While both experiments were essentially failures because there was contamination before I achieved an adaptation of yeast, there were some interesting results from each trial that may lead to some interesting supplementary experiments.
While the links take you to pages that discuss yeast growth, it has been almost conclusively decided that there is bacterial contamination (most likely E. coli) and so the results shown above are not in fact yeast. A brief study must be conducted with my actual e. coli to compare that growth with the results from above. Although, the e. coli time trials of experiments past show exactly what the time trial linked above show: that for some reason E. coli really likes D2O.
Also it was first noticed that yeast may not complete reproduction in 99% D2O, as there were a noticeable amount of “colonies”. These colonies are basically chains of buds that go unseparated. This analysis was repeated in…
The experiment only compared to yeast grown in 20% D2O vs 99% D2O, but the morphologies are clearly different. In 20% the cells were slightly elongated, while in 99% the cells are mostly round and pretty uniform in size.
Not only that but I also noticed that the bud chains I noticed in try 1 were more prevalent this time. The colonies had more time to develop (as the sample was 72h old) and grew into large clumps.
Checking on my yeast to ensure there isn’t any bacterial growth, I noticed they look very different in different D2O concentrations. In 99% D2O the cells appear larger and more circular, while in 20% D2O the cells appear to be elongated. I’ll have to grow some cells in normal water for comparison.
Also I noticed that in 99% D2O the cells seemed to form small colonies of about 10-20 cells, while in 20% D2O I saw almost no evidence of colonial formation. I also saw no yeast tea party (no? NO? oh well…). I think the colonies aren’t so much clusters as they are chains of buds, because they all seem to be attached. I didn’t analyze very thoroughly though.
So it seems that my plants of 3 weeks growth somehow stunted their growth and are beginning to die. Because of that I’m starting a second round of plants. Hopefully this time I get some growth. If not, I may need to buy a grow lamp.
I’ve also reached out to some plant biologists here at UNM in hopes they could teach me the basics of plant growth. Let’s see what turns up. Later today I’ll write up my experimental details once I get the experiment started.
It’s the start of the third week of arabidopsis growth. Sadly there hasn’t been much growth difference from last week. Some notes:
I noticed that the seedlings in just about every sample aren’t doing too well.
Generally, planting on top of the agar wasn’t the ideal way to plant the seeds.
The seedlings in the most media and planted in the agar are doing alright.
The seedlings in the small jar (with about 4ml of agar) are all dry and the agar is all dried up as well. That sucks.
It’s hard to find a balance between keeping the plants covered and exposing them to air. I fear the lack of air flow is hindering their growth, but leaving them exposed also increases the evaporation rate. Having to constantly replace the water makes this experiment expensive for D2O/DDW studies.
It was discovered that individual cells of D2O adapted yeast are very rod like and potentially fissionable. This indicates either one of two things: (1) there has been contamination and this is either a fissionable yeast or bacteria, (2) D2O fucks shit up really messes with cells and these are really distressed. I’m inclined to believe it is contamination since I wasn’t personally overseeing the yeast propagation for almost 3 weeks.
So to check, (1) I will regrow the yeast cells from the beginning with antibiotics, (2) grow a sample of this stuff with antibiotics, and (3) reintroduce these cells to DDW for a few days to see if the growth reverts back to wt yeast. Any of those experiments could reveal the truth, but I don’t think my yeast is antibiotic resistant so I’d have to figure out some way to achieve that.
The biggest issue is that money is getting tight and D2O and DDW is expensive, so I’ll need to develop some cost cutting methods.
The yeast colonies grown in D2O agar are finally big enough to compare to the other samples. It took almost a week to grow this much!
Up above is an image of a single colony, and another of a few colonies that have merged together. It seems that in the presence of D2O, the colonies grow quite smooth still, but a little asymmetrically. Since we know (from unpublished research) that D2O stabilizes microtubules, it would be interesting to compare these results with the morphology of colonies grown in taxol (a cancer drug known to stabilize microtubules).
These images and the final comparison image (and ALL the original raw files from the camera) are available via the figshare link above. These images were also taken after this post, ie the conditions are the same.
Images were acquired at 10x magnification and the scale is 1um/px. The largest image is 1959×1925 px (the larger D2O yeast on normal agar) and the smallest image is 1462x1749px (the D2O yeast on D2O).
As you can see there is quite the interesting phenomenon here. The interesting thing is that there is something morphologically different about the D2O adapted yeast. The reason for that thinking is because the yeast colony retains its brainy shape when placed on normal media, as compared to the wt yeast on the normal agar (smooth circle). Before I make any bold claims about what may be happening here, I need to read some papers about yeast morphology.
Also if anyone wants a glycerol stock of this strain of yeast to run some tests on it, I’d be happy to send it to you. I personally don’t have the means to perform any advanced tests so any experimentation is welcomed!