Category Archives: Water Type Effects on Organism Growth

Yeast Colony Morphology

I’m not sure what to make of these colony morphologies, but I thought I’d post them for the world to see. I started them a couple weeks ago to compare the growth to that of the E. coli morphology experiment, then just kept them growing to see what would happen.

Arabidopsis Growth Try 4: Setup

I’ve got larger test tubes (1in diameter and about a ft in height), I’ve got plenty of water, and I’ve got a PhD. Looks like I’m ready to grow some plants! Here is the protocol ( adapted from Jan 22):

Cleaning the seeds (protocol provided by Pedro Nunes):

  1. Place seeds in microcentrifuge tube.
  2. Wash with 4:1 ethanol to bleach solution. (I used 1ml of this mixture)
  3. Let sit for 10 min.
  4. Pipette out mixture.
  5. Wash twice with 100% ethanol, and discard ethanol.
  6. Let the ethanol evaporate.

The seeds will sink to the bottom so it is fairly easy to pipette any liquid in the tube. After step 6 I’ll add some water so I can pipette the seeds into their growth media.

Preparing the growth media:

I growing seeds in 10 different mixtures of D2O/DDW: 0% D2O, 5% D2O, 10% D2O, 20% D2O, 30% D2O, 40% D2O, 50% D2O, 60% D2O, 70% D2O, and 80% D2O. I’m not doing a 99.9% D2O sample this time. Each sample will have 20ml of water total.

  1. I used 1 bottle of D2O (100g), 1 fresh bottle of DDW (100g), and one old bottle of DDW (~50g, from 1/22/13, stored in desiccator).
  2. Add 0.22g per 50ml of water of MS media
  3. Mix water in 20ml amounts in test tubes
    • 0% D2O – 0ml D2O, 20ml DDW
    • 5% D2O – 1ml D2O, 19ml DDW
    • 10% D2O – 2ml D2O, 18ml DDW
    • 20% D2O – 4ml D2O, 16ml DDW
    • 30% D2O – 6ml D2O, 14ml DDW
    • 40% D2O – 8ml D2O, 12ml DDW
    • 50% D2O – 10ml D2O, 10ml DDW
    • 60% D2O – 12ml D2O, 8ml DDW
    • 70% D2O – 14ml D2O, 6ml DDW
    • 80% D2O – 16ml D2O, 4ml DDW
  4. Add 0.2g agar (to make 1% gel)
  5. Heat to dissolve agar
  6. Allow to cool for gel to solidify

Planting the seeds:

Normally, I use a pipetter that one would use for small volumes to deposit the seeds, but because the tubes are so much bigger than the ones in the past I need to alter my method. Today I used a glass Pasteur pipet with a very nice long tip. I used the wrong kind of bulb, so it was more challenging than expected, but worked well enough.

Also because my test tubes are much bigger than before, I made the mistake of not purchasing a test tube holder. So I had to fashion one from spare lab components. Check out my system:

Test tube holder fr 25mm diameter tubes.
Test tube holder for 25mm diameter tubes.

It’s made from the breadboard that has become my plant station, screws, and nuts. Pretty simple, and works amazingly well.

The Biophysical Effects of Heavy Water – My Defense Presentation

Defense Outline

Just over a week away now…

  1. Introduction
    1. What is D2O?
    2. The history of D2O
      1. Gilbert Lewis:
        1. purification
        2. biological effects
        3. The hypothesis
      2. Joseph Katz
        1. various experiments
    3. Uses of D2O
      1. NMR, mass spec
      2. The need for a D2O adapted organism
    4. Experiments in DDW
      1. use for space travel
      2. cure for cancer?
  2. The effects on life
    1. Tobacco Seeds
      1. The Crumley experiment and repeating the experiment
      2. Tobacco seed germination rate
      3. tobacco seed growth rate in low deuterium concentration
    2. Arabidopsis
      1. arabidopsis growth rate
      2. arabidopsis morphology
    3. E. coli
      1. growth rates
      2. adaptation and adapted growth
      3. morphology
    4. Yeast
      1. growth rates
      2. adaptation – can’t adapt
      3. morphology
        1. stall during cell division
        2. microtubule stabilization in D2O
  3. Molecular effects
    1. Stabilization of biomacromolecules
      1. DLS experiments
        1. Catalase
        2. Ovalbumin
      2. YPD longevity
    2. Investigation of HD exchange
      1. mechanism and exploitation for protein struture studies
      2. FT-IR analysis
      3. Cavity ring-down analysis
        1. low cost measurement of local atmosphere isotopic composition
    3. Effect on DNA
      1. The pursuit of shotgun DNA mapping
      2. optical tweezers
      3. methods
      4. overstretching data
  4. Future Work
    1. Arabidopsis
      1. adaptation
      2. seed growth in low deuterium
    2. Tobacco growth in low D2O
    3. Yeast morphology in taxol
    4. E coli protein expression in D2O and protein structure analysis
    5. DNA
      1. overstretching in D2O with intercalators

Well there is my idea of how to present my dissertation. I’m not sure if/where I should put my discussion on open notebook science. Also there are a couple things that I could see going elsewhere. I could describe the yeast and e. coli stuff in parallel instead of one after another. Also the HD exchange stuff could easily go right after the yeast, e. coli, or even the tobacco seed stuff. What to do…

Otherwise I think the story is pretty compelling: history of D2O and the unanswered question by Lewis. Investigations into D2O effects and trying to understand low D2O concentration effects, effects on macromolecules, and the understanding of large volume/long-term HD exchange.

