The Presentation

For the last time...

WOOHOOOOOO!!!!

The presentation was last period. It's always nice to not have it looming in the distance, but I am sad to see this project end. It was BY FAR the best project I have done in any class. Ever. I just want to make that clear. So, how would I grade myself? Well, I'll be totally honest. I forgot my notecards at home, which was pretty terrible preparation on my part. However, the way it turned out I didn't really use the new ones I made. I stumbled pretty badly a couple times, it is really hard for me to onstage, which is something I have to work on. And I did deviate from the speech I had written for a few parts, but I maintained some kind of organization I think.

Logistics aside, the purpose of this presentation was to convey a general idea, and how my project had been my epiphany. I spent a lot of time drafting my speech, and I think that I accomplished that pretty well. I tried to display the message throughout the explanation of my process, something I did not do last year for our Inventor Presentation. I'm proud of the improvement. Actually, there have been a lot of changes since the beginning of last year to my presentation skills. Today I found myself laughing, onstage, at the ridiculous chicken article. It was a connection with the audience that I couldn't have had a year and a half ago. This being the last solo presentation (I think) I will ever give for Mr. McDaniels and Mr. Perlman, I think improvement can be factored into the grade.

So, I have 30 possible points to rate this project by. I'll divide it up and allot points as follows.

Clarity and delivery of intended message: 14.5/15 (97%)

Description of project/process: 5/5 (100%)

Poise and preparation: 4/5 (80%)

Improvement: 4.5/5 (90%)

Total: 28/30 (93%)

There's always room for improvement. But I am really happy I had the opportunity to do this project and the way it turned out.

Seeds...

Well. That was terrible. Yesterday I collected seeds from the Drosera burmannii I have been trying to clone. Let me just say these seeds are extremely valuable to me, as they are my last tie to this plant before it dies. I had one jar of the tissue culture gel left. Showtime.

So everything started out well. I modified my sterilization process a little, because I was working with seeds and didn't want to use as many chemicals. I hauled the box upstairs and sprayed everything down with bleach. I put the seeds in a piece of coffee filter and paperclipped it shut. I placed this in a baby jar filled with 10% bleach for 13 minutes. I added a little dish soap so that the bleach would have a lower surface area and better cover the seeds. As I shook the jar to make sure the seeds were thoroughly cleaned, I noticed about ten seeds slipped out of the packet. This was ok, I had sort of planned for this. Not that big a deal.

So then I transfer the packet to a vial of hydrogen peroxide for about 4 minutes. After that I swirled it around in two different jars of steril water for 10 minutes each.

For the whole sterilization process, you aren't that worried about the seeds because they are about to get cleaned again and again. However, at the last stage where you have to transfer the seeds, you have to work FAST to make sure you don't compromise the sample and the jar. I pulled the filter paper out of the final jar quickly and started to unfold it. Unfortunately, it ripped. Stupidly, I just decided to keep ripping the filter paper apart to get at the seeds as quickly as possible. Emphasis on stupid. I had folded the paper enveloped multiple times to try and seal the seeds inside. So when I forcefully tried to rip it apart, all the layers compacted into a solid pulpy mass, trapping my seeds inside. I pulled and pulled and pried, but I could never get the seeds out from inside the filter paper.

I managed to get about four seeds of the forty seeds I has started with onto my finger (in hindsight I realized I had forgotten to wipe myself down with alcohol, so they were definitely compromised). These I transferred onto wet tweezers and tried to flick into the jar of nutrient gel. However, they stuck to the tweezers. After about four unsure bangs on the rim of the open jar (meanwhile millions of bacterium, algae, and mold spores are falling inside) I moved onto the other seeds and repeated the process. At the end of this miserable attempt, I found one black speck (which still might not be a seed) lodged in the nutrient gel.

Ya.

Well, I refuse to let this hinder me. While I do not have time to redo the entire project (plus I'm out of baby food jars), I would like to retry the process further down the road. The important thing is to learn from yesterday. Here is what I could come up with.

1. Always unfold the filter paper.
2. Practice flicking wet seeds into a jar, or sterilize a thin wire, get them onto that, and then carefully lower them down onto the gel (lab scientists use a thing called an inoculating loop).
3. Practice with seeds you don't care about.

Hopefully, I can get this right the next time around. While I still considered yesterday fun, I really hope the third time's the charm.


I almost forgot the good news! If you watch my vlog, you can see my previous plant grow area. Over spring break, I bought and built a GROWSHELF, complete with reflective bubble wrap I rigged up around the plants.

YEA BUDDY

That was fast...

Alas, the most likely outcome in this experiment has arrive. Contamination. I checked last night and every single leaf had some form of white/grey fuzzy mold on it. But hey! At least I could recognize it. And the problem was on the leaves, not the gel, which means my project isn't a total failure. I just need to reconsider. I will be redoing the experiment tomorrow with some new jars, and seeds instead of cuttings. These sundew leaves are particularly hard because of the hundreds of nooks and crannies created by the tentacles.

This was the best photo I could get. See the white smudge on the green leaf? That's the mold.

Seaweed Chutney

Now that I'm done tissue culture, I felt the need to do a little more reading to improve on my process. Remember a little while ago how I mentioned the need for plant growth hormones? Well I did a couple hours worth of reading on these substances, and it is really, really cool. Basically, these chemicals come in two forms, auxins and cytokinins. Both are responsible for cell enlargement and increased division, which makes for more plant in a short amount of time. Which is perfect for tissue culture.

