By Taylore King
There I was, ten minutes from departing for Lovefield Airport in Dallas, Texas. Prepared with six suitcases of clothes and shoes and readying myself to board a plane headed for Baltimore, Maryland where a college adventure at Johns Hopkins University awaited me. When suddenly…an email! From Dr. Schildbach, advising us Phage-Hunters-To-Be to take a few photos to incorporate into our future blog posting about the Phage-Hunting course. So with great haste, I grabbed my mother’s finest dinner spoon, a hot pink ruler, and a camera and scooped a heaping sample of fine Texas dirt into a Ziploc bag. Specifically though, my collection of Texas soil came from our backyard pumpkin planter, an oasis for microbes, that was fertilized with Miracle-Gro and watered daily. And so, with my soil gathered and stuffed into my bright orange Hawaiian suitcase, I headed to Baltimore to begin my journey at Johns Hopkins University.
The next few days were a blur. My parents and I arrived to BWI late that night and spent the next day gathering items for dorm life — Clorox wipes, silverware, a shower curtain, all the necessities for college. On move-in day, with my soil sample far from my concern, I unpacked boxes and suitcases, moved furniture, met floor-mates, and began adjusting to a new chapter in my life. Everything was exciting, living in a dorm, walking around campus, meeting other freshmen and upperclassmen. And we still had several days before classes started.
After four days of orientation activities, though, I was ready for structure and routine. The Monday night before class began, I retrieved my soil sample from the depths of the desk drawer I had tossed it in and prepared for my first day as a Phage-Hunter.
Though I was aware that we would “jump right in” to hunting phages on the first day of class, little did I expect to begin and complete the process of direct plating by the end of a two and a half hour class session. The procedure of direct plating involves extracting phage from the soil sample by using a 10-mL pipette to add 4-6mL of phage buffer (PB) into a test tube one-third full of the collected soil sample. The mixture is shaken vigorously and allowed to settle for 10-15 minutes. Once the mixture has settled, a distinct liquid layer will have formed and, using a syringe, 1.0-1.5mL of the liquid layer is extracted, run through a syringe filter, and dispensed into a sterile, microcentrifuge tube. Three test tubes containing 0.5 mL of an M. smegmatis culture are then labeled, two as positive samples and one as the negative control. 50 μL of the filtered phage fluid is added to the two positive samples and 50 μL of sterilized phage buffer is added to the negative control test tube. The tubes will then sit for 10 minutes as any present phage(s) infect the M. smegmatis bacteria. After the tubes have sat for 10 minutes, 4.5 mL of top agar (TA) mixed with a calcium solution is added to each culture tube using a sterile, 10 mL pipette. The entire mixture of the culture tube, bacteria, phage, and top agar is then pipetted out and dispensed onto an agar plate evenly. This process is repeated aseptically with the remaining two culture tubes. The plates must sit for at least 24 hours to allow bacteria to grow and phage(s) to lyse them.
Once I had completed the above process of direct plating, I knew I would have to wait another 48 hours before I could inspect my plates for any phage plaques. When I walked in for our second day of class, Katie, our TA, announced that one set of plates had definite plaques. Dr. Schildbach then added that it was unlikely for direct plating to yield successful plaques, especially on the first attempt and that the process of finding phage plaques can often be a lengthy one. So, not wanting to get my hopes up, I walked to the lab under the assumption that the definite plaques were not mine and that I was in for a long search for bacteriophage.
When I found my agar plates, tore off the tape attaching them, and compared the negative control to the two positive culture samples, I noticed a very distinct difference between the control and the samples. Two different phage morphologies covered the two culture samples: large, opaque circles and tiny, clear dots, while the control simply contained a large smear of M. smegmatis. The definite plaques were mine. In that moment, my inner nerd shone through and glowed with excitement…I had found TWO phages.