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By jht-union
Posts:  3205
Joined:  Tue Apr 13, 2010 11:43 pm
#111259
Today for the first time i did TC, something i have been posponing for so long, and even today i thought about it. But i think it was worth for the first time.

I started at 3pm and finished at about 8pm, so it took me more than 4 hours to finish. Whether or not if i did it correctly time will tell.

I was going to write everything i did, but honestly with all the things you need to do at once, it's very hard to write it out, or even explain it, i'm saturated with all the time it took, so everything is mix in my head. Matt- now i understand you more than ever, so those people that ask matt when are they're going to be available :lol: (including me), do TC, and then talk to Matt about it hehehe!

Well sounds like i did everything right, but the time will tell hopefully! :)

One of the biggest mistakes i did was:

I put seeds first in 10% bleach rather than 91% ethanol alcohol, so i had to repeat everything again.

Probably the most difficult part was cleaning the tissue, one of the things i did 3 times and forgot about, was when putting the tisue from the sterile water to the media, i forgot to rinse the tweezers 3 times, what i did was: keep the tweezers in 91% ethanol, some sterile water, another sterile water for the tissues, so i did from alcohol to sterile to sterile water pick tissue and put in media then alcohol for tweezers again......and so on, but i forgot to do it 3 times with 3 tissues i don't even remember what they were. So i pick tissue put in media, pick tissue put in media, pick tissue put in media, that was a terrible mistake, but they might work out!

Well wish me luck! :)

Here's some pics:

I used my tank as my working area, the plastic was to protect even more from particles. Something i might do next time close the tank or aquarium completely with plastic, and make just two holes for the hands, and i'm able to keep everything more sterile!

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Oh and yes, i cleaned the kitchen with bleach the night before, and also before starting, all the doors i close, and everything.

Pressure cooker:
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Some materials:

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I labeled everything:

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Everything in their own bag:

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labeled more :) :

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Some vessels out of the pressure cooker:

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I sprayed 50% alcohol inside the working area:

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I'm keeping everything by a #, since i decided to start backwards i started with 86, the reason i'm keeping it with #
s is because i have created a table in excel in order of: #, name of the tissue, date put in TC, days in culture, swelling, shoots or roots, callus, contamination, other observations. and it also keeps everything more organize with just a single number and not the name of the tissue. ;)


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Some VFT seeds, the few i got from my plants! there is Czech giant x Akay Ryu, and Dutch X Akay Ryu, a really nice cross i would say, so i do hope the babies grow and germinate!:


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Some tissue:

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Thanks for watching and i'll be posting here if anything happens.



Thanks for the Dionea for helping me a lot and giving me some advices! Also to matt for the well and wonderful TC FAQ, and all the info about TC. And last but not least, thanks to the knowlegeable members who have answer my questions, and have helped me a lot!

Now is just a matter of waiting, my next TC date will be sometime next week, or this week.
Last edited by jht-union on Thu Jul 07, 2011 3:15 pm, edited 1 time in total.
jht-union, jht-union liked this
By Shimizoki
Posts:  975
Joined:  Tue Jun 07, 2011 3:12 am
#111260
wow JHT... seeing all that stuff terrifies me. Now I get why people say its complicated. There are tons of steps (I'm sure far more than you are even mentioning)

I really hope they work out for you. Luck.
Shimizoki liked this
By Daniel_G
Posts:  5472
Joined:  Thu Mar 25, 2010 7:27 pm
#111279
Wow! With all that, i would be very shocked if you fail!

Best of luck! I hope you get some nice plants from those seeds!
Daniel_G liked this
By SnapSnapIOM
Posts:  635
Joined:  Mon Apr 18, 2011 5:44 pm
#111284
LOL i hear you JHT

I started last week and yes all those photos looked like my house :)

I did similar mistakes as you lol

My fish tank is 100% like yours but i modded using my tank by using lots of plastic sheeting over the tank then another plastic sheet made like a tent over everything ( Nearly killed myself with alcoholand bleach fumes O well it would have been a worthy cause hehe)