I do not have a PhD in Chemistry and so was not able to decipher all of the jargon, but basically what I learned is that these chemicals are manufactured by the plant to help growth. They start at the roots and work there way up the stem to the apical growth (or middle stem). If this apical growth is removed or conditions are very nice, these plant hormones will trigger growth in the dormant nodes surrounding a plant (those funky little nubblets that surround a plant). This is why your prune plants- by severing the top you get those surrounding nodes to grow, making for a larger, bushier plant.

So where are these found? Well they are made by the plant. There are four auxins created by the plants themselves, though the most notable is called IAA (this is just an acronym, but because I don't want to type out the name and don't understand it at all, I'll leave it at that). However, this chemical is extremely unstable and frequently breaks down. In tissue culture, scientists have manufactured synthetic auxins and cytokinins that are cheap and way more powerful. Good for us!

However, I don't like to buy things from labs. It's too easy and still too expensive. So what if I make some chutney? As I was doing my reading, I came across "seaweed extract" as containing a potent mix of natural plant growth hormones. I tried to figure out why this is. It turns out that seaweed (or kelp) is one of the largest and fastest growing organisms on the planet. Some can grow two feet per day and top out at 260 feet long. Ya.

Now how does one "extract" these chemicals from seaweed. Well some further research showed that IAA is somewhat soluble in water, and one paper showed that these hormones can either be easily freed from the plant or are seriously locked inside proteins and won't leave. An extract is created when you mash the seaweed up and steep it in water and other solvents, and basically let it break down into a concentrated seaweed tea. Here's a video I found hysterical about a guy outlining his process, including the infamous "seaweed chutney."

So then I thought to myself, is there anything better than seaweed. Well as it turns out a lot of these extracts rich in plant growth hormones are also marketed as rejuvenating skin lotions. This is one of the primary selling points for coconut water, which I talked about earlier. But there is one plant on this earth that grows at an even more ungodly pace than seaweed.

Bamboo.

Bamboo is the rat of plants. With a growth rate of nearly three or four feet per day in some species, bamboo tops the charts of growth. So, what if I extracted the wicked cocktail of growth hormones in bamboo and applied it to samples in tissue culture. I couldn't find studies that had done this, though coconut water and seaweed had both been tried. I need to do a little more research into exactly what makes bamboo so hopped up on crazy dust and how one goes about extracting it. If this is possible, then here is a natural, flip floppin' unending source of growth hormone. Or I could always buy the laboratory stuff. WHOA. What if I could replace the growth hormone in bamboo with the ones we've made? Would it grow even faster? Or is bamboo nature's perfectly mutant result of 'roid rage?

I wanted to add one more note that just occurred to me. Seeds are very rich in growth hormones, which means that the seedlings do not produce very much, if at all. So whatever is substituted in the media (nutrient gel) of tissue culture is its main source of "inspiration" for growing.

I wanted to add another note. In an experiment based on the above logic, I could compare the effects of bamboo extract hormones vs. synthetic hormones on bamboo seeds. If the extract came from the same species, it would be a proper comparison.

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.

VIDEO BLOG

Here I talk about the process and detail my inspiration. It's all explained in the video, but with this project my main goal was to bring a complicated science to my home. Though it may seem like it, I've really simplified the process by synthesizing a lot of research.

 

Tissue Culture Vlog from Drew E on Vimeo.

Halfway There!

This week has been full of lots of fun research and concluded in me finishing my first five jars. But I'll start at the beginning. I spent the week learning about these chemicals called Plant Growth Hormones, or PGRs. They're pretty self-explanatory, they are natural chemicals found in plants that make them grow. Certain combinations either make plants grow new shoots or roots. In real tissue culture, you can buy ones made in a lab. However, I'm doing cheap tissue culture! So what I found was that coconut water contains a number of these PGRs, but is extremely unreliable. Here's a study I found explaining coconut water's various chemicals. Here's one showing how it applies to tissue culture. It was some really interesting reading. If you want to learn about PGRs, I found this, this, and this to be very helpful. In the end, I determined that seaweed extract would actually be the most consistent supply of PGRs, but I ended up not finding any. Maybe I'll make a new media over spring break that contains some seaweed. It'll help if I transplant some of the samples and make stimulate some real growth.

So now onto the real news. I continued to play around with corn starch, and cooked three jars with different amounts of it in my pressure cooker for about 10 minutes. I determined that 6 teaspoons per 1/2 cup of water worked the best.

4 teaspoons per 1/2 cup water. Notice the clear spot of liquid.

5 teaspoons per 1/2 cup water. Notice the small dots of liquid.

6 teaspoons per 1/2 cup water. Notice the white jelly goodness.

A quick note about pressure cookers. They are very thick metal pots that have a very tight lock on the top. They have one valve to let just enough steam out to maintain 15psi. They are HORRIFYING. The steam screams out of the valve, and I refused to stay in the same room as it. Pressurized shrapnel from glass jars would not feel good ripping through my face if it blew up. I dropped a baby food jar this weekend, and when they shatter, they are sharp.

Pressure Cooker from Drew E on Vimeo.

After these tests, it was time for the real deal. I mixed my nutrient liquid with some corn starch (see video blog), and added it to the pressure cooker. I cooked it on high for about 2 minutes and then let is simmer to make a total of 30 minutes. I pulled the jars out, and the liquid had gelled perfectly! One jar opened as I pulled it out, which is a big no-no because any bacteria that entered the jar can quickly spoil it. But I closed it up again and am going to leave it, just so I know what contamination looks like. The other jars I covered with Saran-wrap to further seal them and keep out any nasties. I plan to sterilize the tissue samples and add them to the jars on Tuesday. I still have five more jars to prepare for Spring Break with the high-quality gel, agar.

WOOHOOOOO!!!