by Alannah Lejeune
Dear phage blog reader,
Let me tell you the tale of one of the biggest emotional journeys I’ve undergone this year. No, I’m not talking about leaving home and going to college, I mean my search to isolate a phage. And while I may exaggerate a little, I’ve had a great time this past month learning about elusive bacteriophages, M. smeg, and laboratory techniques.
I started the course with high hopes, a vial of dirt from near a river, and no idea what was meant by the terms direct plating or enrichment samples. However, my excitement began to fade as I went through five samples and tens of plates as I enriched, plated, or streaked for potential plaques and still found no tell-tale circular patterns indicating phages on my plates. As I was about to go out and collect yet another sample with the high tech soil collecting apparatus of a plastic knife, Dr. Schildbach walked in with a microcentrifuge-tube tray full of already-filtered phage buffer and potential phage solution. In the tubes were samples taken from a compost heap–a place known to be crawling with soil bacteria like M. smeg–and hopefully the phages that infect it as well.
I dutifully mixed 50 microliters of the purified compost sample with 0.5 mL of M. smeg and let the tube they were in sit for 15 minutes so the phages would have time to infect the bacteria. Then, the bacteria and phage mixture was added to 4.5 mL of top agar and plated. Next class period, when I checked my plates, I saw 4, very very tiny spots, which I though at the time were probably bubbles, but decided to streak anyway. Streaking, for those of you who aren’t experienced phage hunters, involves poking a wooden stick into the center of a putative plaque and then dragging it across an agar plate, using three different sterile sticks and streaking in different directions to get different dilutions. To my surprise, when I glanced at the plates next class, on plates #1 and #4, there were actual, albeit very small, plaques covering the plate! I learned, however, that finding a phage is only the beginning. I needed to continue streaking in an attempt to isolate a single phage species from each plate, and along the way I faced such tribulations as the great bacterial infection of 2011, wherein about a quarter of the class’s plates became infected with a foreign bacteria. One of my streak plates also became slightly infected, but luckily, the bacteria hadn’t engulfed all of my phage, and I was still able to get a viable sample from it. Shown here is a picture of my lovely #1 (named Fry after the Futurama character) after three streakings:
Now that I was in the possession of two potentially distinct phages (#1 seemed to grow faster and than #4) I was in the position to help out a fellow phage hunter and adopt out one of my little phages (*sniff*, they grow up so fast!). Now my classmate Eleni has my #4 plate phage, which seems to be doing great under her care. The feeling of being “mommy” to a possibly yet-unknown phage is amazing and definitely worth the effort. I’ve just recently finished doing a phage-titer assay on my original #1 to determine the exact concentration of phages I have, and soon I’ll be calculating pfu/mL (plaque-forming units per milliliter) to see just how numerous my phage is. To titer-assay, I picked an isolated plaque, touched the middle of it with a micropipet tip, and gently shook the tip in 100 microliters of phage buffer in a microcentrifuge tube. From this 100 tube, I transferred 10 µL to another tube that contained 90 µL of phage buffer which became my 10-1 dilution. I repeated this process until I got to my 10-4 dilution and then plated each dilution.
Getting a chance to work in a lab and find my own phage has been such a different, and far cooler, experience than doing mundane high-school style labs. Plus, we get real lab coats!