Week 5 at the Donohue Lab

Hi this is Meg and I’ll be talking about my fifth week working at the Donohue Lab at Duke. This week has been far less busy than the 2 weeks before. We still have to census every day. However, many of the plate have begun to plateau, meaning that nothing new is germinating. As a result, there are less and less plates to census every day.

In addition to censusing, Lien had to hand in a rough draft for her poster on Wednesday, and her proposal on Friday. As a result, Bri, Tarek, and Lien have been working hard analyzing Lien’s data and attempting to clean it up.

On Tuesday and Wednesday, I helped clean some of Lien’s data. Often after the data is compiled, there is a problem with the order, meaning that an earlier day has a higher number of germinants than a later day, which is impossible since we don’t remove seeds that have germinated. Therefore each successive day should have the same amount or a greater amount of germinated seeds than the previous day. In order to fix this, we attempted to modify the least amount of data as possible. This meant trying to only change one day’s data. However, a couple of times the data was just too out of order to fix. As a result, it couldn’t be used and we had to throw that data away.

Data that needs to be cleaned. Each column represents the day the data is from. The highlighted row is an example of data that needs to be cleaned since on day 3, there were 14 germinated seeds and on day 4, there were 10. Similarly later in the same data, on day 6 there were 14, and on day 7 there were 11. 

Although this week has been a lot slower than I’m used to, it’s been nice to have a bit of a break, especially since my lab manager has been letting me leave earlier than usual. However, hopefully it picks up a little bit so that I’m still fairly active in the lab.

Week 4 at the Donohue Lab

Hi this is Meg again, writing about my fourth week working in the Donohue lab at Duke University, where we are researching the different effects of water potentials on seed germination. As I described in my last post, on Monday we finished rinsing seeds from Lien’s project. Then for the rest of Monday we had to census seeds from both from my experiment and Lien’s.

Tuesday morning, we had to head out to the field site in order to weed so that other plants wouldn’t affect the growth of the experimental plants. However, the field is ridiculously overgrown, with weeds taller than I am. As a result, the weeding was pretty intensive and it took us 2 hours to clear out a tiny area. Also, because North Carolina can get extremely hot and humid by around 9 or 10 in the morning, we went out into the field at 6 am and worked until 8:30. Once we finally stopped working, I went back to my apartment to change out of my muddy and dirty clothes and then headed over to the lab. For the rest of the day I censused our experiments. After lunch though, Lien and I met with the postdoc, Tarek, to discuss the analysis of our data. Basically this ended being a 2 our graduate level statistics class, but I still successfully followed along, and was pretty interested by the end.

Inside of chamber with plates

Chamber where plates are kept

On Wednesday, luckily we didn’t have to go to the field again. However we still had to census all day. In addition, we had another 2 hour meeting about analyzing data.

Then, Thursday morning, it wasn’t required, but Lien and I volunteered to help in the field again, meaning another really long day. When we returned to the lab, we censused some more. Then just like the other days this week, we had another meeting. This one was the longest yet and it went over 2 hours and 30 minutes. By the end of the meeting, Lien and I were visibly exhausted, so Bri let us leave an hour early.

Microscope used for censusing

Sheet used to census

On Friday and throughout the weekend, we just censused during the day since we had finished data analysis and field work for the week.

Once again, this week has been exhausting. However, all this work has been pretty enjoyable and has helped me to stay occupied in the lab. As I said last week, I prefer these busy weeks to the fairly slow weeks that I had when I first began working in the lab.

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.

               

Week 2 at the Donohue Lab

Hey this is Meg Dalrymple, and I will be writing about my second week at the Donohue Lab, which mainly focuses in the evolutionary and genetic mechanisms behind germination at Duke University

The Autoclave

Walking into the lab on Monday of my second week, I felt much more prepared and comfortable than just 7 days prior. We still had one more day of censusing left to do and so me and Lien got started on it right away and finished pretty quickly. After that, we had a lot of dirty petri dishes that needed to be sterilized before we threw them out, since they contained biological waste (the germinating seeds). So, we headed over to the autoclave, which is a big machine that sterilizes things using heat and water. There are different types of cycles that you can run, depending on what you need to be sterilized. For the trash we used a setting called “gravity” which uses heat and water, but other dry cycles only use heat. After about an hour the trash was done so we took it out of the autoclave and to the dumpster. In the afternoon, we realized that we had cut the filter paper the wrong size for our experiment. However, the new uncut filter paper that Bri had ordered hadn’t arrived yet, so we continued to cut weigh boats and punch holes in them.

