D2O raised e. coli, grown in 99% D2O YPD. 60x magnification.
D2O e. coli grown in D2O YPD, 10x magnification.
E. coli raised in D2O, and grown here in DDW YPD. 60x magnification.
E. coli raised in D2O, and grown here in DDW YPD. 10x magnification.
More wt yeast in D2O YPD. 60x magnification.
WT yeast grown in 99% D2O YPD.
Wt yeast in D2O at 10x magnification.
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.
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.
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:
Use autoclaved beakers and bottles for water handling and storage.
Measure water volume: D2O usually is supplied as a little over 90ml per bottle, DDW is about 100ml per bottle, and DI is readily available in whatever quantity needed.
Measure YPD powder and add 5g per 100ml of water.
Add a clean stir bar and stir (using hotplate/stirrer) until YPD is fully dissolved. For D2O and DDW stir at a low speed to minimize air mixture with solution.
Using a syringe and a filter, filter the YPD into the bottles. This step is necessary for D2O and DDW YPD to ensure minimization with H2O/D2O contamination. For DI YPD autoclaving is sufficient.
Label bottles and date.
Daily Prep:
For starter cultures, add 10ml of YPD to an autoclaved test tube. For daily measurements, add 9ml of YPD.
Inoculate yeast in liquid media. For daily measurements, inoculate 1ml of culture from the previous generation’s water type (ie add 1ml of 99% D2O yeast to 99% D2O YPD).
Add to incubator/shaker and incubate at 30C and 185RPM.
Nanodrop Measurement:
Aliquot a minimum of 400ul of sample into semi-micro cuvettes. Normally I measure at 0 hours and at 24 hours (or every 24h thereafter for prolonged experiments) for daily measurements and hourly starting with the 0h measurement for time trial experiments.
Blank the nanodrop with pure YPD and make sure to press the cuvette checkbox (I always forget this!).
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:
Today’s sample (left) compared to the “adapted yeast” which is believed to actually be e. coli (right), from a couple weeks ago.
Yeast as seen yesterday (left) and today (right, both in 20% D2O).
60x magnification of yeast and unknown contaminant in 20% D2O.
10x magnification of yeast and unknown contaminant in 20% D2O.
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.
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.
A colony of non-adapted yeast grown on D2O YPD agar.
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).
I’m forgoing future measurements. I have some data to post (in a few minutes) that may reveal potential contamination. And so to verify if the samples are contaminated or if a cool new phenomenon is occurring, I’m growing the D2O adapted yeast in DDW, to see if they revert back. Although, I don’t think that will reveal much either way.
In lieu of the daily measurements I’ll be taking microscope images of the cells as they grow.
Every day I will inoculate a few colonies from the previous generation (similar to what I have been doing, with an inoculating loop) into 10ml of fresh YPD (both D2O and DDW).
No results today 🙁 I accidentally disposed of the samples from yesterday after inoculating the samples for today. And there is no data today because the inoculation used an inoculating loop, so the absorbance wouldn’t be noticeable. But the setup is 10ml of YPD (3 samples, two DDW YPD and one D2O YPD) with an inoculation (using the loop) from the previous generation. The reason for the 3 samples is because I want to see if after a few generations, the D2O adapted yeast revert back to the wt yeast, so in addition to a sample of DDW yeast and D2O yeast, I’m doing the D2O adapted yeast in DDW. We’ll see what happens.
Another D2O adapted yeast grown on normal agar plate.
D2O adapted yeast grown on normal agar plate.
D2O adapted yeast grown on D2O YPD agar.
WT yeast grown on normal ypd-agar plate.
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!
After some really interesting results from some microscope observation yesterday (to be uploaded shortly), I’m doing some different time trials today. The setup is the same, but today I’m switching the growth media. D2O yeast will be grown in DDW YPD and wt yeast will be grown in D2O. Here’s the setup:
Add 9ml of YPD to 3 test tubes:
D2O YPD
D2O YPD
DDW YPD
Add 1ml of culture to each test tube:
1ml of D2O yeast to D2O YPD
1ml of D2O yeast to DDW YPD
1ml of DDW yeast to D2O YPD
Incubate at 30C and shake
Measure in nanodrop every hour
Yesterday I noticed that the D2O yeast grew more on normal solid media than DDW yeast did (and also more than D2O yeast on D2O solid media). I wanted to get an hourly analysis of this growth. Unfortunately I didn’t have enough made DDW YPD and don’t have time to make a new batch because of timing of the measurements with this afternoon’s activities. So for now I’ll compare this to every other DDW growth experiment. And next week run the trial again with DDW yeast growing in DDW.
I also want to do some microscope analysis of the yeast in the different media because of the observations I made last night. Like I said that data will be up shortly.
