DONE DONE DONE DONE DONE

I doubt this will be the last time I say it, but here it is anyway:

WOOHOOOOOOOOO!!!!

Yesterday I finished the first four jars of tissue culture. It all went pretty well, I cut off the first four developing leaves of my D. burmannii. Then I filled some jars/tubes with the following chemicals and started sterilization.

I was going to try and sterilize some tweezers with a lighter, and then realized I had these covering every surface. An oxygen source, two potent fuels, and a very reactive source of a toxic vapor.

Here's the procedure I followed, modified from when it was listed in the first post.

INSIDE HOOD

1. Dunk explants in 91% alcohol for 10 seconds. Skip this for seeds.
2. Dunk in 3% H2O2 for 4 minutes.
3. Dunk in 10% bleach solution for 7 minutes. Put seeds in here for 4 minutes.
4. Place in sterile jar of water and rinse for 5 minutes. Repeat.
5. Pull samples out of jar with sterilized forceps.
6. Loosen cap on TC jar.
7. Place explants in jar extremely quickly.

8. Wrap containers in Saran wrap.


STERILIZATION:

1. Using tiny spray bottle, spray 10% bleach solution on every surface.
2. Generously spray jars with 10% bleach solution.
3. Wash hands with soap and water, then wipe with 70% alcohol.


To prepare the steril water, I filled loose jars with distilled water and pressure cooked them for 25 minutes. For the bleach solution, I used 2/3 cup of extra-concentrated bleach in 9 1/3 cups of tap water. I figured any impurities in the tap water would be washed off in the final rinse. 

If I were to do this again, I would use flat forceps (tweezers) the whole time, for this I used surgical forceps with teeth that I am sure damaged the cuttings. I am really happy with how everything went. The only danger is that by using those toothy forceps some of the chemicals killed off the entire leaf. We'll see how it goes over this coming week, where contamination is the new hurdle.

I still have five jars left and some agar, so I may do the entire process over again later in the week. I wanted to get this done so that I have a solid two weeks before the presentation for something to grow. If I do it again, it will be with agar and not cornstarch.

Pictures!

The aftermath. My parents are lovely people.

The river of bleach that ran down my kitchen counter. I cannot describe the smell of bleach, alcohol, and peroxide mixed together.

The little green thing is the cutting. It sank into the cornstarch perfectly.

The cutting with terrible aim. I hope it grows.

Another cutting in its new home.