The next day on Tuesday, the filter paper came. However, I busy helping the other group in the lab prepare for later in the week so I couldn’t help cut the paper. I was busy preparing plates with which the group would seed (put seeds on the plates). This meant that I counted and laid out 900 plates. Next an undergraduate, Aman, made agar to be poured into all 900 plates. As I went to lunch he started pouring. When I returned, he told me the other agar was in the autoclave and that it would be done in about 5 minutes, and then he headed off to lunch. I went up to the autoclave and got the agar so I could begin pouring. About a half hour later, Aman returned and we continued to pour. In addition to pouring agar, the plates also had to be labeled. So as we poured, we labeled and organized the plates into the different seed genotypes. After we finished pouring the agar at one point, we realized that some of the plates we not solidifying. Aman figured out that because the liquid agar had been sitting for a while, the solution had separated and the top was mainly water, and the bottom was mainly agar. Therefore, we had to redo about 200 plates and make new agar to insure that we did it right. We finally finished pouring and labeling a couple hours later.

Cut filter paper

Then on Wednesday, Lien and I began to assemble our plates. This meant that first we would sterilize the cut weigh boats with ethanol (they couldn’t be autoclaved because they would melt). Next, because we cut slits in the strip of filter paper, we were able to create a loop out of it by locking the 2 ends together, using the slits. In this loop of filter paper, the weigh boat would slide in upside-down so that the bottom of the weigh boat was facing up. Then the weigh boat and filter paper would be put in a petri dish, lying flat. On the first day, we made about 120 dishes and then helped the other group seed.

On Thursday, our only goal was to finish making the dishes. We spent all morning and some of the afternoon working on it. Eventually however, we ran out of weigh boats and had to stop. This meant that we had to order more weigh boats, and eventually will have to cut and poke holes in them. We still have around 250 dishes that we need to make. Once we were unable to make any more dishes, we decided to autoclave some distilled water because we will need it for our experiment. For the rest of the day, we read articles.

The next day we wanted to make the solutions that had different water potentials since we will be seeding our experiment on Monday on dishes with those solutions. However, we needed to first autoclave empty bottles that would hold solutions. Therefore, while we were waiting for the autoclave to finish, we helped the other group seed until lunch. After lunch we began making the solutions. In order to change the water potential of solutions, we added a powder called polyethylene glycol (PEG). PEG is actually commonly used in medicines, in chemical spills, and in toothpastes and creams. It smells a lot like glue and when mixed together with water, it creates a soapy solution. We made 5 different water potentials by weighing out really large amounts of PEG and then mixing it with water. Once we were finished, Lien and I had to go to another discussion about a paper. However, this time the paper was far easier to understand, and I had already read it earlier in the spring. Therefore the meeting was a lot less stressful than the one we had the week before.

Overall I enjoyed this week because although I’m still not doing very exciting tasks, there is more variety to them now. I’ve learned how to do many new things this week, such as autoclaving and preparing dishes, making my research more interesting and enjoyable. In addition, everyone in the lab is still really friendly and enjoyable to work with, making the overall experience a good one.

