I needed an image of wt yeast in DI YPD to compare with the morphology of later strains. As I ramp up the D2O concentration, I expect to see some differences and this is good information to have in general.
I don’t think there is much to infer from the 20% D2O comparison image, but if it’s worth anything there are a few cells in that image that are elongated, and I never saw that morphology in DI water.
Back in the day I was calling the bacteria grown here D2O adapted yeast. Boy was I stupid. Anyway, here is the data I took when trying to compare the morphologies. Despite my naivety, I think there can be interesting studies done with E. coli in D2O based on these experiments and the colony morphology experiments I did.
Also I’ve included actual yeast in D2O images, which show that yeast form “chain gangs.” Basically the buds never seal off and grow new buds and large clusters of bud chains grow. Hopefully I can analyze this more in the upcoming experiments.
I was curious to see what the yeast start out as before I add YPD to the equation. So I took a sample and brought it to the microscope. I then wanted to try and analyze the process that took the yeast from what it was as a starter “colony” (I have no idea what to call this) to become the buds that we all know and love. While I doubt that I’ve really learned anything, I think I took some fun pictures:
It appears the cells start as spores or some kind of egg. And after the addition of YPD the center expands and the yeast cells begin to awaken from their hibernation. I have no idea if what I’m saying is true, but that’s the only thing I can think of from my observations.
I had 3 samples, two of which are imaged here: the first is just the starter volume right out of the fridge, the second is the starter volume with an equal volume of YPD added, and the third sample (not imaged above) is after incubation for about an hour.
It should be noted that I saw quite a few rods in my samples. I hope they aren’t bacteria, because if so that indicates the “infection” is in my stock. After some brief reading across the web, it seems I can just inoculate my yeast in the presence of an antibiotic like ampicillin (which I have) to hinder bacterial growth. Then I can grow on solid media and isolate a colony of yeast.
I almost kinda want to let bacteria take over and add antibiotic to see what happens…
Today is the start of the starter culture. It should be ready for inoculation by Friday morning.
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.
This experiment was interesting for 2 reasons:
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…
While this experiment was relatively young, there was still a lot to be learned and a lot that can potentially be revealed:
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.
Now that the lab is clean, I’m going to collect my thoughts and write a plan to follow. While the first two experiments were unsuccessful, there were some interesting results that can’t be disregarded. But in the pursuit of science more results are required! So let’s get this party started…
D2O adaptation: Based on the paper I read yesterday, it seems that a slow adaptation is the right way to go. Yeast grown in pure D2O seemed to endure too much stress to adapt and the adaptation might be way slower compared to a progressive adaptation plan. So each week I’ll increase the amount of D2O by 20% starting with 20% D2O YPD:
Plan:
These are my protocols for the D2O adaptation experiments. I’m making this post so I don’t need to write a post daily unless something out of the ordinary happens.
Making YPD:
Daily Prep:
Nanodrop Measurement:
This morning while setting up for a time trial experiment, I noticed the 20% yeast sample didn’t smell like yeast anymore. It smelled like a mixture of yeast and something else. So I setup the experiment, took initial measurements, and then analyzed the sample in the microscope. This is what I saw:
Surrounding my slightly modified yeast are these tiny things, that look like super small e. coli so they are probably some bacteria or perhaps they are some kind of spore. Regardless that was not at all in the sample from yesterday (see above), and looks nothing like the e. coli that I temporarily believed was adapted yeast.
So I began a mission to decontaminate the lab. After the cleaning I just did, nothing is alive! Not even myself! In fact I’m not even writing this… (Note to dead future self: Sorry :-\)
Anyways, I began by bleaching the fuck out of everything. The incubator got it the worst as I basically drowned it in bleach. I scrubbed real hard with this super awesome huge bristle brush. I let the bleach sit for about 10 minutes and then wiped it down with clean rags.
Next I used the Activeion Ionator EXP, which was loaned to me by the custodial staff here at CHTM. It’s a spray that ionizes water to clean and disinfect, and supposedly can kill viruses and bacteria. After the bleach treatment on the incubator, I Ionated it and wiped it down and allowed it to air dry.
Then I used the Ionator EXP to clean all the bench tops and all my lab equipment (pipetters, racks, scales, hot plate, etc). I finished by emptying my current supply of YPD and made new stocks for use tomorrow. I feel sad that I had to throw about $80 worth of D2O down the drain, but I gotta be careful in the lab and so it had to go.
Tomorrow I will start the D2O adaptation experiment again (Round 3!) and let’s hope the contamination issues are behind me.
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.