Tag Archives: d2o effects on life

D2O1, DDW1, Microorganisms, Water Type Effects on Organism Growth, Yeast

Yeast Growth Trial 1 Data

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Here is the data courtesy of figshare:

S. cerevisiae growth in DI Water, DDW, 50% D2O, and 90% D2O. Anthony Salvagno. Figshare.
Retrieved 22:01, May 22, 2012 (GMT)
hdl.handle.net/10779/bfceb213ffb663b34149d97141755232

I would say this data is pretty exciting. Unlike the E. coli data (which was all over the place), there is a clear difference in growth rates between the D2O samples and the H2O samples. Also the growth between the DDW and DI (the H2O samples) were almost identical! That’s pretty solid.

D2O1, DDW1, E. Coli, Microorganisms, Water Type Effects on Organism Growth

A cool figure I forgot to share: E. Coli growth in Water

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the e.coli data reimagined in illustrator

I made this figure for my rockethub proposal and forgot to post it here in my notebook. Look how pretty it is!

I made this in Adobe Illustrator and if anyone is interested I would be willing to host an online workshop to teach others how to use Illustrator for science. This particular image took almost no time (maybe 30 minutes), which is a marvel because I obsess over everything I do in Illustrator (which is why there isn’t anything tangibly Illustrator on this site).

D2O1, DDW1, Microorganisms, Water Type Effects on Organism Growth, Yeast

Yeast Growth in D2O, DDW, and DI YPD: Setup

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Yesterday I created a starter colony of yeast from the lyophilized yeast that I ordered from ATCC.org. Today I’m doing growth in different water types and checking the growth every hour just like the e. coli experiments. Here is my setup:

  1. Make YPD broth with DDW and D2O.
    1. the bottles of both come in 100g quantities which means there is 90-ish ml of D2O and 100-ish ml of DDW
    2. I had 91ml of D2O –> 4.55g of YPD powder
    3. 100ml of DDW –> 5g of YPD powder
  2. suspend powder in water types and filter via syringe. DO NOT AUTOCLAVE
    1. the reason is because of deuterium exchange. The powder is ultrapure and the water is pretty pure itself, so filtering should be just fine.
  3. To make diluted cultures:
    1. I have 4 samples: 1) DI water 2) DDW 3) D2O and 4) 50% D2O in 50% DI water
    2. Put 9ml of each water type into a test tube (except for the 50/50 mix which should be 5ml of d2o and 4ml of di water)
    3. Add 1ml of starter culture (which is made from DI water)
    4. put in incubator
  4. take time points every hour in semi-micro cuvettes using nanodrop. I fill them with 0.5ml of each culture.

Simple!

D2O1, DDW1, E. Coli, Microorganisms, Water Type Effects on Organism Growth

E. coli growth in different water types data

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Via figshare:
E. Coli Growth in DI, DDW, D2O, 30% D2O, and 60% D2O. Anthony Salvagno. Figshare.
Retrieved 23:35, Apr 27, 2012 (GMT)
hdl.handle.net/10779/51fcd2f94fd7464449ee0f794642214c

I’ll post some interpretations here later…

D2O1, DDW1, E. Coli, Microorganisms, Water Type Effects on Organism Growth

24h e. coli growth in di, ddw, d2o, 30% d2o, and 60% d2o results

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24h Growth of E. coli in D2O, DDW, DI, 30% D2O, and 60% D2O. Anthony Salvagno. Figshare.
Retrieved 19:23, Apr 27, 2012 (GMT)
hdl.handle.net/10779/5e1e8fdaeeb4d7161c1f73990d42147e

D2O1, DDW1, E. Coli, Microorganisms, Water Type Effects on Organism Growth

E. coli growth in different water types: Methods

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Today I’ll be taking time points of the e. coli growth every hour in LB suspended in DI, DDW, D2O, 30% D2O, and 60% D2O. I’m going to follow my protocol from yesterday, where I blank for the water type, before I measure the absorption for that water type.

  • This morning I recorded the values from the starter cultures
  • Then I diluted 1ml of each culture in 9ml of it’s respective water type (so 1ml of culture from LB-DI goes into 9ml of LB-DI, 1ml of culture from LB-30% D2O goes into 9ml of LB-30% D2O, etc).
  • Next I measured 500ul of each sample in the nanodrop.
    • Blanked for water types DI, DDW, and D2O. The readings for the blank 30% D2O and 60% D2O were pretty close to the reading for 99% D2O, so I used the 99% D2O sample as the blank for these two cultures.
  • Results will be up on figshare later, but I’ll put the starter culture results up now.

I’m not sure what to think about the values for the overnight growth. E. coli grew in 99% D2O, which was rather surprising. Maybe it isn’t. All there needs is to be 1 cell that grows and then there will be a colony. This makes me think that growing e. coli in 99% D2O and switching that colony to DDW wouldn’t reveal too much. I may have to do multiple generations of growth in D2O before I switch growing medium to make sure the colony is fully adapted to life in D2O.

Also that makes me think that my setup from yesterday wasn’t so optimal. Perhaps by growing cells in each water type initially, I’ve just insured that they will all grow at the same rate and there will be nothing to reveal here. Hmmm, I’ll have to think about this. I feel like I’m trying to trick e. coli, but it somehow is outsmarting me.

