Category Archives: Microorganisms

D2O adaptation, D2O Adaptation 2, D2O Adaptation 3, Microorganisms, Water Type Effects on Organism Growth, Yeast

Daily Yeast Growth Protocol

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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:

  1. Use autoclaved beakers and bottles for water handling and storage.
  2. 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.
  3. Measure YPD powder and add 5g per 100ml of water.
  4. 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.
  5. 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.
  6. Label bottles and date.

Daily Prep:

  1. For starter cultures, add 10ml of YPD to an autoclaved test tube. For daily measurements, add 9ml of YPD.
  2. 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).
  3. Add to incubator/shaker and incubate at 30C and 185RPM.

Nanodrop Measurement:

  1. 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.
  2. Blank the nanodrop with pure YPD and make sure to press the cuvette checkbox (I always forget this!).
  3. Measure each sample.
D2O adaptation, D2O Adaptation 2, KochLab Stuff, Microorganisms, Uncategorized, Water Type Effects on Organism Growth, Yeast

Lab Contamination!!!

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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.

 

D2O Adaptation 2, Microorganisms, Water Type Effects on Organism Growth, Yeast

Yeast morphology in D2O

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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.

D2O Adaptation 2, Microorganisms, Water Type Effects on Organism Growth, Yeast

Yeast Adaptation Day 3

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My original yeast is still going in D2O. There hasn’t been much growth in the past 48 hours. And to hopefully achieve a faster/more efficient form of adaptation, I’m growing yeast in slightly increasing amounts of D2O. Today I started growing in 20% D2O (mixed with DI water to save money).

After 48 hours of growth the yeast in DDW measured 3.327 in the nanodrop.

Also I noticed that the YPD in DDW aggregated. This took about 12 days. I’ve never had this happen in D2O, except one time it did in a 1ml amount in a cuvette. That took about 14 days. And it was next to a heat source during that entire time. So it would be interesting to test the aggregation affects of YPD in DI/DDW and D2O. If I can achieve an adapted form of yeast, growing in D2O YPD could be beneficial all around (cost effective).

D2O Adaptation 2, Microorganisms, Water Type Effects on Organism Growth, Yeast

D2O Adaptation Try 2: Day 1

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Because I’m 90% confident that the adapted yeast was actually e. coli (based on images and smell) I’m going to restart the experiment. I started by cleaning the incubator/shaker. Might as well try and minimize e. coli outbreak…

Setup:

  • Add 9ml of D2O YPD (all that I had left) to test tube.
  • Inoculate a colony from yeast grown on D2O solid media.
  • Add 9ml of DDW YPD to test tube
  • Inoculate colony from yeast grown on D2O solid media.
  • Place in incubator at 30C and 185rpm

I also made a fresh batch of D2O YPD:

  • 4.6g of powder YPD
  • 92ml of D2O
  • stir until dissolved
  • filter (2um) into bottle
D2O adaptation, Microorganisms, Water Type Effects on Organism Growth, Yeast

Individual cells grown in DDW and D2O

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These images were acquired last week (Thursday evening).

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.

D2O adaptation, Microorganisms, Water Type Effects on Organism Growth, Yeast

Non-adapted yeast grown on D2O YPD Agar

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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).

D2O adaptation, Microorganisms, Uncategorized, Water Type Effects on Organism Growth, Yeast

D2O Adaptation Day 54

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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).

D2O adaptation, Microorganisms, Water Type Effects on Organism Growth, Yeast

D2O Adaptation Day 51

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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.