This post was contributed by Francesca Foltz, a senior at Loyola Marymount University and a 2020 Burtt Undergraduate Mentoring Grant recipient.
Two years, seven months, and ten days ago I sat in my professor’s office, nervously picking at my nails and wondering what was about to happen. My only indication lay in my email inbox with the subject title “Research” and a brief message about “discussing opportunities.” I couldn’t stop wondering what she wanted to talk about—even though the meeting was just another box on the calendar for the person across the desk, I had a premonition that it would be a turning point in my undergraduate career. So there I sat for one minute and thirty-eight seconds, destroying my cuticles with anxiety as Dr. Covino wrapped up a phone call. She hung up, grabbed a whiteboard marker, and proceeded to—for the first of what would be many, many times—draw diagrams of the project overview and goals for what she had dubbed “Project Poo.” The project aims to use fecal samples from breeding Great Black-backed Gulls to determine the testosterone levels of individual adults and determine if they’re correlated with the birds’ aggression levels. As someone with a lifelong love of birds and a budding appreciation for lab work, this piqued my interest immediately. Without hesitation, I jumped on board this project with Allie, another student, and we began the process of applying for funding.
The fecal samples had already been collected from a study site on Appledore Island, Maine, so our job was to conduct a literature review of existing fecal hormone studies and develop methods to extract and quantify the hormones from the sample. I created a spreadsheet organizing the methods from these papers, making a reference sheet that would allow us to quickly compare focal species, hormone of interest, homogenization procedures, extraction solvent, and types of assays used for determining final hormone concentration. After three weeks of filling out the spreadsheet, we found that lyophilization (freeze-drying) followed by grinding with a pestle was the best way to homogenize fecal samples (though the amount of time drying varied in each study), and that there was absolutely no single, agreed-upon set of instructions for hormone metabolite extraction.
Fast-forward to the summer of 2019, after I had jubilantly accepted funding from Loyola Marymount University’s Summer Undergraduate Research Grant—my first success in the realm of research. On my first day, Dr. Covino showed Allie and I the freeze-dryer in the common equipment room, mentioned that no one in the building knew how to use it, and directed us to find the manual and figure it out. Two hours later, we had changed the oil on the vacuum pump, and the machine was up and running for the first time in years—a success! The next step was to determine the optimal amount of time to freeze-dry the samples, which meant checking sample weights to monitor water loss every four hours, including overnight. Everything was going great until a few partially unfrozen samples burst out of their containers from the pressure changes, resulting in what we decided to call “poopsplosions.” Back to the drawing board we went! After trials with “fake poo” consisting of frozen applesauce and yogurt, we figured out how to cap the vials with a porous cover that allowed for water loss but prevented these defecation detonations. Finally, we had cracked the freeze-drying and homogenization aspect of the project.
Next we had to tackle the hormone extraction. Sure, the hormone was in the dried and ground sample, but how could we get it out in a concentrated form so we could put a tiny amount onto an enzyme immunoassay (EIA) plate, and how could we do this in a way that would not affect the biochemistry of the EIA? Even though extraction procedures in the studies we’d reviewed varied, there were a few common threads that we used as a foundation for our procedures. First, extraction—using a solvent to dissolve the hormones in the sample—was mentioned in all of the studies we found, cementing it as a necessity. Second, many of the studies did the extraction with a ratio of 2 mL of solvent for each 0.1 g of dried fecal material. Finally, a few of the papers mentioned doing multiple rounds of extraction on the same sample then combining the extracts. With these consistencies—and a fair amount of time drawing on that whiteboard in Dr. Covino’s office—our next few months of running experiments to optimize extraction protocols were planned. We agreed on the 2 mL/0.1 g extraction ratio using 80% methanol (which consistently had the highest percent recovery in reviews) and extracted the same samples with up to five rounds each. In theory, after extracting a certain number of rounds, we would reach a point of diminishing returns.
I was ecstatic to have solidified at least some aspects of the extraction, and it was at this point that I had received the news that Dr. Covino and I were selected for the WOS Burtt Mentoring Grant—a strong vote of confidence in our research and our process. With the promise of presenting our findings at the 2020 meeting (which later became the 2021 meeting), my tenacity and drive to find answers were renewed. This propelled me forward to the next step of developing our methods: conducting the enzyme immunoassays to actually measure the amount of testosterone in each sample.