No sir, I don’t like it…

D2O1, DDW1, E. Coli, Microorganisms, Water Type Effects on Organism Growth

DDW and 30% D2O starter culture analysis

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Data on figshare:

24 hour growth of e. coli in DDW and D2O. Anthony Salvagno. Figshare.
Retrieved 22:57, Apr 26, 2012 (GMT)
hdl.handle.net/10779/6a26c1c4c7e487101f894fe53c5e8473

Setup

Notes:

  • So I mentioned several times that these cultures aren’t optimal, which is why I resetup the experiment today. But I did get some interesting results.
  • The absorption readings on the nanodrop were:
    • 30% D2O – 0.782
    • 99% DDW – 1.239
  • So the growth over 24 hours in 30% D2O was ~63% less.
  • Interestingly the number in 30% D2O is similar to yesterday’s time sampling data, both in number and consistency. The D2O sample reached 0.7 the fastest, but started to decline at that point, whereas the other samples continue to increase.
  • My data collection technique may be a bit weird to those looking at the raw data.
    • I analyzed samples of lb-water (where water is the water type of the lb media) to see how much difference in the absorption spectrum there were between the water types. There wasn’t much difference except from the DI sample, which had a much larger absorption reading than the other 4 readings.
    • I also blanked the nanodrop of each water type before measuring that water type. Example: I would insert the LB-DDW and blank it, and then measure the culture labeled DDW. Same went for the 30% D2O sample.
    • I did one measurement of the DDW sample after blanking in LB-DI to compare to the LB-DDW blank reading. The comparison is off by about as much as the absorbance of the LB-DDW and LB-DI which is a 0.017 difference.
  • All that leads me to think that for some reason growth in D2O isn’t slower, but has a peak meaning for some reason the media can only sustain so much culture after a while. Tomorrow I’ll try for a longer time series study to see if the decline is real or if that was some weird glitch that happened.
D2O1, DDW1, E. Coli, Microorganisms, Water Type Effects on Organism Growth

Starter Cultures in DDW, DI, and 30%, 60%, and 99% D2O

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Tomorrow I’ll be running the same experiment I ran yesterday, only this time I did a much better job preparing the cultures and the liquid media. The broth setup can be found here.

Today I initialized the starter cultures in each water type. The liquid media was made in 99% DDW and 99% D2O and the partial medias are a mix of each water type in the appropriate ratios. Here is my setup:

  1. 5 test tubes were filled 10ml with each media type.
    1. 10ml – 99% DDW
    2. 10ml – DI water
    3. 10ml – 99% D2O
    4. 3ml 99% D2O, 7ml 99% DDW – 30% D2O
    5. 6ml 99% D2O, 4ml 99% DDW – 60% D2O
  2. Single colonies were plucked from this plate, and put into each test tube.
  3. Cultures were put in incubator/shaker at 37C at 150rpm for overnight incubation.

Tomorrow I’ll be repeating yesterday’s experiments with each of these samples, taking time measurements every hour all day long. Happy, happy, joy, joy!

D2O Effects on Life, RCD, Water Type Effects on Organism Growth

RCD: Day 28

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Still nothing growing and the old sample that had one sprout stopped growing as well, which leads me to believe it started growing a long time ago, possibly while still in the package and not in D2O, and I just never noticed.

DOI: 10.15200/winn.142801.12160 provided by The Winnower, a DIY scholarly publishing platform

D2O1, DDW1, E. Coli, Microorganisms, Water Type Effects on Organism Growth

E. Coli Growth in DI, DDW, and D2O Data

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From FigShare:

E. coli growth in D2O, DDW, and DI. Anthony Salvagno. Figshare.
Retrieved 01:18, Apr 26, 2012 (GMT)
hdl.handle.net/10779/e186403d944367bcbf5bdd9ed6977a88

The handle is a broken link for some reason so here is the direct link until that gets resolved.

And some notes:

  •  Right when I was collecting data for the last set, I noticed that the incubator/shaker wasn’t running. Basically it wasn’t shaking. The reason for this is that the lid technically was open. Usually I have to lean on the shaker to get it to close properly, but for some reason that wasn’t working. I then had to spend about 15 min trying to fix it (which I did), and I’ll post my fix for it tomorrow. Bicep kiss on that (Note: don’t google bicep kiss with safe search off).
  • For some reason the data at hour four regresses. By that I mean the e. coli absorption was less than it was at hour 3 for the D2O sample (and for the other two I believe). It continued to regress for hour 5, but the other two samples did not.
  • Also I haven’t analyzed the growth, but based on the numbers it appears that the cells divided faster in 33% D2O than the rest of the samples. I have no idea what’s going on here.

For tomorrow, I don’t think I’ll be redoing this experiment. So I’ll just do another starter culture and try it for Friday. I’ll also do cultures in DDW, 30% D2O, 60% D2O, and 99% D2O.

Now big important question. Should I autoclave the DDW and D2O LB Broth mixes? Keep in mind the major fear of deuterium exchange.