Ok folks i didnt want to leave my friends that are still here in the dark. I have been talking about setting up my tissue culture lab. Well i have done it and completed the lab and have made my 1st tissue cultures.
- Laminair flow hood for aseptic sterile work
- High temp sanatizer box.
- Graduated beaker set.
- Media containers and agar and hormones
- 2 types of culture jars and tubes.
- Graduated glass droppers.
- Magenetic heated stiring plate.
- Stainless steel work bench.
- Rolling stool.
- Complete culture lab kit.
- Spatulas and measuring tools.
- Scalpels and forceps and hemostat.
- Body bio suit.
- 14" surgical gloves
- Face mask
- Enclosed grow tent as a mini lab.
- 14 living strains to clone
- Plant preservative
- Bleach
24 Likes
Damn right @preybird1some day just don;'t have space at the moment…
6 Likes
So you need a pressure cooker unless you have a autoclave. This is needed to steralize the media the vessels and paper towels and to make steralized water.
Next step is media preparation.
Media prep is best done in a clean kitchen. Media is mixed, distributed into vessels and sterilized by autoclave, pressure cooker and or microwave **micro wave is the last choice. Not very good but works in a pinch.
Extra water, paper towels, instruments, tools are steralized before transfer into the laminar flow bench or hood. Give yourself enough room to mix and move around freely.
You need MS or media salt hormone and a PPM preservative mix, and Gel-agar powder, ph control kit and ph test paper or ph meter. Vessels or tubes or jars that are autoclavable .
To make the media gel
2. 1 quart or 1liter ot 1000ml pyrex beaker.
3. Magnetic stirring plate
4. Distilled water.
5. 2 tablespoons white sugar.
6. Ms media salts-ppm preservative liquid
7. Agar gel
Take the beaker and put on the magnetic stirrer plate turn it on. Add 900ml of distilled water. Then add the 2 tablespoons of sugar. Let the sugar disolve then add the ms-hormone-ppm liquids. The most common establishment and multiplication media is called B1 and is made from 1mg BA, 0.1 mg NAA and 1ml PPM per liter. This is a standard mult formula and is the most common starting media.
Then make sure the water level after adding solution is now at 1000ml if not add more water. Now ph this to 6.0 using the ph control kit. because adding the gel agar will decrease the PH a few 0.1 of a point. Now turn the heating plate on if your magnetic stirring plate has that option. Now get your rack of liquid tubes-jars ready if so make sure you PH the water before adding the agar.
Agar is special because it must be added to cold water warm water makes it clump up and doesnt dissolve in hot water. When added to the solution the agar will fall to the bottom and separate from the solution. And must be stirred while adding to the vessels. This is why a magnetic stirring plate is so awesome and the heat makes the agar mix with the solution more uniformly.
7 Likes
Dispense at least 10ml into each tube. Or if using baby food jars 22ml for 4oz and 33ml of solution for 6 oz. The recipe make plenty of media for any of the 3 vessels described. Then take foil sheets and make squares and wrap the top of the vessels only if your making media for later use.
5 Likes
Media shelf life is 2 weeks before discard. Now put 2 liters of purified water only into the pressure cooker and turn on the fire to preheat while you getting the foil on the tubes. This is so the solution doesnt boil away while steralizing. Put the rack into the pressure cooker after you foiled up all the vessels.
5 Likes
Put the tubes and foil into the pressure cooker And seal up the lid get the pressure cooker up to 15psi and set a timer for 20 minutes.
5 Likes
Stay with the pressure cooker in case never leave it unatended. After 20 minutes let the cooker cool 20-30 minutes. Then spray your gloves with rubbing alcohol 70% and take the rack and vessels out and directly into a running laminar flow bench to cool make sure the fan is running inside the hood to aid in cooling the media and this keeps the hood sterile.
Then once the tubes are cooling in the flow hood prop them up on the side because any water that condenses on the tube inside will run down and could drown your explants.
Ok update folks. Make sure you use tops or lateral shoots for your cuts. And make the cuts at least above 2 nodes of leaves and then cut off all the leaves and leaf stems. Then take the cuts and rinse them under tap water for 5 minutes using a rinse jar. That has a screen top so the cuts dont overflow up and out into the drain
Then immediatly take the cuts drain them to 1" of water. Then take long tweezers and pick them up and drop them into a solution of steralized water and bleach at 9 to 1 ratio.
I like a 500ml beaker for this and 50 ml of bleach and 3 drops of dawn dish soap original unscented. Or tween wetting agent if you have it.
5 Likes
Your wash solution should look like this now. And now it can be used to disinfect the plant material.
6 Likes
I use a magnetic stirring plate to wash the plant material.
Makes it a lot easier than trying to shake and aggitate the solution.
The plant material needs to be washed for 15 minutes to disinfect the plant material before. Trying to cut it up.
Ok on the rinse part. You need to take the material and
- Rinse
- Alcohol dip and swish for 5-10 seconds
- Wash for 15 minutes
- Sterile water rinse.
6 Likes
Next you take the final rinsed plant material that is brought into the hood after rinsing and spray the beaker and your gloves with alcohol before it goes into the laminar flow hood. So you dont contaminate the the hoods insides.
Then take the cut out of sterile water with long tweesers. Do not handle material without tweezers this is the most critical part of cleanliness.
You must now put the material onto the plate and cut off all the ends you cut before the bleaching. All cut points like leaves and stem base.
Then after its trimmed it needs to be immediately put into the vessel. Grab the cutting perpendicular to the vessel and use the tweezers to insert the plant into the gel 1/2" then cap the tube and clean your plate off and spray with alcohol again and repeat.
6 Likes
Then after all the cuts are finishedbthe tubes need to be sealed with parafilm.
When done it should look like this.
Then put the rack of tubes under a low intensity light for 4 weeks .
8 Likes
Well done @PreyBird1. Thank you for all the information! I’m still pretty far from being able to do this, but this definitely peaked my interest.
4 Likes
After a week check on them. The nice straight cut bottoms should start to callus up. And look like a bell bottom .
you can see the difference a week made
7 Likes
@preybird1 so how long until you plant those in solid media(soil coco turpur etc)… Good to see you back bro… You’re knowledge and technique is greatly appreciated brother
7 Likes
The meristems at this level will take approximately 3-4 weeks. Then i will show the next step of making a rooting media mix. Then it will go into hardening the plants to come out of invitro. This is one of the tuff parts bringing a sterile plant out of a bubble thats completely safe into humidity variances and light changes and open air.
7 Likes
Absolutely amazing man… So in theory you could preserve let’s say 200 mother’s or phenotypes you want to carry on??? With a very very small amount of space
6 Likes
After this we will be going even smaller cloning. into true meristem harvesting at the microscopic level using a binocular microscope and micro scalpel. And adding the meristem buds to the petri dish and growing 5-6 cultures per dish.
9 Likes
Yes i can essentially have hundreds of mothers in perpetual growth. And i can keep them in 1/12th of the space required. Also i want the strains mobile/ portable so if i need to take the genetics . Then they will be safe in a lighted case i have been making.
10 Likes
that is amazing man. Great work.
6 Likes
Thankyou! @happyhippy
I wanted to share it with everyone! (All) the people here and help others maybe try and and dabble in this part of cloning. Eventhough…I am an ASS. (You regulars know who im talking to) I have no ill will for anyone. I never did. So I wanted to finish what i said i was going to do. This way other people can see its not terribly hard to do it. I go overboard on everything i do. Hence the laminar flow hood and equipment and the get up. But if anyone wants to see how i do it. feel free to pop by and ask some questions.
Love my pinstripe lab suit😉
7 Likes