Anywhere got my fingers crossed for you and hope you get a strike
SnapSnapIOM liked this
By jht-union
Posts:  3205
Joined:  Tue Apr 13, 2010 11:43 pm
#111299
Shimizoki wrote:wow JHT... seeing all that stuff terrifies me. Now I get why people say its complicated. There are tons of steps (I'm sure far more than you are even mentioning)

I really hope they work out for you. Luck.
Oh this is nothing, the protocols and all other things you need to have ready or do, it was a good experience but time consuming.
Dubstep13 wrote:yea this is so cool! ill be tuning in here when u update i wanna do some TC in the future when i get my own place.
I'll try to keep this updated! ;)
You should do TC, for now just read and watch learn from the experts as well as beginners! ;)
That's exactly what i did about a year ago, starting to get familiarize with the names, terms, just looking and reading, and honestly i didn't knew anything, even when i had the kit i didn't know pressure cookers had to be use with water :lol: , but after reading, and reading and reading and reading more it made sense at the end, i would say is worhty! :)

Daniel_G wrote:Wow! With all that, i would be very shocked if you fail!

Best of luck! I hope you get some nice plants from those seeds!
I hope so too daniel, i can't to see what will come out of those coats! :)
SnapSnapIOM wrote:LOL i hear you JHT

I started last week and yes all those photos looked like my house

I did similar mistakes as you lol

My fish tank is 100% like yours but i modded using my tank by using lots of plastic sheeting over the tank then another plastic sheet made like a tent over everything ( Nearly killed myself with alcoholand bleach fumes O well it would have been a worthy cause hehe)

Anywhere got my fingers crossed for you and hope you get a strike
Thank you SnapSnapIOM, i wish you good luck as well! :)
Next time i will try to cover my back and the top as well. The idea of covering the tank and making the two holes for the hands sounds about right for me.
Did you have a dust mask? they work really well!
Let me know how it goes! :)
By jht-union
Posts:  3205
Joined:  Tue Apr 13, 2010 11:43 pm
#111310
NelsonR wrote:A way to make it less time consuming is to make the media one day an then put the explants or seeds on it another. Thats how I do it.
Hummm, that's an excellent idea, and i put the vessels in the refrigerator right with the cap on?
By corners
Posts:  25
Joined:  Sat Jun 18, 2011 11:05 pm
#111390
I don't know much about tc(interested in learning though), but i do know you want to make sure the area is well ventilated when using bleach and alcohol products.Especially the 91% alcohol being very flammable.
By jht-union
Posts:  3205
Joined:  Tue Apr 13, 2010 11:43 pm
#111392
corners wrote:I don't know much about tc(interested in learning though), but i do know you want to make sure the area is well ventilated when using bleach and alcohol products.Especially the 91% alcohol being very flammable.
Something i did don't know if it was helpful, i turn the air conditioner on before starting at 35F and it got down to 60F( it was at 80F), that was first to kill any particles since most live well at a temperature of 70-85F, and that also reduces the chance of flamable products like alcohol at high temps.
corners wrote:And i cant wait to see how this turns out.
I can't wait either how it turns out! :)

Thanks for looking! :)
By NelsonR
Posts:  157
Joined:  Tue May 24, 2011 3:29 am
#111394
You shouldnt have any ventilation in the room, that would improve the chances of contamination. And i neer put my vesels on the fridge.
By jht-union
Posts:  3205
Joined:  Tue Apr 13, 2010 11:43 pm
#111395
Dionae wrote:Looking good jht! I'm sure you'll get some strikes with that many explants. Like Nelson, I make my media the day before and put the vessels in the fridge until the next day. Good luck!
I hope i did everything correctly and i hope i get something out of it!

And that's a really good idea, i'll do that next time! I still have like a liter of 1/2 MS in the refrigerator, and i also have some vessels ready with agar(just 3) in the refrigerator. When do i use them, can they stay long enough in the refrigerator before i clean more explants?


Thank you!!!!
By jht-union
Posts:  3205
Joined:  Tue Apr 13, 2010 11:43 pm
#111396
NelsonR wrote:You shouldnt have any ventilation in the room, that would improve the chances of contamination. And i neer put my vesels on the fridge.
sorry i forgot to put that when the temp was at 60F, i shut down the air conditioner for that same reason to avoid floating particles! ;)

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