Week 1: Developing and Troubleshooting a Standard Operating Procedure
Riya d -
**To stay consistent with my peers, this blog update will discuss my experiences during the first week of my internship (February 3 to February 7)**
Beekeepers face significant challenges in managing bacterial diseases in their hives, often resorting to antibiotics, which, if over-used, can lead to bacterial resistance. Exploring natural, alternative treatments can help beekeepers combat such diseases while also preventing the issue of antibiotic overuse. My senior project focuses on using natural and hive-derived substances against bacteria that cause disease in honeybees. The substances that I have chosen to investigate, at least to start with, are essential oils — specifically thyme, spearmint, and lemongrass.
During the first week of my internship, I completed the training required to work in the lab. My knowledge of laboratory safety from chemistry classes at school (such as interpreting NFPA diamonds and using common lab safety equipment) proved useful in navigating the safety protocols at the lab.
Next, in preparation for my project, I cultured some of the bacteria that we would test the essential oils against on agar plates. This gave me a chance to learn proper plating technique and etiquette, a skill that came in handy later in the week as we began to prepare increasing amounts of plates. We plan to test the essential oils on 24 bacterial strains, so it is important that I can properly plate bacteria.
Most importantly, I worked with my on-site advisors to develop a SOP (standard operating procedure) for my project before beginning any lab work. There were a few important things we had to figure out prior to conducting my planned experiments. Something that proved to be more challenging than we had initially anticipated was solubilizing the essential oils. In my project, the effectiveness of the oils against a given bacteria will be determined using zone of inhibition tests. These tests involve inoculating an agar plate with a given bacteria, cutting wells in the plate, filling the wells with the oil being investigated, allowing the bacteria to grow on the plate, and then observing if their growth is inhibited around the wells containing the oils. In order for the oil to have any effect on the bacteria’s growth, it must be able to diffuse through the agar on the plate. It is difficult for a non-polar substance like oil to diffuse through a water-based substrate such as agar — the oil must first be solubilized with a solvent that will allow it to diffuse through the agar. My on-site advisors and I reviewed literature to determine how other researchers have solubilized essential oils for similar experiments in the past. We initially decided to use 70% ethanol (based on its success in the lab’s previous experiments) to solubilize our first essential oil (thyme), however, this did not work well for our purposes. We then tested different concentrations of ethanol and also varied the amount of liquid being put into the wells, but this still did not allow the oil to diffuse through the agar. We looked through literature for a better solvent, and decided to try using Dimethyl sulfoxide (DMSO), with proper precautions due to certain personal hazards associated with it, for our last round of trials for the week.1,2
We planned to look at the results from these trials the following week and determine which solvent to use. Finalizing the solvent would allow us to start testing the oils against the desired bacteria.
Stay tuned for my next update, in which I will share our conclusions.
References
1. Naik SS, Thilagaraj WR, Gangadharan P, Leela KV. Comparative Study of Antibacterial Activity between Selected International and Indian Essential Oils against Selected Pathogenic Bacteria. J Pure Appl Microbiol. 2024;18(1):401-409. Published 2024 Feb 24. doi: 10.22207/JPAM.18.1.23
2. Puškárová A, Bučková M, Kraková L, Pangallo D, Kozics K. The antibacterial and antifungal activity of six essential oils and their cyto/genotoxicity to human HEL 12469 cells. Sci Rep. 2017;7(1):8211. Published 2017 Aug 15. doi:10.1038/s41598-017-08673-9
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