This is the setup for the second trial of deuterium depleted water effects on plant growth. For the most part the experimental setup is identical to the first trial which you can find here. There are a few notable differences though so let’s run through the prep:
5 seeds of two differing varieties from The Tobacco Seed Company are used in each sample (trial 1 had 3 seeds per sample). I am using Dark Virginia and Virginia Gold #1 variety seeds.
there are 5 different water types: 99.9% deuterium depleted water (ddw), deionized water (di water), tap water, 33% deuterium oxide (d2o) mixed with di water, and 33% d2o with ddw. The d2o mixtures are new additions to this experiment.
The samples are stored in semi-micro cuvettes and sealed with a silicon top (maybe it’s rubber).
The seeds are added to a cuvette and then 2.5ml of a water is added to the sample. This volume minimizes the air bubble in the sample to minimize hydrogen/deuterium exchange with the water.
There are 10 samples of seeds and 10 more samples of seeds that are presoaked in their respective water buffers.
Seed growth is tracked using a Nikon D40 DSLR with a 10x magnifying lens. The samples are arranged on some Thor Labs optomechanics to make image taking simpler.
These pictures are setup as in picture 1 (with a computer attached to the webcam). The webcam is a Logitech HD Pro Webcam C910 with the capability to take 10MP images and record in full 1080p.
I took one picture of each sample and then discovered that I could increase the image resolution and manually control the focus and some other properties, so I took one more (the second picture of the 66% D2O sample). From the looks of it, the image quality is more than enough for this experiment. And also between the identical pictures (with different settings) the resolution difference doesn’t make that much of a difference.
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I’m setting up the Crumley Experiment now that I have most of what I need. I ordered some more tobacco seeds but I have no idea when those are set to arrive so I’ll begin a preliminary experiment with what I have to work out the kinks. If you are too lazy to click the link above then I’ll recall for you that the experiment involved putting tobacco seeds in different percentages of D2O and tracking seed germination.
Their setup was to put seeds in petri dishes on moist cloth. I found these slide/petri dish hybrids called Analyslide that seemed perfectly suited to this and I’m going to omit the moist cloth and instead just fully submerge the seeds. The volume of the dish portion seems pretty low (less than 5mL is my guess) and the lids create a nice seal which will hopefully keep deuterium exchange to a minimum.
I built a photography system for the dishes which works a little like an inverted microscope. The slides sit up high on some cage system rods (from Thor Labs) and the camera is mounted on a rail below aimed upward. Currently I have to use a mirror to see in the eyepiece, but I’m thinking of equipping a webcam under the camera so I can see precisely what I’m looking at.
You can see the setup below with some dummy slides and some test images. I wanted to see how easy it would be to focus on something in the Analyslide so I wrote “HI!” on a piece of paper and taped it inside. It worked pretty well.
Update: I forgot to mention where the water comes from! It is very easy to overlook that fact since water is the most used chemical in the lab. When Koch talks about setting up osmotic pressure experiments he always mentions that water is the most overlooked variable because it is everywhere and we take it’s effect on the experimental world for granted. Anyways:
All water is purchased from Sigma-Aldrich, except DI water which comes from a Thermo purifier in the lab. I purchased D2O in a 100g amount bottle product number 151882. Deuterium depleted water (DDW) is also purchased in 100g amounts and has the product number 195294.
The DI water comes from a Barnstead EasyPure RoDI filtration system from Thermo Scientific, with part number D13321. I took a picture of ours for your viewing pleasure below.
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.
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.