Any feedback you may have would be GREATLY appreciated. I’ll send you a figshare t-shirt, or if you are XL, I’ll send you a hoodie (but I only have one).

Feb 28 Yeast Growth

I’ve been scratching my head about this for the past two days. I could have sworn that I did a measurement of yeast growth after adaptation and it turns out I did. Here is the data:

I’ll graph this data and publish that when I’m done (after I defend).

Tobacco Seed Growth Rates

I got some awesome new data to show. The first is the compilation of all the Repeating Crumley experiments. And the second is some new data that I’ve been meaning to create and now have with the help of Koch.

Tobacco seed germination rates
Tobacco seed germination rates

The data above is the compilation of all the RC data. Each trial had different water types, but I combined the samples that were the same in every set (DDW, DI water, 33% D2O, 66% D2O, and 99% D2O). Steve adapted his R-code that applies binomial confidence intervals to a data set and used it on this data. If that makes no sense, then just know that the dotted lines are the most probable range of germination rates. For instance, in 66% D2O there is a ~70% likelihood that seeds will germinate at a rate within the dotted yellow lines.

Now it’s time for some brand new data:

tobacco seed root length
tobacco seed root length

Here we went through the pictures from Trial 5 and compared the growth rates of the roots. We calculated the lengths of various seeds in each image and tracked the changes from image to image. We chose DI, DDW, and 1% D2O, because the D2O concentrations are relatively similar and because we wanted to test a hypothesis from a while ago. It’s interesting that the seeds in DDW and D2O grow at the same rate, while seeds in DI water grow at roughly half the speed.

WT E. coli colony (on D2O LB agar) morphology

Yesterday I posted some pictures of E. coli colony morphologies. This was one of the colonies, but it wasn’t as developed, so today I’m adding the extra day’s growth image.

WT E. coli grown on DI LB agar
WT E. coli grown on DI LB agar

Looks great! It’s interesting to note that the colonies grown on D2O agar grow out. Instead of getting thick like it normally does, it grows in an outward direction. I guess I would attribute that to the stress induced by being in D2O.

Comparing the results from today to WT E. coli grown on  DI media and D2O adapted E. coli grown on D2O media, it seems there is an interesting mix of morphological behavior. The adapted E. coli is very “brainy” and obviously the normal WT is “smooth,” but today’s specimen is in between smooth and brainy. Unfortunately I can’t make out the topographical features because the E. coli (as I mentioned above) is very flat. But the contour is very feature rich.

E. coli cells in D2O

I’m not going to make many comments about these cells. It seems that in D2O, E. coli is more likely to say fused but it’s not as obvious as it is with yeast. I make no other observations.

Last Arabidopsis Update

The media is dwindling and the plants will die shortly. I’ll be restarting this experiment in larger environments with more media. Hopefully the plants can do better.

Yesterday Koch noticed that the roots in 10% D2O were much longer than those of the plants in DDW. Something interesting.

E. Coli Colony Morphology in D2O

Here are the results of yesterday’s setup. Here I’m comparing 4 samples:

  1. Wild type (WT) E. coli grown on DI LB agar
  2. WT E. coli grown on D2O LB agar
  3. D2O adapted E. coli grown on DI LB agar
  4. D2O adapted E. coli grown on D2O LB agar

All 4 samples were incubated for the same amount of time, and taken from starter cultures of similar absorbance. The absorbances of the starter cultures are as follows:

  1. Wild type (WT) E. coli grown in DI LB – 0.641
  2. WT E. coli grown in D2O LB – 0.325
  3. D2O adapted E. coli grown in DI LB – 0.489
  4. D2O adapted E. coli grown in D2O LB – 0.112

The D2O adapted E. coli took over the DI LB plate! I’ve re-inoculated those cells from that plate to get a picture that would show a typical colony and compare that morphology to the rest. I’ve also allowed the two D2O media samples to incubate for another 24 hours.

It should also be said that the D2O adapted colonies grown on D2O media look distressed compared to the WT colonies on DI media. But compare the two D2O adapted colonies and it’s tough to discern. Whatever mechanism gives the colonies a distressed appearance on D2O media, seems to be completely uninhibited on DI media. It’s tough to tell if the cells are distressed or just out of control.

Still these results seem pretty comparable to the last time I did these experiments (except using YPD). I’ll update again tomorrow, and insert these results into my dissertation.