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.
Influence of ordinary water, 33%, 66%, and 99% deuterium oxide on tobacco seed germination. Counts made at daily intervals for 39 days. (From Crumley, Fig 3)
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.
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.
Barnstead EasyPure RoDI
Another view of the setup
sample and image acquistion setup
Test image at closest zoom
Test image at farthest zoom
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.
Barnstead EasyPure RoDI
As demonstrated in the setup of the previous post, I have setup 5 samples of Dark Virginia tobacco seeds with each sample being in a different water type: DI water, 33% D2O, 66% D2O, 99.9% D2O, and for poops and giggles 99.9% deuterium depleted water (DDW).
The percentages are by volume (for example 33% D2O is 2mL of D2O and 4mL of DI H2O). Each sample contains 6mL of its respective water type. I’ve determined that the volume of each cell is a little less than 7mL and 6 comfortably fits with no spillage (there is a small air bubble in the chamber). Each sample also has 30 seeds.
The sample cells were setup as follows: Seeds were counted and placed on one of four prefolded sheets of weigh paper. Airflow was a problem so I placed relatively heavy objects on the edges of the paper to prevent a seed catastrophe. After seed sorting, I prepared the water samples in centrifuge tubes for temporary holding and mixing (in the case of the 33% and 66% D2O samples). 6mL of each water type was then poured into prelabeled Analyslides. The seeds were then added with the lid of the analyslide placed on top to “seal” it. Slides were then placed on the pre-assembled photography station.
Today marks day 1 of the experiment and I am proud to report that in the 5 minutes it’s been, 0% germination is seen across all 5 samples. So far results are as expected.
If you don’t want to look at pictures of gradual change every day then you can just come back to this spreadsheet to see the numbers change and then you can look to see what days something new happened and find the corresponding images through the blog. If I remember to, I may link blog posts with activity to the day number in the spreadsheet, which would be a great idea (but like I said, if I remember).
Update: slide all the way over to the right to see links to the image notes from this notebook corresponding to that day.
Update 2: I added the original Crumley Data on a separate sheet, and I began plotting the percent germination in real time. Currently I’m not sure how to label each line in Google Spreadsheets. I can’t find a way to do it, but I’ll mention it here:
- Blue – DI water
- Red – 33% D2O
- Orange – 66% D2O
- Green – 99.9% D2O
- Purple – DDW
- Not shown – di water, no seeds
BTW: It took me a while to figure out how to do this because Google Docs changed their appearance and formatting yet again. In order to embed a Google Doc in the new format there is a “Collaborate” menu along with the usual “File, Edit, etc” menus. No longer can you go to the “Share” button and select “Publish as web page” because that button has been removed (it is not just the original share button that let’s you set documents as public and determine who you want to share with).