I’ve come across a decent flaw with my setup. It seems the airflow is a little more than expected. Well at least it is enough to notice. The air bubbles in a few samples have been getting noticeably larger, in fact in one sample there is more air than water. So in an effort to stop this I’ve done one thing and am trying a few new things:
Upon Koch’s suggestion I attempted a seal with nail polish. I have experience with this before when working with DNA tethering. I would make a micro-channel from a slide, a coverslip, and two pieces of double stick tape. This would make a rectangular channel with two ends open, to flow DNA and other components. After all flowing was finished I would seal the chamber with nail polish. In my experience there is still some air flow, but a sample would last for days and we are talking a volume of ~12ul. Here I am dealing with a volume of 6ml which is roughly 500 times more. If I get comparable air flow, the effect could be negligible.
As an idea, I’m trying to see if beeswax creates a decent seal. I filled the lid of a glass petri dish with melted wax and coated it. Then I poured out the wax that wasn’t solid. I filled the bottom with water and seeds and pushed the top onto the bottom. We’ll see what happens over time…
As an aside of an idea, I’m playing with measuring the seeds via the cellattice microruled coverslips I have. I’m also seeing whether seeds can grow in nail polish. I added some polish around the rim of the cellattice slip to bond it to the inside surface of the bottom of the analyslide petri dish. Then I added a very thin coat of polish to the top of the celllattice slip and dropped some seeds on top. I let it all dry for about 30 minutes before adding water to the sample. The idea was that just slight contact with the polish should hold the seeds in place over the ruler and it worked… so far.
I’ll let you know how these little experiments work out over the next few days.
Pretty soon this experiment will be developing into something more and I’ll be looking to write the results up from this experiment in multiple formats (stay tuned). Anyways onto the notes:
As I mentioned in my other post from today I sealed these samples with nail polish. How that works out remains to be seen, but as you can see in the 99.9% D2O sample and the 66% D2O sample this was becoming an urgency.
Because of the movement involved with the sealing, the seeds are now in different locations then they have been in the past pictures. This presents one new challenge, but shouldn’t affect the results. It should be mentioned that I further agitated the seeds because in several samples (like the DDW sample) there were multiple bubbles and I wanted the seeds to be away from the water-air interface.
In the DDW sample there are plenty of seeds that have shed the seed coat and this fact, along with the fact that I moved everything makes it hard to find the remaining one or two seeds that may/may not have begun germination. So until I notice little radicles I will keep the count the same. This goes for the DI Water sample as well.
Finally make note that there is a new picture of a new sample which I also mentioned earlier. This sample is seeds in DI Water in a glass petri dish (much bigger than the analyslides) and sealed with beeswax. My folly is that I’ve discovered it is really hard to remove melted beeswax from other glass surfaces and I’ve tried boiling water and it is just not doing it. Oh woe is me!
Update: I also wanted to add one final note. I noticed yesterday that it seemed most of the seeds in the ddw sample were a little further along in their development than the seeds in the di water sample. The roots appeared longer and there are quite a number of seedlings without their seed coat (first leaves and such) in the ddw water.
Nothing new to report. It looks like the 99% D2O seeds still haven’t sprouted which is what should happen (contrary to Crumley’s report). Everything else is growing as expected. And as usual there is nothing in the sample with no seeds.
I’m leaning towards doing this trial for 2 more days because all the seeds are at/approaching max germination. Beyond day 15 I’ll keep my eye on the D2O sample to see if there is any sprouting, but it looks like it is becoming a race against evaporation.