The Root of the Problem

This last week of summer research has flown by. I can’t believe it’s all over! After returning from Maine, I was quickly put to work entering all of the data for newly recruited trees into a spreadsheet, which took about two full work days. Not the most glamorous of jobs, for sure, but I’m happy to have gotten it out of the way early.

The rest of my week was dedicated to performing a single, simple task: perfecting the root-clearing step of our staining procedure. The goal of this is to eventually be able to observe and quantify micorrhyzae in our American chestnut root samples under a microscope. We will officially begin collecting this data in the fall, after the growing period ends. Root clearing is an important step in this procedure. It essentially flushes out all pigments in the roots. This allows a stain to be added to the sample that only highlights the miccorhizae and makes them visible under a microscope.

There is  a good amount of literature on this process, with lists of clearly defined materials and instructions. Based on this, we decided on a method using Potassium Hydroxide. I got up early in the morning and dug up practice- chestnuts at the Sullivan Greenhouse. After returning to the lab, I cut and washed the roots from these plants, while also mixing and boiling a solution of 10% KOH. The roots were divided up into little cassette boxes, and dropped into the boiling solution. I immediately saw plumes of brown coloring rise up from the cassettes, and I felt assured that after a few hours these roots would be translucent and  ready to go.

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I was wrong. After two hours, the solution had turned the color of dark coffee, yet when I looked at the roots they were all still a dark brown color. In fact, even after leaving them in solution for more than 6 hours and changing out the boiled-away solution twice, I could still only see about 50% of the roots being cleared. By 5pm, I felt tired and frustrated that there was not enough time or materials to continue testing before the summer session ended. However, after a conversation with Dr. Dalgleish and Olivia, we decided the best option was to take a break and try looking in to for efficient clearing methods for what we assume are heavily pigmented roots, such as bleaching. Luckily, there is still time at the beginning of next semester to figure all of this out.

In retrospect, this is an infinitely small setback in what I feel sure will be a successful project. While it’s never ideal to leave on a less-positive note, its much preferred over rushing to finish, and making mistakes or wasting chemicals. As a whole, the summer has been a great success. I had the opportunity to fully immerse myself into field work and watch the growth of our little chestnut trees, which we will get to begin experimenting on very soon. These will be enlightening experiences to look back on- and I’m truly excited for future endeavors in my plant ecology lab, as this Chestnut project reaches completion.

Comments

  1. I know I hate it when my work momentum is thrown off by something that doesn’t work the way I think it should. Fortunately, it seems like you and your professors handled this setback pretty well, giving you a chance to finish this project at a later date. If possible, could you perhaps post a photomicrograph of any mycorrhizae you see once you start the project up again during the school year?