For the enzyme immunoassay to work, all the methanol used for the extraction had to be evaporated off before we proceeded with the analysis. Even here, our literature search offered little guidance apart from offhand comments about drying in a fume hood overnight or using nitrogen. Following the somewhat unclear path from literature, we left extracts out in the hood overnight to dry and were frustrated when the liquid levels had barely decreased by morning. Once again, we headed back to the drawing board!
A nitrogen manifold, heat vacuum, and hot block later, we found that the most effective method of drying the extracts was placing them in a 50ºC water bath for up to 48 hours. It’s a tedious, time-intensive method that requires overnight checks in lab, but it is the most effective method with the lowest risk of sample contamination or denaturation. Even though we didn’t particularly enjoy this part of sample preparation, the fact that we were confident in its efficacy was a success following a series of setbacks.
Next came the assays themselves, the technique we would use to actually measure the amount of testosterone in each sample. These were the final step in the methods we were developing and the last step we needed to do to gather our data, but coaxing the assays to work led to an entirely new set of problems. We had to learn new lab techniques, try different ways of adding reagents, determine optimal dilution factors, and maximize precision in all aspects of the procedures. One major challenge is that since there are no published studies that quantify fecal testosterone levels in this species, we have no reference point for what levels to expect. As you could imagine, this made the initial “trial-and-error” stage very frustrating. Each of these challenges came with their own setbacks and triumphs. Finally, after all of the assay-related wrinkles were smoothed out, I got to run the procedure for our first rounds of extracts so we could receive our long-awaited data. The full set of single through quintuple extractions for six focal test samples was run, and we found… well, not “nothing,” but no clear effect. Our hope was that after enough rounds of extraction, we would identify the point of diminishing returns, where any additional testosterone we could get out of the sample would be negligible. However, after running several statistical analyses on the data, we found no indication that the amount of hormone we recovered leveled off after a certain number of extractions. We had chanced this experiment with a low sample size of just six, so clearly needed to…do more!
In our master lists of finalized lab protocols, there’s no mention of the tried-and-failed methods, only the tried-and-true. For anyone joining the lab after I graduate in December, the time, effort, and patience that have gone into the protocols will not be known. In my time as an undergraduate, I’ve realized that viewing research from this success-only lens is a phenomenon that occurs frequently but is rarely talked about. Every time something went wrong with our procedures, my friends in other labs would have just completed the measurements on their 150th mouse skull or run their 14th protein gel of the week. It was hard to not get discouraged when after two years on a project, they were publishing their results in scientific journals, and I was still trying to get my methods to work. Pity parties ensued: it wasn’t fun, it wasn’t fair, and it certainly wasn’t encouraging.
It took me a while to realize that their projects were not any better or worse than mine, simply due to the number of obstacles they faced. From honest conversations with them, I learned that although their procedures are solid, their professors and the students who’d preceded them had faced the same series of hindrances that I had. Not to mention that the fact that their procedures were set did not mean that they didn’t face their own difficulties and setbacks—like me, they had to troubleshoot their own problems in research every week, if not every day. And like me, they did not always get the results they had expected or hoped for.
Often, in our pursuit of significant results, we forget that the lack of a results can also be valuable information. Procedures that did not work are just as important to share as those that did. Behind every research venture is a blazed trail, with numerous obstructions cast aside. I’m just an undergraduate who’s not sure what her future holds, but in light of what I have learned in my undergraduate career, I would like to humbly ask you, seasoned ornithologists, to share a little more about your trials, your tribulations, your angers, and your frustrations.
I still have my first lab notebook for Project Poo. It’s been full for a while now, but it has all the drawings, diagrams, and dilemmas that we faced—including my notes from Dr. Covino’s outlines on that first day in her office, with chips of my picked-off nail polish stuck between the pages. That journal is a record of what worked, but also what didn’t work. At this point, the updated protocols are digitalized in our online library, rendering my notes relatively useless. I keep the notebook, though, because it is a reminder of all the obstacles we have overcome. We may not have found those less-than-0.05 p-values yet, but I have faith that the answers we’ve been looking for are out there. And when I’m struggling to keep moving forward, all my inspiration lies in how far we have come.