow hood + Hepa filter, see below:

Image
Image

I tried to make the media from planttc... as shown in the pics above.
I used the same method as described in faq but I think that I have to learn more....see my shoot from Drosera below:
Image

Can I save my shoot?

Thanks in advance
Regards
Ricardo
By coline
Posts:  1230
Joined:  Wed Feb 27, 2013 1:57 pm
#181531
Well, in case of fungi, there is no escape, the tissue would be infected and is not viable in TC so it is a jar that goes to autoclave.

Also, try to do a plating experiment:

Get 3 jars with medium
open them in the laminar flow hood.
Leave them for 30 seconds.
close them.

If the jar contaminates then the hood is the problem.
If not, it is the dissinfection protocol.

Also, did you see where the infection started?
By Highlander
Posts:  27
Joined:  Mon May 06, 2013 1:20 pm
#181536
coline wrote:
Also, try to do a plating experiment:

Get 3 jars with medium
open them in the laminar flow hood.
Leave them for 30 seconds.
close them.

If the jar contaminates then the hood is the problem.
If not, it is the dissinfection protocol.

Also, did you see where the infection started?
Hi Coline,
I'll do the experiment, thanks.( I think that my desinfection process was bad)
I can't see the point that the infection started because I´ve been busy with my job, I was 2 days away from my home.

Thank you
Saludos
By Highlander
Posts:  27
Joined:  Mon May 06, 2013 1:20 pm
#191304
Hey All,

I'm sorry but I've been busy at my job so I had to let my experiences stopped.

Updating some things, just bought some supplies:

Supplies


- MS media with macro and micro nutrients *new
- kinetin *new
- IAA *new

- PPM
- MS powder from planttc as multiplication labeled
- MS powder from planttc as stabilish labeled
- NAA
- IBA
- pure agar
- KOH (ph + )
- HPO4 (ph -)
- food coloring
- alcohol ethanol 70%
- alcohol ethanol gel 46%
- alcohol isopropyl
- tween 20
- H2O2 6%
- captran(fungicide)
- bleach 10%
- bleach 5%
- bleach 3%
- bleach 1%

Coline, I did the test into my laminar flow hood and after 1 week had 0% contamination on the medium :D

Yesterday I tried to make a batch of MS as described bellow.

With a magnetic stirrer+becker I put 900ml of distilled water adding the following supplies:

2g MS with Macro/micro nutrients
30g suggar
1ml IBA (1mg/ml)
1ml PPM
colouring food
7g pure agar

I adjusted ph to 5.7 and add water up to 1 liter.
Put the medium into a autoclavable bottle and let it to sterilize.
After sterilize, I transfered the medium to small tubes(sterilizers) into my flow hood.
Now I have to wait about a week to see if occurs any contamination.

Please tell me if I did something wrong in the process.

Thanks in advance
Regards
Ric
Last edited by Highlander on Thu Nov 21, 2013 10:52 pm, edited 1 time in total.
By coline
Posts:  1230
Joined:  Wed Feb 27, 2013 1:57 pm
#191306
Good you have no contamination!
Just 1 step had a problem, not a big deal if you are not in scientific experiments, but, first you add every ingredient and water to 1L then adjust pH.

And it is not necesary to wait 1 week to use it, from the moment it is room temperature you may use the medium.

But in this first time it is good, to see if it contaminates or not.
By Highlander
Posts:  27
Joined:  Mon May 06, 2013 1:20 pm
#191563
Hey friends!

Today(8 days after made the medium) I saw my tubes and all is ok, without contamination :D

Coline,
I was wrong....first I add water until 1L mark and after got the ph in the medium.
Thanks, you always helpped me :)

So I have nepenthes,sarracenias,heliamphoras,droseras and dionaeas to start my tests.
Can I follow the faq desinfection method to any specie from my list or somebody recomends me a step-by-step based in my products list?

Thanks in advance!!

Best regards
Ricardo
By rellenburg
Posts:  241
Joined:  Fri Aug 10, 2012 2:57 am
#191567
Highlander wrote: Put the medium into a autoclavable bottle and let it to sterilize.
After sterilize, I transfered the medium to small tubes(sterilizers) into my flow hood.
Now I have to wait about a week to see if occurs any contamination.

Just notice this... Hopefully you will be ok cause you waited a week on contamination to show up, but typically you should add medium to vessels THEN autoclave them. This way you are not risking picking up contaminants while transferring the media. Also, this sterilizes the vessel at the same time. Sorry, if I didn't read your procedure correctly.

And for the new question... With flytraps and the FAQ procedure you may have a problem with the length of time in the bleach solution killing the explants, especially if you use small tissue, so you may find you have to use a little less time. However, with seeds... really you can't go wrong. Also, I notice in your list that you have 6% H202. The FAQ and most published protocols call for 3%, so you may have to dilute it. Good luck!
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