Tag Archives: recap

Yeast hourly growth in DI, DDW, and 30%, 60%, 90%, and 99% D2O

Results:

Yeast growth in DI, DDW, and 30%, 60%, 90%, and 99% D2O. Anthony Salvagno. Figshare.
Retrieved 21:20, May 24, 2012 (GMT)
hdl.handle.net/10779/caa57855586213d79eea5576f8da23d0

Notes:

  • I don’t think this is a reliable data set. Several of the samples actually read a lower absorbance after the first hour than they initially do. And then they all dip again at hour 4.
  • I noticed a considerable amount of cell settling after hour 3 on the bottom of each test tube. I mixed prior to reading the absorbance values, but this is likely to skew the results. Next trial I will have to mix before reading every hour.
  • The data between DI, 30%, 60%, and 99% D2O look consistent with Tuesday’s results, but it scares me that the DDW and 90% D2O are completely out of whack.

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…

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.

RC5: Results

di water (first blue); 33% d2o in di (red); 66% d2o in di (yellow); 99% d2o (green); ddw (purple); 1% d2o in ddw (second/light blue); 33% d2o in ddw (pink)

I don’t know what to make of these results. As usual the 99% d2o seeds didn’t sprout, a good sign. The DDW seeds sprouted first, but then kinda stalled. The seeds in 1% d2o/ddw did the best and quickly reached the max germination percentage. The good news is that there is a clear gap between {di, ddw, 1% d2o in ddw} vs {33% d2o/di, 66% d2o/di, 33% d2o/ddw} (a little math language for y’all where brackets designate sets). Opinions?

RC4 Review

Let’s just get right into rapid fire bullets:

  • With the fourth trial of the Repeating Crumley experiment, I added the development of arabidopsis to the mix. This was not done by Crumley et al but I figured the data should be similar. Unfortunately I carried out the experiment poorly and this hindered the success of this aspect. The problem was that there were too many seeds per sample and after about 5 days it became increasingly hard to count sprouted seeds. Some seeds were still germinating while others had already shed their seed coat, making it hard to tell which seeds were old and which were undeveloped.
  • The tobacco growth was more like expected, but the 66% D2O sample developed slower than in previous trials. Interestingly though, I think the seeds in this sample developed more steadily, meaning on pace with what should be expected.
  • No seeds grew in 99.9% D2O in 10 days, and from the 3rd trial set there are still no developed seeds.
  • I think the percentages from this trial aren’t a good indicator of the results. Only because saying that only 70% germinated in a sample doesn’t sound like good recovery. But that’s just me.
  • It seems the rates are right on with Crumley’s analysis. DI seeds grow first and fastest, then 33% d2o seeds, then 66% d2o seeds, and 99% d2o seeds don’t germinate at all.

I’m going to add time-lapse slideshows to the blog later to show the seed development. And I’ll keep watch over the seeds to see if anything else happens. I won’t be around next week (Florida vacation!) so I’ll start RC5 (and DDW4 as well) as soon as I return.

Repeating Crumley: Seed Growth vs Time

As you know I posted pictures of the seed growth every day. Well a couple of days ago I decided to organize the pictures by sample in one post as opposed to by day. This way you can see the growth of each sample over the 15 day period. The posts are already posted but I figured I would compile the links here with some commentary so you know what’s up.

As you can see trial one wasn’t a huge success in this regard because every so often I would shift the seeds around. It makes it hard to track each individual seed, but with that said there are some obvious things you can take away:

  • You can really see the growth over time. It’s hard to keep track on any given seed in each sample, but on an average you can really get a feel for how quickly these seeds develop compared to the other samples (ie the stuff in di water grew first compared to the stuff in 66% D2O).
  • You can track the evaporation rate decently enough. This was a major problem towards the end of the experiment and you can really see its effect.

Overall I’d say I did a decent job with this experiment given that it was a first try. I’ve managed to improve some things for Try 2 and I’ll be making more improvements when it comes to Try 3, which hopefully will be good enough for an official experiment.

BTW: The plugin I used to create the slideshows is called Portfolio Slideshow.