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.
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.
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!
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.
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.
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.