Category Archives: DDW Effects On Life

Paper Summary: Adaptation of Arabidopsis to D2O

C. R. Bhatia and H. H. Smith, “Adaptation and Growth Response of Arabidopsis thaliana to Deuterium,” Planta, vol. 80, no. 2, pp. 176–184, 1968.

Looking for papers on yeast adaptation (of which I’ve found precisely one!) I came across this paper where they attempted to adapt arabidopsis to D2O. I was instantly intrigued and downloaded the paper for brain consumption. I will now take notes on the paper with the intent of setting up an experiment that can run with minimal effort on my part.

  • A paper that is mentioned in the intro that reviews all the genetic and cytological effects of D2O, seems like an interesting place to check out next for work with the yeast project.
    • Flaumenhaft, E., S. Bose, H. L. Crespi, and J. J. KATZ: Deuterium isotope effects in cytology. Int. Rev. Cytol. 18, 313–361 (1965).
  • Concerning the possible biological action of deuterium, it is known
    that substitution of deuterium in hydrogen bonds of essential macro- molecules (nucleic acids and proteins) changes the properties of these molecules by increasing bond strength and also by a general retardation of reaction rates.
  • Methods:
    1. seeds were surface steralized in 3% hydrogen peroxide and 90% ethanol (a 1:1 mixture) for one minute
    2. seeds were sown into mineral media mixed with 0.78% agar – I’ll have to find out what mineral media is available in 2012. Also I find it strange that in this paper they say what products they use, except for the mineral media. If it’s made in house, why don’t they say what the composition is?! Argh!! It’s always the important detail that is left out…
    3. seeds were grown in test tubes, and closed with glass caps.
    4. samples were cold treated for 5 days at 4C and then moved to a climate controlled box maintained at 24C and under constant illumination (they use some fluorescent lighting and I’m assuming they are 1960’s grow lamps)
  • Results:
    • they did 2 experiments: one was a germination experiment much like the experiments I did in the repeating crumley series, the other was the full adaptation experiments, in all experiments seeds were grown at 0, 10, 30, 50, 70, and 90% D2O
    • in both experiments they looked for morphological differences from normal plants
    • in the first generation of plants, they found germination rates to be increasingly delayed with increasing concentrations of D2O. This is pretty consistent with with the results I linked to above, although they counted germination from the emergence of the first leaves, whereas I looked for any emergence from the seed coat.
    • they also discovered that the green-ness of the plant decreased with increasing D2O conc. and the survival rate of the plants also decreased. No plant produced seeds above 50% D2O in this first generation.
    • flowering rates were also lower in samples with higher d2o amounts. the authors subtracted out the germination delay to determine an average flowering time and saw that that increased with increasing d2o concentration.
    • as for the adaptation experiment, they say they screened over 850 plants, but only have growth information for plants grown in 50% D2O. They find that after 6 generations of growth on 50% D2O seeds can survive to maturity at 70% D2O and seeds obtained from these plants grow normally in the first generation on H2O media. They conclude that the D2O adaptation is not genetic because of this. I would like to add that I’m highly skeptical of the adaptation efforts included in this paper. There are no counts of the number of plants per generation, the number of seeds planted, etc.
    • they hint that they would are taking measures to develop mutants that can grow at concentrations of D2O that are above 70%, I’m guessing they failed because the papers that cite this paper are not by the authors.
  • This paper has been cited twice according to the internet:
    1. Brown, B. T. (1972) A new screening procedure for detecting plant growth regulating compounds. Pesticide Science 3(2)
    2. Foston, Marcus B. (2012) Deuterium incorporation in biomass cell wall components by NMR analysis. The Analyst 137(5)

This paper must have been one of the last studies on organism development in D2O, especially because the papers that cite this paper are in unrelated fields and this is one of the most recent papers I’ve seen in this field. With that said, I’m a little skeptical of the effort to obtain D2O adapted arabidopsis.

I am impressed that they wanted to answer the question of whether H2O affects D2O adaptations in similar ways that D2O affects H2O adaptations. I don’t think their study was thorough enough and growing plants in 50% D2O still leaves the room for plants to get the H2O requirements. I think growth at levels above 70% would be key to obtaining true D2O adapted plants.

