Category Archives: DDW Effects On Life

DDW5, Water Type Effects on Organism Growth

DDW Effects on Life 5: DI Water Edition Setup

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After 4 trials and talking with Steve, there is no way to ensure that the results we are getting have anything to do with deuterium content. We can’t tell at all that our DI water is just not the same kind of “pure” that the DDW is. So I bought deionized water from Sigma (same people who make our DDW and D2O) to compare with our water and to compare to the DDW results to determine if the root hairs grow based on water purity or if it has something to do with deuterium content.

Here is the setup of the experiment:

  • I am using 4 different “types” of deionized water. We have a Easypure RoDI from Thermo (see Experiment Product Page at top) that I get DI water from. CHTM also has their own deionized water filtration system and I’m using that water in this experiment as well. I also purchased two different kinds of pure water from Sigma, one is molecular biology grade water and the other is double purified water for tissue cell culture. I honestly don’t understand the differences, but this chart says there are some.
  • I also used pure DDW for one sample and a 1% D2O mixture with DDW for another (because why should I exclude the DDW results from this study?)
  • I am keeping this experiment to just the tobacco seeds since the arabidopsis results are perplexing and not as obvious. So I am using both kinds of tobacco seeds (Havana and Virginia Gold #1, see Experiment Product Page).
  • I chose to do four samples per water type per seed type. With 5-7 seeds per sample. That means for each water type there are 8 samples, 4 for each type of seed.

Today’s protocol was really easy. I used my macro cuvettes (see Experiment Product Page) and set up 4 cuvettes for each water type giving me a total of 24 cuvettes. I did this so I could set up samples for one seed type at a time, and because I only had enough racks for this many cuvettes. I also prepared the D2O/DDW mixture (29.7mL of DDW, 0.3mL of D2O).

I poured 5-7 seeds in each macro cuvette until all the setup cuvettes had seeds in them. Then I added 3mL of water to the samples (again making 4 samples per water type). Then I sealed the cuvettes with PE caps.

I repeated this setup for the next seed type (24 cuvettes, 5-7 seeds per sample, 4 samples per water type, with 3mL of water per sample) and sealed and labeled all cuvettes.

I placed the cuvettes in the 4C fridge to synchronize the growth among all the samples. Tomorrow I’ll remove the seeds and take Day 1 pictures.

DDW4

DDW4: Day 27

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I asked Koch to take pictures of the plants for me while I am out of town. Here are his notes regarding the experience:

Wasn’t sure of your camera settings. I had trouble with depth of focus, so I chose aperture priority (“A”)
and F=22, which seemed to work OK. I didn’t want to change your zoom, so I didn’t. I took pictures of bottoms
until half way through when I realized to take tops too (so two pictures each for arabidopsis). This was a
mistake, but since I don’t know if the photos are useful, I’m stopping now.

Photos are labeled TYPE CUVETTE_NUMBERS UP/DOWN and I left in the actual photo number, as follows

1 VG cuvettes 1-3
2 VG cuvettes 4-6
3 VG cuvettes 7-9
4 VG cuvettes 10-12 (DI, TAP)

5 H cuvettes 1-3
6 H cuvettes 4-6
7 H cuvettes 7-9
8 H cuvettes 10-12 DOWN (DI, TAP)
9 H cuvettes 10-12 UP (DI, TAP)

10,11 CA cuvettes 1-3 UP, DOWN
12,13 CA cuvettes 4-6 DOWN, UP
14,15 CA cuvettes 7-9 UP, DOWN
16,17 CA cuvettes 10-12 DOWN, UP (DI, TAP)

 

Thanks Koch for the help!

DDW4, Water Type Effects on Organism Growth

DDW4: Day 15

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I can’t tell in the havanna seeds sample, but the virginia gold tobacco seeds in di and tap have tiny hairs. This is not alarming because I noticed this in prior trials. It is obvious that the hair growth in the ddw samples far exceeds that of the growth in these two samples.

Ecoli1, Water Type Effects on Organism Growth, Yeast1

Project Outline for DDW Effects on Microorgansims

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Another page for project outlining that will change over time.

UPDATE: I changed the embed code so the map has live update capabilities.

Water Type Effects on Organism Growth, Yeast1

Today I learned how to synchronize yeast cells

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protocol with a bunch of extra info

I spoke with Kelly Trujillo about cell synchronizing after Koch had mentioned to me that we may need this for ddw yeast studies. He mentioned that he was going to be synchronizing for a study that he was setting up and that I could come learn the process. So I took him up on his offer.

I have to say, the protocol is surprisingly simple. Kelly grew a colony, diluted it to about 0.1 OD and then let it divide up to 0.4 OD. We looked at the asynchronous growth and then he added some mating pheromone (details to come) to some of the culture to halt the cells in G1 phase of cell division. From here, he could synchronize them to enter S phase by using hydroxyurea (HU) after washing out the mating pheromone. The remaining culture was arrested in G2/M phase with the drug Nocodazole.

We paused during each step to look at each culture (asynch, G1, S, and G2) but I have no pictures. I do have the knowledge to do it myself and hope to incorporate this in the DDW experiments shortly. A preplanning thread will come soon.

DDW4, Water Type Effects on Organism Growth

DDW4: Day 7 cropped photos of tobacco seeds

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And here are the cropped pictures of the tobacco seeds (no past photos). Also courtesy of JPEGcrops.

DDW4, Water Type Effects on Organism Growth

DDW4: Day 7 cropped photos of Arabidopsis (and some pics from DDW3)

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Here are the cropped photos. I used a program called JPEGcrops to mass crop the images from day 7. It was sweet and quick. I also included (uncropped) photos of the DI and tap water samples from Trial 3 of the DDW experiment. I don’t know if the curviness is real or not so I’ll let you comment below if you think it’s real. My money is on yes!

DDW4, Water Type Effects on Organism Growth

DDW4: Day 7

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Focus on the middle sample in each image.

I have to say I’m convinced. I’m convinced that the root hairs are a real phenomenon of growing in ddw. All of the tobacco seed samples (havanna and virginia gold) growing in ddw have at least one or two seeds that have root hairs. ALL. The DI and the tap water samples are barely growing hairs. There is a clear difference between these hairs and the DDW samples.

As for the arabidopsis. I don’t know. The only thing I’m noticing is that the stems are growing very crooked. So crooked that most of the seedlings have intertwined. This is tough to confirm because the DI sample barely sprouted, but the tap water sample appears to have longer persistance lengths (to borrow a word from DNA). By this I mean the angle of curvature is greater in the tap water than in all the ddw samples which tend to curve like no ones business.

I can confirm this by comparing to the previous batch of samples. The tap and di water samples had similarly longer curve radii, while the ddw (which had to start after) seeds got all tangled together.

What should I make of this?!?!?!?!?

DDW4, Water Type Effects on Organism Growth

DDW4: Day 2

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CA = columbia arabidopsis

VG = virginia gold

There is at least one seed in every CA sample that has sprouted (in fact in many of them most seeds have sprouted). Not only this but I’d say overall that over the past two days, there is more growth in the DDW samples than there is in either the tap or di water samples. By more growth I mean the length of seedling (radicle).

As far as the tobacco seeds go, there are a few sprouts here and there, but they generally don’t sprout until days 3/4.

Ecoli1, Water Type Effects on Organism Growth

Preplanning for DDW with E. coli: Thread

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Since I have no concrete method for performing the DDW experiments with E. coli, I’m starting a thread to discuss possible steps that should be taken. I’ll summarize the results of this conversation (hopefully not with myself) in another post that will lay out the plan. If you have anything to contribute please don’t hesitate to add to the discussion.