# Preliminary Results of How Water Effects Organism Development

This is a little backwards because I’m announcing some preliminary results of a project that I haven’t even discussed yet. It’s also a little awkward because I still don’t know what to call this project (at least in a way that would fit in a blog post) and I have even less of a clue what to categorize this project as. With all that said, I say let’s forget all that and just get into what we all came here for:

The results!

I put some tobacco seeds in different water buffers (types of water?). My next post will explain all this, but the short is I have seeds in tap water, filtered 18MΩ (deionized water), and deuterium depleted water (water contains $H_{2}O$, but it also contains a hydrogen isotope called Deuterium that can form $D_{2}O$). The lab has been working with $D_{2}O$ a lot and found (along with research done in the 1930’s) that life is greatly hindered under pure $D_{2}O$. So Koch has been thinking that life evolved to find a use for $D_{2}O$. The hypothesis is that the seeds won’t grow as fast in water that has no $D_{2}O$ compared to regular water. Let’s see what happened in the first trial of this experiment.

Now remember these are preliminary results so I don’t want to make claims that aren’t accurate, but it appears that the roots have sprouted tiny offshoots in the Deuterium Depleted water (DD water). The other option is that it is mold, I’m not sure. Hopefully this will be more clear in a few days.

# Effect of Deuterium Depeleted Water on Life

Hydrogen has several isotopes and one of them, deuterium, exists quite naturally in water to form $D_{2}O$. In previous experiments and several papers by Gilbert Lewis, it has been found that life is hindered in the presence of $D_{2}O$. While this may be true, my PI Steve Koch wondered if life had found a use for it because naturally occurring water has about a 17mM (millimolar) concentration of deuterium.

To put that number into perspective, when I do a typical polymerase chain reaction of DNA I add 10mM of each base of DNA (which is less than the amount of naturally occurring deuterium) to create millions of copies of a DNA template from an amount that is 1000x less then what the reaction yields. In fact most chemicals in most of my buffers on the order of the amount of naturally occurring deuterium.

So you can see it isn’t a stretch to think that nature has found a use for $D_{2}O$ since it is quite abundant and life has been constantly evolving for billions of years. I want to test this hypothesis in a variety of different organisms:

1. Tobacco Seeds – to act as a foil to Lewis’ experiments in which he grew tobacco seeds in pure $D_{2}O$.
2. Mustard Seeds – from what I’m told mustard seeds are the powerhouse of the botanical genetics world much like Drosophila and S. cerevisiae are in their respective genetic fields.
3. Escherichia coli – another molecular biological powerhouse that is very easy to grow and may be easy to see results with. We just got the facilities to be able to grow E. coli and damn it I want to use them!
4. Saccharomyces cerevisiae (Yeast) – I know a guy who grows yeast for his experiments and I’m sure it wouldn’t be a stretch to get him to do so in deuterium depeleted water.

So the idea would be to try to grow these in regular water and in deuterium depleted water (no $D_{2}O$), and in the case of E. coli and yeast, perhaps in pure $D_{2}O$ because I don’t think those experiments have been carried out yet. Hopefully I will be able to conclusively state whether or not life has developed a need/use for $D_{2}O$ which would be a very interesting discovery indeed!

# Setup of Tobacco Seed Growth in Different Types of Water

I am growing 2 different species of tobacco seed (Virginia Gold #1 and Dark Virginia purchased from The Tobacco Seed Company, as an aside I find it strange that we bought seeds from a company in England that gets their seeds from the United States) in different water buffers: regular tap water, 18MΩ deionized water (DI water), and deuterium depleted water (DD water).

I place 3 seeds of each species in a cuvette (I actually have no clue what company these are from because they are from a former student in the lab, but USA Scientific has a comparable type) and add one type of water. So there are a total of 6 cuvettes (3 for each species).

The experiment has way more to consider than I initially suspected which presents some interesting challenges. On top of that I don’t know all that much (right now) about how deuterium interacts with the environment and the seeds, and I don’t know the biochemistry of seed growth in general. Because of this I started a second set of experiments that are identical except that the seeds were presoaked in their respective buffers in the refrigerator in case the initial stages of growth dramatically changed the water solution.

Finally I’m in the process of figuring out how to accurately record growth rates and right now I’m using very primitive macro photography (my camera phone and a big magnifying lens), which will develop into more advanced macro photography (Dr. Koch’s personal DSLR with 10x magnification lens) and hopefully eventually evolve into the microscope and camera system. Here is my current photography setup:

I have a 2in lens with a focal length of about 3.5in setup on an adjustable post (which is mounted on a rail). I place cuvettes on a cylindrical lens holder (the clampy thing in the back) and adjust the lens height and distance to get the best picture. Most parts are opto-mechanics purchased from Thor Labs.

