Phages, phages and more phages!

by Bettina Wunderlich

Phages, phages, phages. That’s all we hear around here, and it makes sense given the name “Phage Hunters”. But if I were reading this for the first time, I would have no idea what a phage is, so I’ll just give a quick recap to those of you who don’t know. I would have probably ended up on wikipedia, so I’ll start off giving you their definition: “a bacteriophage is any one of a number of viruses that infect bacteria. Bacteriophages are among the most common biological entities on Earth.” Phage would just be a shortened form of the word bacteriophage, which I would have learned from there, too. Their definition is simple, yet precise: a phage is indeed a virus that infects a certain type of bacteria. Literally, it is an eater of bacteria and, in our case, the latter would be M. smegmatis. We are “hunting” to purify one of these phages, in hopes of maybe even calling one our own. Phages are the most abundant life-form on Earth and they can survive in almost any environment, which is why we all started our journey in a place where we hoped would be infested with them: dirt.

I can very happily say that I have recently been able to purify my own phage – two actually – and the plaques they form are morphologically very different (one clear and one cloudy). Thus, I can hopefully venture to say that I am on my way to success. However, it was by no means an easy process, quite the contrary, getting to where I currently am.

Actually, it was after many unsuccessful attempts that finally I had a small clearing – literally. But, as we have been told, how to deal with failing is one of the lessons we learn in this class, and boy, how we have been learning a good deal. After many failed direct platings, a failed enrichment, and many trips out to get more and more samples of dirt, I finally managed to have a sliver of luck on my second enrichment – a putative plaque on the 10^-1 and two on the 10^-3 dilution plates! This might not seem like a lot, but to a phage hunter with nothing, its more than we can ask for. My partner, Ben, had the same thing: three putative plates, and that same day we eagerly performed a spot test on them. However, as it wasn’t very promising we were mentally preparing ourselves and also getting ready to adopt a phage, as luck is not always on our side and we have to be prepared for anything. But luck was indeed on my side that Monday afternoon, when I saw three beautiful clearings on the spot test. There is a three letter word that describes this exact feeling: YAY!

Ben and I struck these plaques – unfortunately he wasn’t as lucky with his as I was – for our first streak, and we were disappointed to see our plates contaminated the next lab. Nevertheless, on we went flooding, filtering and re-plating them. We weren’t giving up that easily, and happily, these rendered us beautiful results.

We then continued on to streaking our second and third plates, all thankfully filled with plaques, giving us positive results and confirming what we been hoping for: we had purified our phages. With this in our hands, I chose my two best phages, a cloudy one from my BW2 sample and a clear one from my BW3 sample and continued on my phage journey to carry out the titer-assay. This is necessary to determine the concentration of the phage particles, and to do this I picked a plaque from each and diluted them until the 10^-4 dilution. After plating each of these dilutions and incubating them, I was able to count the number of plaques in my BW2 10^-4 dilution plate and my BW3 10^-3 dilution plate, hence calculating their titers: BW2 8.2 x 10⁷ pfu/mL and BW3 9.0 x 10⁶ pfu/mL.

I am now on my final plaque purification stage and edging closer to our goal. And that’s where I’ll wrap up as this is the story of my phages so far. I have truly been loving the experience and the ups and downs we encounter – the thrills of actually seeing a plaque and now being a proud parent of two beautiful phages (who I now have to name – which is very hard considering how much they mean to me), but also learning that we cannot give up even after many failed attempts because sooner or later something will work out, and believe me, there is no better feeling! Hopefully luck will continue to be with me as I continue on this awesome journey in the weeks to come!