Since I had leftover MS salt-water mixture leftover from the first experimental setup, I decided to use the remaining bit to make this batch. I did add some water since last time I had over 1% gel. I added about 5ml to the remaining solution, which is some unknown amount. Obviously this isn’t the best experimental setup, but I essentially just want to see if I can grow plants so accuracy isn’t too important.
I have received some replies from some UNM Biologists and hopefully can meet with some students this week to work out some experimental details. More information to come as I know it.
So it seems that my plants of 3 weeks growth somehow stunted their growth and are beginning to die. Because of that I’m starting a second round of plants. Hopefully this time I get some growth. If not, I may need to buy a grow lamp.
I’ve also reached out to some plant biologists here at UNM in hopes they could teach me the basics of plant growth. Let’s see what turns up. Later today I’ll write up my experimental details once I get the experiment started.
It’s the start of the third week of arabidopsis growth. Sadly there hasn’t been much growth difference from last week. Some notes:
I noticed that the seedlings in just about every sample aren’t doing too well.
Generally, planting on top of the agar wasn’t the ideal way to plant the seeds.
The seedlings in the most media and planted in the agar are doing alright.
The seedlings in the small jar (with about 4ml of agar) are all dry and the agar is all dried up as well. That sucks.
It’s hard to find a balance between keeping the plants covered and exposing them to air. I fear the lack of air flow is hindering their growth, but leaving them exposed also increases the evaporation rate. Having to constantly replace the water makes this experiment expensive for D2O/DDW studies.
Here are the pictures of my samples after setup. Note that there are three additional samples of seeds in water. These samples are basically the equivalent of my original DDW Effects on life experiment. But now that I think of it, I didn’t use DDW so I don’t know exactly what I’m looking for… d’oh!
It was discussed a long time ago that adapting arabidopsis to D2O could reveal some interesting finds. So in an effort to do that I’m going to grow some example plants to test the setup and try and produce seeds.
The one issue I worry about is evaporation. Since this cannot be stopped, evaporation could become a costly component of the experiment (D2O and DDW cost about ~$100 per 100ml). The first experiment will be using DI water. Here is my setup:
This is based on the combination of these two protocols:
I mixed 0.2192g (4.3g/L) of MS salt with 50ml of DI water.
I then put 30ml in a beaker with 0.5g of agar, heated, and stirred. I was supposed to make 1% gel and measured 0.5g for the 50ml that I made instead of for the 30ml that I added to the beaker. When the agar dissolved, I added 10ml of MS salt water to the solution to reduce the gel percentage (now at 1.25%).
I then put the MS agar solution in test tubes: 2 @ 1ml each, 2 @ 2ml each, 2 @3 ml each, and 2 @ 5ml each. I also put ~4ml into some random (but clean) glass jar thing. I bought these a long time ago when I was looking at clear, glass jars for the tobacco and arabidopsis seed DDW experiments. The reason for the various volumes is to determine how much medium the seeds need to grow efficiently and healthily.
While I waited for the MS salt-agar solution to cool (and thus solidify) I poured a bunch of arabidopsis seeds into a petri dish and added some DI water to it. I did not steralize these seeds, because I’m just timing the growth and learning about the pollination process and judging how well the seeds will grow in the lab. For the actual experiments I will steralize the seeds.
Once the growing media cooled, I used a pipetter to collect a few seeds (~5 seeds per sample) and place them in each sample. There are two sets of test tubes (one volume of each set, ie 1ml, 2ml, 3ml, 5ml).
For the first set, seeds were placed on top of the growing medium.
For the second set, seeds were plunged into the gel.
For the random 4ml jar, seeds were plunged into the gel.
Once seeds were set, rubber stops were placed over top of the test tubes to minimize evaporation.
I’m sure this process has tons of kinks, which will be worked out when the real experiment begins, but this is the purpose of an experiment right?