Week 3 at the Donohue Lab

Hi this is Meg again. I just finished my third week of working in the Donohue lab at Duke University. Finally after about 2 weeks of setting of for my experiment, we were able to begin on Monday. To summarize my experiment, we will be putting seeds from different genotypes and different maternal conditions (some maternal plants were grown in hot temperatures and some were grown in cold temperatures) in dishes of different water potentials. Water potential (Ψ) helps to describe where water will move due to diffusion and more specifically osmosis; the area with the more negative water potential is where the water will move to. After the plates have been seeded, we will then census the dishes every other day to count how many have germinated. Once the all the data has been collected, hopefully we will find a threshold or base water potential (Ψ_b), which is the lowest water potential at which germination will still occur, for each of the genotypes and maternal conditions.

So this week on Monday, we started to seed both my experiment and an undergraduate’s experiment. Lien, the undergrad, is also working with different water potentials but her experiment focuses on a different aspect of water potential. However, she has a very similar setup, and therefore we have been working together on both projects. Overall, on Monday, we seeded 270 plates, 120 of those with 24 seeds, and 150 with 12 seeds, which made for a pretty tiring day.

On Tuesday we ended up seeding 50 more plates than we had originally planned, which meant that we seeded 270 plates again. Then on Wednesday, luckily it was a little less with 220 plates. On Thursday, even though it was the Fourth of July, we had to come in and finish seeding for our experiment. However, because we had done extra on Tuesday, we only had to seed 170 dishes, which meant that we were all able to leave the lab by around 1.

Buchner funnel attached
to Buchner flask

Funnel and Flask attached
to vacuum

In Lien’s experiment, the seeds are kept at in dishes with varying water potentials, just as mine are. However, after 4 days, the seeds are switched from dishes with negative water potentials to dishes with distilled water (water potential of 0). The goal of this is to see if the first treatment with varying water potentials helps the seed to progress towards germination, without actually germinating. If the negative water potentials do help the seeds progress towards germination, the amount of time it takes for the seed to germinate will decrease. In order to transfer the seeds to dishes with water, we first needed to rinse the seeds to ensure that all of the solution from the original treatment is washed off. On Friday, we began this process.

Cutting filter paper
with seeds on it

 To rinse the seeds, first we cut out the filter paper that the seeds were resting on in the plate and placed it on paper that absorbs the excess solution. Next, we placed a piece of filter paper in a Buchner funnel, which fed into a Buchner flask. The funnel is made of ceramic and has many small holes. The outside of the funnel is surrounded by a rubber stopper to create a seal. The flask has a hose barb so that a hose can connect the flask to a vacuum. Then we placed the now dry filter paper with the seeds in the funnel on top of the other filter paper. 

Rinsing seeds. The soapy-
looking water in the flask is
the dirty water that is sucked
in due to the vacuum

Next we rinsed the seeds twice with 10 ml of distilled water. Because there is a vacuum in the flask, but the paper cannot pass through the funnel, only the water that the seeds are rinsed with moves into the flask, leaving the seeds clean and sitting on dry filter paper. Finally, we placed the original filter paper in weigh boats filled with 20 ml of water, which is where they are seeded from onto the new distilled water plates. This whole process is really time consuming and stressful because the water scatters the seeds everywhere so that some are lost in the process. We couldn’t lose more than 12 of 24 seeds each time in the process or else there wouldn’t be enough seeds to complete the experiment. 

Rinsed seeds in weigh
boat, ready to be seeded

Also, because the time the seeds are placed in the dish is important to the experiment’s results, we couldn’t take any breaks until we had rinsed all the seeds for that day. As a result we had to work from 9 am to 3pm with no breaks. In addition to this, because plants don’t understand the concept of weekends, we had to work through the weekend, rinsing seeds for 6 hours straight. Finally this Monday we finished rinsing, meaning that for now on we only have to census the seeds since all seeds are in the correct plates and solutions.

Although my third week felt extremely long and stressful, and I haven’t had a day off in a while, this week was actually pretty enjoyable. In the weeks prior, seeding was pretty annoying. However, this time around, because it was my own experiment and we had been preparing so much for this week, the seeding was actually pretty exciting. I thought it was interesting that the in first 2 weeks of the lab I had just been helping everyone around me, but now everyone was actually helping me. In addition to this, the busyness of this week helped the days pass by quickly. I remember my first week in the lab the days seemed to last forever because all I really did was read articles since there wasn’t much else to do. Now that I’m a lot busier, my days feel a lot quicker and are a lot more interesting, making my overall experience a lot more enjoyable.