# Repeating Crumley et al (1950)

Koch asked me in a comment to replicate an experiment done in 1950 by Helen A Crumley et al demonstrating Tobacco seed growth in deuterium oxide (D2O). The experiment is rather simple (and the figure from the paper is shown below) as Crumley placed 100 seeds in differing amounts of D2O (double distilled water, 33%, 66%, and 99.8% D2O) and analyzed the growth.

So here I am planning the experiment. I will change some things from their experiment. First they placed the seeds on wet cloths (paper towels?), and I will submerge the seeds in the water amounts they used. They also used a variety of plant species (tobacco, clover, radish, Kentucky bluegrass), where I will just use tobacco seeds (but I will try two different species). Finally they talk about their results in terms of percent germination, but it isn’t clear from the paper if they mean number of plants that have exhibited germination, or if they are referring to some amount of growth exhibited by each plant. I will look for both possibilities and report the results as I find them.

In a preliminary experiment I will submerge the plants in water in petri dishes and seal it with parafilm. I will be looking into a more airtight solution as time goes on. I also won’t do 100 seeds but probably on the order of 33 seeds per sample. And in the future I will look into figuring out a way to measure the seed growth.

Original Crumley paper can be found here.

# 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?

# 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.

# Mindmapping Proposed Projects (Updated)

Mindmeister and WordPress apparently do not work well together. I spent a good hour trying to figure out how iframes work in WordPress and it turns out they don’t so I had to install a plugin. I used Embed Iframe and it works, but it turns out that (well at least for me) Mindmeister wants to stick a big panel in front of the mindmap blocking at least 50% of the map. Booo! There is a mindmeister plugin, but I don’t know if I want to play with it right now. Maybe later, and then I’ll tell you all about it.

Back on track, if the above mindmap embed doesn’t work for you then the link is here to see it in full page glory.

Update: I found out that there was a plugin for Mindmeister and replaced the previous iframe coding with the new plugin. The process of discovery can be found here.

# 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.

# 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!

# 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.