By Alison Tretter
When I told my parents on our weekly phone call that I would be streaking in the lab on Monday, they were less than thrilled. It took a very hurried explanation that I wouldn’t be arrested for public indecency, but rather, I’d be purifying my mixed phage population in hopes of isolating a single strain of phage. I was thrilled to reach this phase since I lost heart after my two direct plating samples came up phageless. However, my enrichment sample (taken from under a water spigot in the Hopkins sculpture garden) really came through and has made me the proud parent of two unique strains. My path to phage motherhood was as follows:
After diluting my enrichment sample using serial 10-fold dilutions, screening the five plates and the negative control, and incubating them for the weekend, I observed my plates the following Monday with bated breath. It was a great feeling observing my enrichment samples and seeing one unmistakable plaque on the surface of the 10^0 plate and two plaques on the 10^-1 plate. Once I intelligently exclaimed, “I did Science!!,” I went on to follow plaque streak protocol. I selected a clear-looking plaque from the 10^0 plate and two smaller, more turbid plaques from the 10^-1 plate. After a rocky start,—I somehow thought that to make a negative control streak, I needed to transfer the M. smegmatis from a negative control enrichment plate to my streaking negative control, but was stopped by a slightly frantic-looking Dr. Fisher—I got the streaking strategy down.
To streak a plaque, you must use the aseptic technique. First, you remove a sterile wooden stick from its test tube. Next, you open the Petri dish that contains the putative plaque and touch the center of the well isolated plaque using the sterile end of the stick. Once you return the cover to the Petri dish, you open the lid of the Petri dish to be streaked. Gently streak the top 1/3 of the agar with the tip of the wooden stick that was dipped into the plaque. After returning the cover to the Petri dish and throwing out the used stick, you remove a new, sterile stick and streak the adjacent un-streaked portion. Make sure to overlap the first streaking area only on the first few swipes and cover another 1/3 of the dish. To make the final, third streak, follow the same process of removing a sterile stick, streaking the adjacent portion, and covering the Petri dish. The purpose of streaking is to dilute the phage so that you find individual plaques on the new plate and can be sure that only one type of phage resides in that plaque.
For the most accurate results, I decided to create two steaks per plaque. Thus, I had 2 plates from the same 10^0 plaque, 2 plates from one isolated plaque on the 10^-1 plate, and 2 plates from another isolated plaque on the 10^-1 plate. I fondly named them 10^0 #1, 10^0 #2, BP1 (Big Plaque), BP2, SP1 (Small Plaque), and SP2. Just like with kids, you pretend to love them all equally, but in reality, you have a favorite. Don’t tell the others, but I like 10^0 #1 the best! 7 fresh agar plates, 21 of wooden sticks, 31.5mL of TA, 3.5mL of M. smegmatis, and at least 5 terrible phage-based puns later, I was on my way to finding a pure population.
But, in order to get a truly pure population, the streaking process must be repeated at least three times. I selected 10^0 #1, BP1, and SP2 to continue on to the second round. I created one second streak plate for each of the three and put the original streaking plates up for adoption. It was a hard choice, but I knew they would go to good homes. The Second Streakers© came back with consistent results. 10^0 #1 produced a strong lytic population while BP1 and SP2 both produced temperate plaques. Because BP1 and SP2 both came from the 10^-1 plate, it’s quite likely that they carry the same strain of temperate phage. For the third round last Monday (third time’s the charm!), I streaked the second streak plates of 10^0 #1, BP1, and SP2. Once again, I saw lytic, temperate, and temperate plaques respectively. Three streaks is the minimum, however, here at JHU Phage Hunters, we do 110%. So I’ll be moving on to my fourth streaking session on Friday.
Some people might tire of the endless flow of agar plates and wooden sticks but, if Old School has taught me anything, it’s that streaking is a pivotal college experience. Bring on round number four!