Tag Archives: e. coli

Open Notebook, Water Analysis, Water Type Effects on Organism Growth

Project planning notes via Handrite

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Here are some notes I took using handrite for android regarding my project plans. I don’t mean to advertise for anything, but I use this app ALL THE TIME and it is super helpful. Typing on my phone is annoying sometimes and Handrite allows me to use my finger to write (albeit sloppily). These notes were taken yesterday while I was meeting with Steve (on my phone) and now I have time to paste them in my notebook. So here is some background on my ramblings:

  • I would like to effectively analyze the amount of hydrogen-deuterium exchange in my samples. This is a phenomenon I’ve talked about a lot, know very little about, and stress a lot over. We tried FT-IR of water samples to see if we can determine differences in the water types. We’ve had mixed results in this regard. It boils down to the fact, that I don’t trust anything. How do I know that deuterium isn’t sneaking into my unopened bottles of DDW, or that hydrogen isn’t getting into my D2O? What about after it’s been opened? What about in my samples? If I’m going to get reliable results with yeast and e. coli I’ll need to know if I can trust commercial products.
  • NMR (nuclear magnetic resonance) seems to be a promising route to try and there may be some equipment either in the Chemistry department or Biomedical Engineering here at UNM that I’ll need to look into.
  • Mass spectrometry may be another avenue to determine precise amounts of deuterium in water.
  • Something not written but that I talked with Steve about yesterday and just remembered is that I need to talk with Sigma to find out how they measure the purity of the DDW and how reliable those measurements are long term. If something sits on a shelf for a year, how do I know it isn’t really just pure natural water?
  • I also have some stuff in my notes about the Repeating Crumley experiment. I just setup a D2O only experiment to show definitively that tobacco seeds do not grow in D2O. Previously I would only track this data for 10-15 days and Crumley went up to a month (if I remember correctly). I’ll write the setup in a minute, but I want to do 30 days so I have some pictures for my open access, self published RC paper (spoiler alert!).
  • Also to show that I’m no slouch, I want to try and repeat Crumley’s original experiment with paper towels to show that the growth of the 100% D2O seeds was due to H-D exchange and other environmental factors and not because they eventually grow in D2O. Yea I’m a badass.
  • The rest of the notes are some ramblings that are not really private but thoughts I would prefer not to elaborate on now.

Tata for now!

Ecoli1, Open Notebook, Water Type Effects on Organism Growth, Yeast1

#SciFund Challenge Accepted!

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If you watch “How I Met Your Mother” you’ll get the title of this post. But if you don’t then I’ll let it be known that I have just completed my submission for the second #SciFund challenge which is hosted by RocketHub.com. Here is my submission:


Effects of DDW on Microorganisms

Ecoli1, Water Type Effects on Organism Growth, Yeast1

Yeast: Day 2

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So my overnight culture grew, that’s cool. The E coli did not though. That’s not. To make matters worse, the -80C freezer had some weird malfunction and it was nearly RT in there. Hopefully nothing is ruined. I’ll restart experiments on Monday and make new batches of yeast and e coli spreads and liquid media as well. Then I’ll make some glycerol stocks so that I can be ready for all eternity.

On top of all that I’ll reacquaint myself with doing transformations and purifying plasmids because there are some vectors that I need to replenish. Not for the purposes of these experiments, but just because.

I’ll try and do some preplanning over the weekend and through Monday.

Ecoli1, Water Type Effects on Organism Growth, Yeast1

The bread is ready

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The yeast has spread into colonies (yay), but the E. coli have not. I don’t get what is happening there, but I’ll try growing them in liquid media and seeing what happens. Tomorrow I’ll check on the e. coli and decide what to do from there. In the mean time, I’ll also start a liquid media batch of yeast to grow side-by-side with the e. coli.

Let’s get it…

Ecoli1, Water Type Effects on Organism Growth, Yeast1

Baking Bread!

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and poop to some extent…

Ahem! I started cultures of e. coli and s. cerevisiae streaking cells on some plates to be incubated overnight and beyond. From here I will pick colonies and grow flasks of them to get familiar with the growth rates so I can take data. If all goes according to plan, I’ll be getting some preliminary results by the end of the week!

Here is some info about my cells:

E. coli: XL1-Blue MRF’ competent cells that are designed to replicate pBluescript

Yeast: S. cerevisiae of the variety g160/2d

Water Type Effects on Organism Growth

E. coli day 2

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Nothing to report, and since this is an open notebook I have to say that so there is no gap in the data. Actually the thing I have to report is that the overnight culture I started didn’t yield anything. And since I didn’t leave any starter culture I can’t even compare the absorbance profile (but I doubt it will do anything) so tomorrow I will try again bright and early.

And once my water stuff finally gets here I will be able to move forward with the experiment. But in the mean time I will get acclimated with taking time points and using the cuvette function of the nanodrop and just play around in the lab.

Water Type Effects on Organism Growth

Beginning E. coli growth

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It’s been a while since I’ve had to grow e. coli and I’ve never really had to measure E. coli growth, just because I would vaporize the cells immediately to get at their precious nectar (plasmid DNA), but now I have to do all that. So I’m learning on the fly, the best way to learn.

Yesterday Alex and I started two colonies from E. coli that is designed to grow pBluescript and anther designed to hold pALS (my custom plasmid). Here are her notes on the matter (scroll down to read some comments from myself).

This morning we got acquainted with the cuvette reader on the Nanodrop and here are Alex’s notes on that. In summary we got an OD of 0.714 on the one test tube that grew bacteria. Now I have to figure out what that number means and how to accurately measure OD in the future. For that I’ll need to find some protocols that explain all about measuring cell cultures and do something with making a standard curve, which is talked about here.

In preparation I’m doing another starter culture that I’ll check on in 36 hours (or so) and begin dilutions and what not. My starter culture is literally just LB broth resuspended in DI water purchased from Sigma, without antibiotics (since I don’t really care what grows as long as something does, for now and because we don’t have any). I put a scrape of the Stratagene competent cells into the broth and put it in our shaker at 37C, 150 RPM, left for overnight and beyond.

Water Type Effects on Organism Growth

Preparing for E. coli in DDW, DI, and D2O

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Sorry for the lack of notebooking today. I had to send a bunch of emails this morning, catch up on teaching work, and then had to write an informal CV this afternoon. Wanna see it?

Anyways, I’m doing the prep work for the E. coli studies and showing Alex how to use an autoclave. I also am getting into sterile technique and Alex will have something up later about all of this to which I will link.

Tomorrow we are going to mess around (and get triple doubles) in a safe environment and grow some e.coli and see what happens. I also hope to get my hands on some yeast. There are a few barriers before we can get any serious data. First I have to successfully grow microscopic creatures in small containers. I have to get acclimated to using the nanodrop as an OD measuring device. Then I have to do all this with different water types.

Fun times ahead.

Ecoli1, Water Type Effects on Organism Growth, Yeast1

Project Outline for DDW Effects on Microorgansims

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Another page for project outlining that will change over time.

UPDATE: I changed the embed code so the map has live update capabilities.