Their methods seem pretty simple and I could work on my own version of this experiment pretty easily and have already looked up product information regarding plant growth in the lab:

  • http://www.sigmaaldrich.com/catalog/product/sigma/a1296?lang=en&region=US
  • http://www.sigmaaldrich.com/catalog/product/sigma/m5524?lang=en&region=US
  • and a protocol: http://www.biosci.ohio-state.edu/~plantbio/Facilities/abrc/handling.htm

I’ll order those things tomorrow morning. And tomorrow I read a paper about D2O-Yeast adaptation!

New Yeast

I have no idea when this will get here, but I put in an order for some new S. cerevisiae. I think on Monday I’ll have Alex grow another starter culture and then on Tuesday, we’ll try and get better resolution under the microscope than we got last time.

Anyways, here is what I’ve ordered (from ATCC.org).

E. coli growth experimental setup and data (on FigShare)

E. Coli Growth over 4 hours. Anthony Salvagno, Alexandria Haddad. Figshare.
Retrieved 20:17, April 20, 2012
hdl.handle.net/10779/b627469dabcd4034053cc53040d4dcbd

I went through the data that I posted on Tuesday and realized it was even less useful to people than I expected. I almost didn’t even know what I was looking at! Anyways, I did a couple of plots in excel with the data (which can be found on FigShare along with both the original data and the revised and cleaned data) and tried to extrapolate some other information. But first let’s discuss the experimental setup.

So on Monday, Alex created a starter culture from the E. coli we grew on plates last week. Then on Tuesday (I realize how not very real-time this post is for me, but the data came out in real-time which is also important) we made 3 dilutions of the starter culture to track the growth of the E. coli over time. We did:

  • 1ml in 9ml of LB broth (1:10)
  • 2m in 8ml of LB broth (1:5)
  • 5ml in 5ml of LB broth (1:2)

Every hour we took an absorbance reading from the nanodrop and read the 600nm value (A600 according to the machine). We also reblanked every hour according to the instructions from the nanodrop. The initial readings were:

  • starter culture – 1.076
  • 1:10 – 0.097
  • 1:5 – 0.23
  • 1:2 – 0.665

It is interesting to note (and I literally just noticed this), that the initial readings are almost exactly what the dilutions are, ie 0.097 is ~1/10 of 1.076. Good for us!

In the FigShare data, you will find the original data (which I linked to in my post on Tues, but in Google Docs instead of Excel) and a revised data set. The data is messy but the graphs are interesting.

Also I tried to link the 3 data sets together into one coherent graph, but the time series doesn’t seem to match up right, or maybe it does and I just think it doesn’t. The 1:5 dilution seems to provide a bridge between the data in the 1:10 dilution and the 1:2 dilution. After about 2 hours the 1:10 sample overlaps with the 1:5 and likewise the 1:5 begins to overlap with the 1:2. Also at hour 3, the 1:10 sample overlaps with the 1:2 sample.

Because of this I tried to graph the data as one continuous set. It seems Like it may be alright, but I feel that the in the 1:2 sample there isn’t much growth in the 4 hours, which is reflected in the 3 data set plot, but it doesn’t look like it peaks in the continuous graph. Hmmm… Anyways check out the FigShare data.

PS I’m including the two plots I’m referring to below.

 

Quick E. coli growth data

I have to leave right now, so I’ll post some methods later, but here is some incoherent data for you all to digest. This is done in DI water in regular LB broth.

And here is the data via Google Docs:

E. coli growth

Please critique my #SciFund proposal

https://docs.google.com/document/d/15y12xbD9tNVdw5taQU9EAiWDHai4215Fx3zLAeXu7l8/edit

Yeast Day 2

These are the results of the “experiments” we did yesterday and Tuesday. The difference between the samples is that the cultures from Tuesday were streaked from a colony on another plate whereas the cultures that grew last night were streaked from liquid media. So it seems this yeast doesn’t grow so well from a single colony on agar. Further evidence to prove that I should just get a more common strain.

Yeast and E. coli Day 2

First off, how come yeast has a common  name but E. coli doesn’t? Can we call it shitus?

Anyways yesterday Alex and I did some follow up work after starting some cultures. She did a good job notebooking the experience so I won’t double up on her thoughts. Check that out here.

We took some pictures of the e.coli and the yeast which turned out pretty bad. I’ll have to try and take some better images today or something. In general though it looks like the yeast we have isn’t what we thought it was. It may not be a mutant of S. cerevisiae but may be some version of Schizosaccaramyces pombe (I hope I spelled that right, I like the first name a lot) because it looked nothing like budding yeast.

The result of this is that I will be ordering some new yeast straight up from atcc.org that is of some variety that I commented on in Alex’s notebook (hopefully).