Category Archives: D2O1

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 onĀ  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.

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.

D2O Adapted E. coli Experimental Replication

Yesterday while writing I realized that the images of the D2O adapted E. coli that I’ve taken were grown on D2O YPD. In an effort to figure out if the morphologies are due to the YPD or the D2O, I’ve decided to redo the experiments on LB agar.

Today I made some D2O and DI LB broth:

  • 1.84g LB in 92ml of D2O
  • 1g LB in 50ml of DI water
  • Filtered broth for sterilization

Then from there I made some LB agar

  • 40ml of liquid D2O LB with 0.8g of agar (2% agar)

I already have solid LB plates with normal water (commercial).

I then incubated E. coli at 37C in liquid media so that I can streak the plates and analyze them. I used 2 different E. coli:

  1. Normal competent cells
  2. Cells from Day 33.
  3. I also had made a separate culture from an unlabeled glycerol stock that I’m pretty sure I made when I finished the experiment. I’ll check it out tomorrow after the sample has developed.

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

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).

E. coli growth in different water types data

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…

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

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

E. coli growth in different water types: Methods

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…

DDW and 30% D2O starter culture analysis

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.

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

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!