By Maddie Goodman
Just coming from the womb of high school, fresh from the days of labs with expected results and monotonous data analysis, I was more than ready to start a class whose end result was for the most part unknown. Would I be successful in isolating a bacteriophage? What will it look like? How am I going to be able to do this?
My adventure started with digging up dirt in front of my dorm the night before school started. Not the most conventional start to college, but it was certainly something I will remember for a long time. We started doing work right away on the first day of class, which was a refreshing break in the endless stream of syllabi. We started with direct plating of our samples. This involved mixing our dirt with phage buffer in the hopes that the phage would be released from the dirt and mix with the buffer. Then this solution was filtered and we infected tubes of M. smeg with it and hoped for the best. Unfortunately, my first sample did not yield any phage, but I was not at all discouraged as that seemed to be the general consensus throughout the lab.
The next day in class we went to get more dirt from a community garden. This too was an interesting experience as it almost felt like we were sneaking into someone’s back yard to steal dirt. I harvested my dirt from under a tomato plant; it was very rocky and I took it from the surface. We did enrichment cultures on these samples, with the hopes that the phage would propagate and we would be able to get actual plaques on our plates. From this we diluted the enrichment because there would hopefully be too much phage in a straight enrichment culture that we wouldn’t be able to see any plaques. I was very excited when I saw that I had plaques on my plates after plating the dilution series! From here I picked two different plaque morphologies to pursue, one medium sized and cloudy (b), the other small with very defined edges (a). From here I streaked two plates picking from each plaque using the traditional zig-zag streaking pattern.
This round of streaking was largely unsuccessful for me. The plates from my b plaque were clear, while the plates from my a plaque did have some phage. From here I decided to use a t-streaking pattern to streak my plaques from then on.
On my third round of streaking I got what I had been looking for: clear distinct plaques!
The only problem was that they looked a lot smaller and clearer than either of my plaques had before. However, Dr. Schildbach told me that this was because these were more isolated and so I could see them a lot clearer because of that.
From here I picked a plaque (the one with the heart next to it) to perform my initial titer assay. I simply touched a pipette tip to the plaque, swirled it in phage buffer, and then plated out serial dilutions of it. From here I calculated my initial titer to be 2.8*106 plaque forming units (PFUs) per mL. I then took my plate with the most distinct plaques (10-1) flooded it with phage buffer and let it sit to soak up as much phage as possible to make my medium titer lysate. From here I flooded the liquid and plated another dilution series. From this series I calculated my medium titer to be 5.3*1010 PFU/mL. I could tell just by looking at my plates that my 10-4 plate was a good web plate.
So I started my 6-plate infection, simply plating 6 of the same form my 10-4 dilution. Once I got these plates I flooded all six of them with phage buffer and let them sit for an hour and a half. From there I collected all the liquid to make my high titer lysate. I plated out a dilution series to make sure that it was pure. Unfortunately I got weird results from this plating and got two different morphologies on my plates. We hoped that it was just that the smeg was doing something funky, but just to make sure I did a dilution series picking from both morphologies. Thankfully this test came out that form both plaques I got the same morphology on all the plates.
From here I was able to purify my DNA. This process involved a lot of adding things and letting them sit for a while, and centrifuging things, and filtering them out, but the end result was just a tiny pool of liquid at the bottom of a microcentrifuge tube. The next day I came into the lab I took some of my high titer lysate spun it down for an hour, resuspended the pellet, and then took this amount of my phage to the imaging center so I could see what my actual phage looked like. It was amazing to actually see that thing that I have been working all semester to isolate, it really was like giving birth to a child!
After this I had enough time in class to be able to test to see if my phage was lysogenic, meaning that it inserted its DNA into the bacteria and not propagate within it, but allowed the bacteria to propagate and thus copying its DNA. To do this I simply picked form a plaque and did the traditional streaking method, but I did not add M. smeg to the plate. This is because, if it was lysogenic, there would be some bacteria in the plaque, and this would show up on the plate even if there was no smeg with it. Alas, I did not get anything on my plates, so I did the process again. And still nothing. I then accepted that I did not have a lysogenic phage.
Next I will be running my isolated DNA in a gel to make sure that I have good DNA. I cannot wait to learn more about my phage! Hopefully next time I write I will be creative enough to think of a name for it!