By Heather Han
When I was looking for potential courses during summer, I knew that the Phage Hunting class would be the right choice for me. For how could it not? Throughout high school, my favorite class had been AP Biology, not because of the interesting lectures as much as because of the numerous labs that I had been granted the opportunity of doing. The ability to perform labs in which we did experiments from modifying E. coli plasmids to breeding and learning how to identify the sex of fruit flies (Drosophila melanogaster) fascinated me.
Yet, upon learning about the first assignment, I began to have some doubts about the class. Digging dirt? Why would I ever want to do that? But the first day of class absolved some of my doubts. After spending only half an hour in the classroom going over the syllabus, we moved to the lab and immediately began to prepare the enrichment culture. I was pleasantly surprised. I did not expect to do as much as I did the first day of class (even though we did have to stay after class for a while). But what surprised me the most was that I got my own lab coat! My own PERSONAL lab coat, a lab coat only for me! Having my own coat made everything seem that much more official.
Although direct plating did not initially yield any phages, my enrichment culture soon did. We prepared our enrichment culture by transferring about 2 teaspoons of soil sample to an Erlenmeyer flask, then adding 40 mL of sterile water, 5 mL of sterile 10C7H9/glycerol broth, 5 mL of AD supplement, and 0.5 mL of 100 mM of CaCl2 to it. On the second day of class, we once again transferred our sample, except that this time it was from the flask to a 50-mL conical tube. The TAs then proceeded to collect our tubes so that they could spin the entire class’s sample at the same time at 3000 rpm for 10 minutes. I then filter-sterilized my enrichment sample and transferred 1 mL of it into a microcentrifuge tube. Since directly plating the enrichment sample would most likely produce a plate covered in plaques if phages were present, we were instructed to dilute our sample before plating. In the end, we plated 6 plates, each containing 0.5 mL of M. smegmatis, 4.5 mL of top agar, along with either 50 µL of phage buffer (negative control plate), 50 µL of 100, 10-1, 10-2, 10-3, or 10-4 dilutions of the sample. We let the plates sit undisturbed for at least 20 minutes before the TAs went around to collect them and place them in a 37 degree Celsius incubator for 24 hours.
By the time of the third class, I had phages! While my 100 plate was completely covered with plaques – the entire plate appeared to be transparent, my 10-4 plate contained plaques that were more spread apart. In the end, I chose 2 plaques of different morphology (one of which was big and cloudy, the other small and transparent) from my 10-4 plate and streaked them onto 4 new plates – 2 of each kind – as well as a negative control plate.
We had to repeat the streaking process again on the fourth day of class in order to better isolate our phages. When I began the process of choosing plaques of phages to plate again, I noticed that there appeared to be plaques of two different morphologies on one of the plates. This means that I might have 3 different types of phages rather than the initial 2 that I thought I had. But I guess I won’t find out till the next class! Seems like there will always be new information for me to learn from Phage Hunting!