by John Cotoia
The map to obtaining a pure bacteriophage for Mycobacterium smegmatis is not self explanatory; there are dead ends, confounding obstacles, and Jerry-rigged solutions. Testimony to the previous blogs, everyone has their own map to buried treasure, a map that tends to take the shape of a mutilated maze, used in hopes of finding a treasure only a few nanometers in size … do you see our problem? It is a battle against time to find a speck of dust on the beach, and when you catch traces of potential, you cling to hope like your life depends on it – more or less emotionally. This leap of faith, although terrifying, is quite invigorating when you land on solid(ish) ground. Both Victoria and Katie (our lovely Phage Lab TAs) have expressed a mutual understanding, as their endeavors in graduate school have taught them a simple phrase, “Welcome to research!”
To save you the pity-party, let me just say I was not very successful at obtaining my own phage. I was not even successful at adopting a phage! Two Spot Tests, two Enrichments, and four Direct Platings from six different samples revealed nothing. One adoption lost, one optimistically proceeding.
A very generous offering on the part of Rachel, a neighboring phage huntress, gave rise to my first adoption. As a responsible step-phage-father, I swore to uphold the prospective phage’s interest of purification and identification. Streaking gave way to contamination, which required Phage Buffer (PB) Flooding and Filtration, which demanded a re-streaking of the original sample, which resulted in “FAIL” – as I demark it on my map. The contamination was unique in that it took place directly on the streak marks, and also occurred on the negative plate. I was led to believe that the sticks used must have been compromised – how else would contamination arise on my negative, because I was streaking nothing! But when consulting a Victoria, it could mean contamination was present on the previous plates, so I was instructed to fill the previous plates with a few mL of PB, allow it to sit for 20-30 mins to extract any phage present, filter the solution and streak the filtrate. But upon examining the filtered, flood streaks, there was no phage. This evidence was disheartening, given that it indicates no phage is present – *gasp*. To prove this idea conclusively, I very carefully re-streaked from Rachel’s original donated plate, to conclude that there was indeed phage, but not for M. smegmatis. I am sorry sample R.O.2.0, you were loved. (Sadly, Rachel almost achieved purification of her phage when she came to the same conclusion, and was forced to adopt.)
A successful sample – with the name Final Boss, how could it not be successful?! Another generous donation on behalf of Peter, fellow phage hunter, I was given the dilution plates from an Enrichment. As denoted on my hunting map, I am streaking the phage into bacteria like no one’s business. Currently, I am working on the three tertiary streaks, trying to define whether or not Final Boss has multiple morphologies, or is two separate phages. She (I think Final Boss is a girl, because my mother usually has the Final Say) likes to change things up – on two streaks she changed from big and turbid to small and clear, yet on another she remained large and cloudy. If the results progress as indicated on my map, I could have two phages, and be able to adopt one out – with Peter’s approval, of course!
So the mission may not be impossible, and cautiously optimistic, I (the Phage Hunting Ninja Turtle) will press on
(Oh gosh, I hope the incubator is working today)