Week 1 at the Donohue Lab

My first day in the lab, I was the first to arrive. I had somehow overestimated my total travel time by about 30 minutes, even though it was a simple 10 minute bike ride. As a result, I showed up at 9:30 and waited in the hallway until someone showed up at 10, when I was supposed to arrive. Finally an undergraduate student, Lien, arrived and unlocked the door to let me in. She told me that she was a rising sophomore at Duke and that we would be working on the same project together. Since the lab manager who will be helping me this summer was running late due to a doctor’s appointment, Lien began to introduce me to the project. She gave me a paper that Bri, the lab manager, had given to her to read that was a good introduction to our project since it outlined the procedure. Basically in my project, we will be keeping seeds from five different genotypes, some of which were matured in hot conditions and others in cold conditions in different water potentials for 4 days. After 4 days, they will be moved to water and the amount of seeds germinated will be counted. However we aren’t starting this project until another couple of days, so until then, I am busy helping with another experiment. After I finished the paper about my project, I met with Bri, and she told me I could start censusing seeds. This basically means counting how many seeds on a dish have germinated under a microscope. It actually is harder than it sounds because the seeds are really small and often the root that breaks from the seed is clear. As a result it took me a while to get a hang of it, but by lunch I was doing alright.

Plates with dishes that needed to be censused

When I returned from lunch, Bri had to go to a lab meeting, so I continued to census, which gave me an awful headache after a while. Then at 3, Bri came out and told me that me, her, Lien, Tarek (the postdoc), and Dr. Donohue were going to meet to discuss our project. I headed into Dr. Donohue’s office and waited for the meeting to start. Basically we talked about what the point of the project is and what Dr. Donohue wants us to focus on in the next couple of days to prepare for the project. Then we planned what papers we should read and what days we would discuss them. The meeting ended around 3:45. When we came out, we discovered that one plate had yet to be censused. As a result, Bri asked me to finish it. I finished around 4 and then looked around and everyone was beginning to leave. I turned to Lien to ask if we were allowed to leave and she told me we could so I packed up my stuff and headed home for the day.

On my second day, I showed up too early again, but luckily this time Bri was already here so the lab was unlocked. I asked her if I should begin censusing again. She told me that I should and then showed me to the room with all the plates of seeds. She explained to me the system that they used to know which plates had been censused, and which still needed to be done that day. I started by grabbing one plate and initialing that I had done it. By 1, I had censused about 6 or 7 plates, which meant around 180 dishes, each with around 12 seeds on them. But luckily, between me and Lien, we were able to finish all the censusing for that day by lunchtime. As a result, when I returned from lunch, Bri told me and Lien to review some papers since we will be discussing them on Friday. I spent the rest of the day reading and taking notes.

On Wednesday, another group needed help preparing their project, so rather than census seeds, my group helped them. We had to seed, meaning that we had to put 20 seeds on Petri dishes filled with agar in a 5 by 4 grid. The seeds were extremely small, making the work quite tedious. However, it gave me the opportunity to talk and get to know people from the lab who I normally don’t work with.

Then after lunch, we finished seeding, so I began to set up for my project. This meant cutting and counting 1200 strips of filter paper that the seeds were going to rest on in the dish.

On Thursday in the morning we did more censusing and then I finished cutting and counting the strips of filter paper. After this, I reviewed a paper that we were going to discuss together on Friday. When I returned from lunch, I cut and poked holes in weigh boats for my experiment so that they would fit in dishes where the seeds were going to grow.

Censusing in the field

On Friday, I had to come to the lab an hour early because we were going to go look at the field experiments which are about a 15 minute drive from the campus. There were three different experiments that were taking place. However 2 of them were finished for the summer because seeds were no longer germinating due to the heat; they had entered secondary dormancy. One of the experiments was still going on though so we censused the seeds that had begun to bolt and marked them down on a sheet of paper. It was surprisingly tiring and by the end of the morning my knees were sore from crouching to count seeds. However, it was cool to be able to see the lab’s field experiments, even if they were all overgrown. After lunch I censused some seeds in the lab and then discussed a really confusing paper that pertains to my project. Walking into the discussion, I was nervous that I wouldn’t know anything. However, once it started, I soon realized that I actually did understand most of the paper, and that it was ok if I didn’t understand everything. This reassured me, and by the end of the meeting, I was active in the discussion and actually enjoying myself. 

Overgrown field with weather station in middle
that records weather conditions of the field

This first week has been long, and sometimes tedious with all the censusing and seeding. However, the people in the lab that I work with have been really welcoming and have so far made this experience really fun, even though I was nervous and apprehensive walking in. I’m now excited to get to know everyone better and working on my project in the upcoming weeks.