Tag Archives: e. coli

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 on  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.

Yeast and E. coli Growth: What’s next?

There are several things I can do with the yeast experiments:

  • Another but longer time trial experiment (data every hour)
  • Time Trials in larger quantities: maybe 100ml or 50ml – I want to see if the growth in such small volumes affects the measurements any
  • Adapt yeast to D2O and then grow in DDW – I’ll have to do cultures daily in D2O and see if they begin to grow faster in D2O over time. I’m thinking this will let me know they’ve adapted to their environment. I can also try to grow cultures in less D2O (like 10%) and incrementally increase it over time to see if that causes adaptation. Both methods seem viable.

I’m open to suggestions as to what to do next as well.

As for E. coli, those experiments are very inconclusive even in 99% D2O and I wouldn’t have the foggiest as to how to deal with this. Suggestions would be much appreciated here. Ayuda me!

Using cuvettes in the nanodrop

I did a mini-study this morning to find out what the minimum volume needed is to get an accurate reading in the nanodrop. I have 2 different cuvettes (semi-micro and micro) and I wanted to impact the cultures the least so I would like to use the micro cuvettes.

image
From left: 400ul in semi-micro cuvette, 200ul in micro cuvette, 500ul in semi-micro cuvette

I used 5 semi-micro cuvettes and 2 micro cuvettes. I put increments of 100ul starting at 100ul in each cuvette (100 -> 500ul in the semi-micro and 100 and 200ul in the micro cuvette). At and above 400ul the nanodrop was able to effectively and consistently read the absorbance of the semi-micro cuvette (verified because the readings for 400 and 500ul were identical). For the micro cuvette, I looked at the profile (image above) and saw by eye that the height of the meniscus of the liquid media was roughly equivalent to the height in the semi-micro cuvette with 500ul. So I put this in the nanodrop and got an equivalent absorbance reading.

So in summary:

  • For semi-micro cuvettes (and the Thermo Nanodrop 2000c), volumes of at least 400ul or more are sufficient for consistent readings.
  • For micro cuvettes, volumes of 200ul are sufficient for consistent readings.

The End.

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!

Preparing LB Media in Different Water Types

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:

Well that is what I did and here is my protocol:

  •  I have this easy mix stuff that dissolves pretty rapidly (see Experiments’ Products Page), and it requires 20g/L
  • I want to make 100ml amounts for each water type (99% DDW and 99% D2O), so I need 2g of LB media.
  • Add 2 g to the 100ml of water and stir.
  • Suck up in a sterile syringe.
  • Add 0.2 micron filter to syringe and pump into autoclaved glassware.

Presto!