The most consistent piece of advice given to me by anyone who worked in research was to get into it as soon as possible, and do it as often as I could. As a sophomore biotechnology major, I hope to go into a career in research and took this advice to heart. This past summer, I participated in the Research Experience and Apprenticeship Program (REAP) in the lab of Dr. Kyle MacLea at the UNH Manchester campus. As an apprentice under a faculty mentor, the goal is for me to gain skills like data collection and lab technique, as well as a stronger understanding of the topics surrounding my project, which focuses on bacteriophage resistance and lysogeny.
Bacteriophages, or “phages,” are small viruses that infect narrow ranges of host bacteria. When phages are put with bacteria within their host range, they inhibit the bacteria’s growth by replacing the bacteria’s genetic material with their own. This is important in the medical field because they can be used to help treat bacterial infections in situations where common antibiotics are ineffective.
Bacteriophages have a lytic life cycle or a lysogenic life cycle. In a lytic cycle, the new phages kill the bacteria and are released as soon as they are made. In a lysogenic cycle, the phages lay dormant in the bacteria’s DNA until a given stressor, like a specific temperature or pH, causes it to activate and be released. The phage I isolated during my fall 2023 semester, BenchWarmer, showed signs of being lysogenic, so I am testing to confirm this. Additionally, I am developing a “phage training” procedure, which would expand its host range to related strains of bacteria. This is important because the bacteriophage could then be used to treat a dangerous bacterial infection.
My daily schedule this summer was entirely up to me, with the expectation of spending around 40 hours per week on my project. To keep myself regulated and on track, I created an effective schedule, which I detail below.
I arrived at the UNH Manchester campus at 8:30 A.M. to start my day. I set up in a study nook just down the hall from my lab. My first order of business was to go to a fresh page in my notebook, where I entered the date and my daily plan. Once I created a bulleted list of tasks I needed to complete that day, I entered the lab with my notebook, phone, lab coat, and face shield.
The fifth-floor laboratory is shared between many different members of the biological sciences department, but I was usually the first one there, which allowed me time to play music while I put on my Biosafety Level II PPE (coat, gloves, face shield), sterilized my lab bench with bleach spray, and began my work. The first two points on my daily plan were to check my different bacterial plates and take pictures. For streak plates I was looking for individual colonies of bacteria that all look uniform, while on bacteriophage plates I was looking for small, clear “plaques” indicating where phages are. On contaminant test plates I was hoping to see no growth at all, otherwise that means something is contaminated.
My observations usually dictated whether I needed to rework some sections of my plan or carry on. Contamination was an issue throughout the summer, which resulted in me shifting plans a few times. This was okay, however, as the purpose of REAP is to build lab experience and knowledge rather than produce final results.
My next steps included quad streaking promising bacteria colonies, purifying phages by serial dilution, and performing Gram stains. Quad streaking means I take a colony of bacteria with a sterile loop and brush it onto a portion of a new, clean agar plate. I then turn it, get another sterilized loop, and drag from the lines I just made onto a clean section of the agar. I repeat that twice more, resulting in four “quadrants,” where the first quadrant is very concentrated with bacteria and the last yields a few individual colonies of the bacteria I originally took from.
Phage serial dilutions are like liquid versions of quad streaks. A plaque is touched with a pipet tip and mixed into this specialized medium called phage buffer. The phage is mixed in, then a tenth of that liquid is taken and put into another tube containing the same volume of phage buffer, so this tube contains one tenth the phage concentration of the previous one. This is repeated five to eight times, depending on what concentrations I need. The dilutions are all incubated on new plates with the host bacteria, which results in new phage plaques, the number of which tells me how effective the phage resistance is.
Gram staining, also known as the bane of my existence, involves drying a bacteria sample onto a glass slide, staining and rinsing it with different chemical dyes, then redrying the sample and looking at it under a microscope. The dyes make the bacteria more visible and give information about the type of bacteria. If it comes out purple, it’s Gram positive which means it has a specific cell wall structure that holds onto the purple dye. If it’s pink, it is Gram negative. The strain I am working with is Gram negative and tube shaped, so if I am seeing purple or the wrong shape, I know I have a contaminant. This makes Gram stains incredibly useful, but also very time consuming and finicky.
I usually performed this process on 15 to 20 plates, and finished around 12 P.M.. I would then make a new batch of agar to replace the plates I used up. To prevent contamination and stay as sterile as possible, the agar goes in the autoclave for 45 minutes; however, due to the time it takes for the autoclave to cool down and depressurize, the agar is in for closer to 90 minutes. I used that time to eat lunch, upload pictures and notes to the slideshow I am using to document my research, and look up any protocols or questions I need answered.
This is when I would check in with Dr. MacLea to discuss the results from that morning, hear his advice on anything I should add to my itinerary for the day, or brainstorm ways to fix issues that prevent my project from moving forwards. Afterwards, the autoclave was ready so I could put the final ingredients in my agar and pour new plates.
From that point is where my day tended to vary. Sometimes that was the last bit of lab work I could do, as I was waiting on results that take a few days’ time. I would then perform research on my computer to better understand the next steps in my protocol, and end my day around 2:30 P.M.. Other days I still had a full list of tasks to complete, and finished closer to 5 P.M.. This schedule worked well because most of the bacteria and phages I was working with take close to two days to grow, so the days where there is a lot of lab work to do and days where there isn’t tended to alternate.
I'm very thankful for the opportunity to experience this process through the help of Dr. MacLea and the Hamel Center for Undergraduate Research. The flexibility in my schedule allowed me to develop self-regulation skills, and the ability to learn new lab skills and grow my confidence in the ones I gained over the past semesters will be very beneficial as I continue working on my biotechnology degree. This has been a great experience that I would highly recommend to any first-year students interested in research.
