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).
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.
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.
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:
5 test tubes were filled 10ml with each media type.
10ml – 99% DDW
10ml – DI water
10ml – 99% D2O
3ml 99% D2O, 7ml 99% DDW – 30% D2O
6ml 99% D2O, 4ml 99% DDW – 60% D2O
Single colonies were plucked from this plate, and put into each test tube.
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!
Yesterday it occurred to me that I couldn’t autoclave LB media made with DDW or D2O because of D-exchange. So I had to come up with a solution which prompted me to ask the world (thank you internet). Through the power of open science (victory for ONS!) I was able to get some comments that said I should use a 0.2 micron filter:
@Thescienceofant two points 1. you can grow E. coli static (no shaking) 2. why not make(or buy) 10x LB and use sterile-filtered D2O?