Identifying Bacteria Isolated from Sorcerer’s Cave

Editor’s Note: This blog was composed by Whitney Brantley and Claire Shudde through a research collaboration with Olivia Dahl and Stephanie Sariles.

Sorcerer’s Cave, deepest cave in Texas


As a biology major at ACU, students are given several opportunities to get involved with the department. One of the biggest opportunities students can choose is to participate in is undergraduate research under the supervision of a biology professor. Many of my fellow classmates joined research teams their freshmen and sophomore year, but it wasn’t until my junior year that I chose to get involved with biology research. The fall of 2016, I had the privilege of joining a research team under Dr. Joshua Brokaw and Dr. Diana Flanagan in which we worked to identify unknown cave bacteria isolates that were collected from Sorcerer’s Cave in Terrel County, Texas. Our project was a small part in a larger research project conducted by Dr. Jennifer Huddleston, in which other teams used these isolates to test for antibiotic production or resistance. When I first began working on this research team, I honestly did not think I would find much interest in my work. I thought research would only be another simple extracurricular activity I am involved in. However, I was quickly proven wrong.

Our research was centered on extracting bacterial DNA and amplifying it through PCR. In the lab, Dr. Flanagan taught us several basic techniques of extracting DNA and setting up PCR reactions along with gel electrophoresis. We used these techniques every week for the next two semesters. After weeks of several hours in the lab and attempting many direct DNA extractions, we consistently received no positive bands in our gels to send for identification. To my surprise, I actually began to get frustrated with these results, and wanted to work even harder to receive positive DNA bands. After many more failed attempts by the entire team, we soon switched to a new extraction technique using a KOH-EDTA method. With high hopes about this technique, we spent more hours in the lab and continued to work diligently. However, after a few weeks, the frustration continued as our results only proved to be inconsistent and varying.

With only a few weeks until our fall research presentation and no consistent data to present, Dr. Flanagan decided to run a few PCRs and gels to test the two techniques we had previously used. Surprisingly, her results came back with four positive bands for unknown isolates using the KOH-EDTA method. This proved that our current method actually did work, but our team was making technique errors that were affecting our results. We were given the option to stop research and present Dr. Flanagan’s results, or try one more set of PCR reactions and gel electrophoresis. Determined to obtain positive results ourselves, our team decided to try once more two weeks before our presentation to receive positive results. I clearly remember the change in our attitudes that research day as each member tried to focus a little extra on each pipette attempt made. Two days after our PCR reaction, we checked our results with gel electrophoresis, and all received positive bands of DNA! It was such an exciting moment in our research to feel our hard work pay off. These PCR samples were then cleaned and sent to Yale DNA Analysis to be identified just in time for our presentation.

Continuing into the 2017 spring semester, our team switched to a new extraction technique using a Zymo DNA extraction kit. We have been able to obtain several new positive results in just a short time using this accurate kit and presented eight identified cave bacteria isolates at the ACU Undergraduate Research Festival. Of all the research topics presented at the research festival, ours may have had little importance, but I along with my fellow team members were extremely proud of our work. These past two semesters of working with these cave bacteria have taught me that biology research is not the least bit uninteresting. On the other hand, research has the power to teach you lessons and spark new interests. This time spent involved with microbiology research taught me patience and determination truly do pay off. It also showed me that cave bacteria and gel electrophoresis can actually be exciting and rewarding!

2016 Fall Poster Session


When I was five, I watched a documentary about scientists. All I remember from it were petri dishes with bacteria growing on them, but I knew I wanted to do that when I grew up. A few months before I came to ACU as a freshman, I was put in contact with Dr. Joshua Brokaw regarding the possibility of doing research with the Biology Department. Thankfully, he added me to the list of people doing research in the fall.

The semester had barely begun when Dr. Brokaw broke everyone into groups and assigned projects. My group was working with Dr. Diana Flanagan on identifying unknown cave bacteria isolated from Sorcerer’s Cave. In the group of four, I was the youngest and the least experienced. I was certain that this would be a semester of shadowing and dishwashing. However, I was completely wrong. I was immediately learning techniques right along with the other students. The process took two weeks. The first week we would attempt to replicate the bacterial DNA running a PCR, and the second week we would check to see if we had successfully replicated the DNA by running a gel electrophoresis. The first time we went through this cycle, I successfully replicated the DNA of one of my samples. This was incredibly exciting. The excitement was short-lived, as we were unable to replicate the DNA using that technique again.

After a month of unsuccessful DNA replication, Dr. Flanagan revised the experiment. This, too, was mainly unsuccessful, and I grew to be quite disappointed in both the research and in myself. This is when I learned an important lesson: research is not always easy. It can be somewhat depressing when results don’t always come about the way you would like them. However, this motivated us to find another way to make things work. A setback in research is not the end of the world, instead it is an encouragement to determine how to overcome. We overcame the obstacle of not replicating DNA at the very end of the semester. We fine-tuned our technique and were able to identify four of the cave isolates.

Using a different DNA extraction technique during the Spring semester, we were able to positively identify four more cave isolates after only one try. After preparing our poster for the Undergraduate Research Festival, we presented the research at the beginning of April. It was an exciting experience. I was able to share the things that I had learned over the past eight months, which is one of the main benefits to research. Research is hard, but definitely worth it. Undergraduate research has given me lab experience that will help in graduate school and later as a scientist. It has been a rewarding year of doing research, and I am excited to continue next semester.


2017 ACU Undergraduate Research Festival


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