# Pre-soaked tobacco seeds early stage growth

Note: There is some wordpress gallery error that won’t display the second and third picture captions. They are: 2. Dark V in Tap Water. 3. Dark V in DI Water

These images are from the presoaked tobacco seeds. I soaked them in their respective buffers (deuterium depleted water (DD water), tap water, and deionized water (DI water)), and then stored them in the fridge (4C) for 5 days in case there is any drastic chemical exchange between the seed and the water (which would change the buffer). It appears that the deuterium depleted water has started sprouting first in both cases, which is speculative but noteworthy. Also the branching that I noted in the last post appears again here, but only in the DD water samples, which is interesting. I wonder what that could be.

# Unsoaked tobacco seed growth

In most of these samples there is one seedling that shed the seed coat, and in few that seedling is floating at the water surface. It also seems that the branching in the second DD water sample (the Virginia Gold species) has been shed on one of the seedlings (perhaps the one that shed the seed coat). There is a stringy thing floating in the background, which I’m not sure is visible in the image but definitely there in the sample.

Anyone notice anything else?

# Updated Preliminary Tobacco Seed Growth “Results”

Those are images of the first batch of samples (Dark Virginia seeds, not pre-soaked), I’ll take pictures of everything and post it next week. But here are the preliminary reports of what different water types do to tobacco seeds:

• Every seed submerged in deuterium depleted water (DDW) sprouted little hairs on the initial root (the radicle). The interesting thing is this happened almost immediately after emerging from the seed coat.
• Typically the seeds submerged in deionized water (DI water) germinated the slowest. More will come on this when I replicate the Crumley experiment.
• Little hairs sprouted inconsistently on the seeds in tap and DI water but are more prevalent on the DI water seedlings. They are not as abundant on these seedlings as they are on the ddw seedlings. If I had to give an analogy (and I do) then I would say the ddw seedlings can grow a nice ‘fro, while the other seeds exhibit male pattern baldness.
• The little hairs remain localized on the end of the root and aren’t distrubted along the hypocotyl (early stem, and I almost said axon, lol!) so I’m inclined to believe that this is an early root system that is developing because of a lack of nutrients in the water (seen on ddw, most DI, and almost no tap water seeds). But I’m no botanist so I’m just guessing. The fact that the hairs are really prominent in the ddw seeds might suggest the plant recognizes the lack of deuterium, but I’m not willing to make that leap yet.

I setup a new photography system for these seeds. Dr. Koch lent me his Nikon D40 dSLR camera and I purchased some magnification lenses for it. I have the camera setup on an optical post and use a cylindrical lens holder to mount the seed samples (in cuvettes). The picture quality is much better now, with a much higher resolution. I’ll be looking into microscope images soon. Soon I’ll be developing a reliable way to measure the germination, but let’s not jump the shark now. All will be revealed in due time.

Also I was going to measure the pH of the samples at this stage of their development to gain some insight into whether the germination event drastically alters the pH, but the probe in the lab is too big and I don’t have enough sample volume. So I’m thinking that next week I combine the volumes of the water (of each type) to do one “average” measurement. There are four samples of each water type, two for each seed species, and each is filled about 2ml which would give me 8mL of combined volume for each water type. I’m just waiting for the pre-soaked samples to reach full germination (ie shed the seed coat). Now I’m not saying this will work, and it may not be reliable, but hopefully it is a decent approximation for expectations for now until I learn a little bit more.

# Another Update on the Tobacco Seeds

I was going to use this space to compare the two different lenses, but I was so enthralled with the quality of the one 10x lens (from Opteka) that I ended up just taking a bunch of pictures with the one lens and thought I would share it all with you.

I tried to do some reading on the subject over the weekend, but it appears there is a term called root hairs that appear during growth of the primary root (which is what the radicle turns into). Now the books I read were limited in their germination physiology but at least I now have a foothold.

I did note while taking the pictures that in every case, the DDW seeds had the most root ‘fro, then the DI water seeds, and then the tap water seeds had the least. I still think this is a product of lack of nutrients, but there may be something in the question “Why would the root fro be more prominent in deuterium depleted water than in deionized water?” They are both pure, but maybe ddw is more pure than di water? Something to investigate I think.

I encourage you to enlarge every picture up there and see the amazing resolution one attains with the Nikon D40.