Checking Mammalian Cell Growth Under 10x Zoom

Author: Katie Loethen | Major: Biological Engineering

My name is Katie Loethen, and I am a biological engineering major in the College of Engineering. My research is within the Civil Engineering Department with Dr. Wen Zhang in her Environmental Engineering Laboratory. I have been doing research since my freshman year of college with the help of the Honors Freshman Engineering Program, but this blog post marks the completion of my second semester of Honors College Research Grant funding. I will be graduating in May of 2022.

This semester, I conducted a mammalian cell bioassay to measure the cytotoxicity of a quenching salt. This research is a complement to previous research on the cytotoxicity of wastewater disinfection byproducts (DBP). DBPs are chemicals that naturally develop when chlorine is applied as a disinfectant to wastewater and reacts with organic materials in the wastewater. To create concentrated DBPs for cytotoxicity or genotoxicity assessment, chlorine is mixed with suspended organic materials. Then, a quenching salt is added which precipitates with leftover chlorine; however, the precipitation is not 100% efficient, and some quenching salt remains dissolved in the aqueous DBP solution. To identify whether the quenching salt may contribute to a cytotoxic response during a bioassay, I completed a mammalian cell bioassay with 8 replicates each of 5 orders of magnitude of quenching salt concentration.

This research is related to DBP research I completed as a freshman in the Honors Research Experience where I worked with a partner to assess the cytotoxic effects of a DBP called N-nitrosodimethylamine. It was through this program that Dr. Zhang and I connected, and it was after I took her hydraulics course that I asked her to be my research mentor. I did not expect to do more DBP research when I returned to campus this semester; I had been working on a wastewater epidemiology project which measured the presence of SARS-CoV-2 RNA over time and throughout the wastewater treatment process for the previous two semesters. Due to funding complications and timing issues, Dr. Zhang asked me to dust off my bioassay notes and take on this quenching salt assessment!

Relearning the mammalian cell bioassay procedures was not easy. Before the assay could be conducted, I had to dethaw and grow the mammalian cells. I tried to do this from memory, and with two years between myself and the last time I had done this, I had a lot of misremembrances. The first two vials of cells I tried failed to grow, so Dr. Zhang and I troubleshooted virtually through dozens of Slack messages. We were able to get assistance from a generous previous graduate student who let me know that despite the potential for contamination, mammal cell cultures should not be parafilm-sealed, or they will not be able to breathe. After removing the parafilm from my third cell culture, the cells grew into a healthy culture.

Next, I messed up the assay. One of the last steps of a bioassay procedure is to dye the plate with Crystal Violet so that the number of living cells can be measured with a UV spectrometer. You have to fervently rinse the plate after dyeing, or it is pretty much ruined. Of course, I lacked fervor when I rinsed the plate. The entire plate lit up dark blue under the UV spectrometer, and I never even sent that reading to Dr. Zhang because it was completely unhelpful.

I hadn’t realized how much I would have to relearn after two years away from those materials, equipment, and procedures, but after a few failures, I conducted a successful bioassay. I had to ask for help way more often than I like to, and I had to admit as was not as prepared as I had thought I was. An additional challenge was being mostly on my own in this task with no graduate student dedicated to this project at the time. I plan to run the assay one more time with more assuredness early in the Spring 2022 semester, but soon the data will be available to refine the study of DBP cytotoxicity assessment.

In my 5 semesters in the Environmental Engineering Laboratory, I have had the privilege of seeing multiple sides of addressing clean and safe water resources. I have looked at the how the wastewater treatment process impacts viral presence as well as how treating wastewater can generate additional health factors. This has enhanced my experience in Biological Engineering where I have taken classes on sustainable watershed design, environmental engineering, and hydrology. I don’t know yet what job I want to pursue after graduation, but I believe I have had robust experiences that can contribute to fulfilling access to clean and safe water as a human right.