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By Carnies
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Posts:  1380
Joined:  Mon Oct 26, 2020 4:48 pm
#433845
Just started my TC journey this week, here's how it went:

A week or two before, I placed orders from Phytotech Labs, Plant Cell Technology, and Amazon. I also went to Home Depot to grab a Sterilite Gasket Box 54qt.

Phytotech Lab Order:
20ct Culture Jars+Caps
MS Media
Carrageenan (High Clarity)

Plant Cell Technology Order:
PPM

Amazon:
Sodium Dichloroisocyanurate granules
Sterile scalpels
Digital scale
1ml syringe
Tween 20
Exam Gloves

Other items from my house:
Hydrogen Peroxide
70% rubbing alcohol
Microwave

Items you'll need:
Heat mat controller
Heat mat
Grow lights
Spray Bottles
Magnetic Stirrer (HIGHLY RECOMMENDED)
Turkey Baster (For evenly dispensing prepared media into vessels)


Now that supplies are out of the way, here's my process IN ORDER.

Monday
Media preparation - media is usually prepared outside the still air box
1/3 MS media
5g/l of Carrageenan
1ml PPM
30g Sugar
Calibrate PH to 5.6-5.8

Transfer media to your vessels. I like to have the media either 1/5 or 1/4 of the way filled. Your preference. You'll have to boil the media in the vessels for a minimum of 3 minutes in the microwave, i don't know anything about pressure cookers sorry. After the media has been done boiling, open up a trash bag and spray it down with 70% isopropyl alcohol. Place your vessels with media in and close up the bag, let the media cool down and press the lids on the vessel base. Here's a video that shows it well: https://www.youtube.com/watch?v=qMQwUUliE0k
After your media has been fully cooled, take them out of the trash bag and put them in a container where it's airtight and stored until Wednesday. Spray down with 70% isopropyl alcohol beforehand!

Tuesday
I sterilized explants - make sure to do this entire process in a still air box, except the soak in NaDCC if you have sealed containers - I use deli cups.
Transfer your collected explants to a 20 minute soak/stir in a beaker (250ml) on the magnetic stirrer, two drops of tween 20 and a drop of dish soap does the job well. Transfer explants into a mixture of 0.5g/l of NaDCC. You're supposed to use this solution for 15hrs but I could only do 24hrs, and it was too much. I had to switch to 0.4g/l because the plants had phenolic bleeding.

Wednesday
Initiation!
Spray down your entire still air box and put your prepared media and vessels inside along with your fully soaked explants. Use a sterile scalpel and tweezers to move the explants to and from the NaDCC solution onto the vessels. You don't need a rinse for NaDCC! After you're done, wrap with plastic wrap and place on your heat mat. Make sure it doesn't go above 80 degrees and not below 70.


Items you might be wondering about:
NaDCC - an alternative to bleach, probably a replacement because of how good it is. I linked a great article showing why a nursery switched from bleach to NaDCC. The only downside is that you have to soak it for a while and it may not work for everybody, I know Matt from FTS hasn't had much success with it.
Carrageenan - I like this better than regular agar just because it has more clarity, also the 5g/l makes nice soft but not too liquidy media.
PPM - Plant Preservative Mixture, basically a contaminant suppressant, I recommend for beginners like myself.

My still air box is a 54qt sterilite gasket box, which is airtight. I cut holes in the front and put duct tape on the front and back of the holes, then made an X with my knife so I can slide my hands through, here's a good video: https://www.youtube.com/watch?v=svUq2OeQNC4

If you have any questions, leave them in the comments and I'll answer and add them into an FAQ section! Please note that even though this isn't my 1st time, I'm still learning and I'm just sharing what I've done and recommend. Thanks for reading!
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Camden, -Stanley-, Sundews69 and 2 others liked this
By Kumie Trap
Location: 
Posts:  25
Joined:  Thu Apr 06, 2023 6:11 am
#433959
Great Work! And write up! Please give us an update(s) Periodically!
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