by Jillian Tse
As I’m sure you’ve read from the rest of the blog entries by my fellow phage hunters, we have all experienced our share of highs and lows in searching for, isolating, and purifying a phage. Let me start at the beginning of my phage journey. I had a rocky take-off: I first started off with completing two direct plating samples and one enrichment. These plates did not contain any putative plaques so I went out to collect more samples. After several more rounds of unsuccessful direct platings it came time for me to adopt a phage. My lab partner, Eve, so graciously adopted one of hers out to me; however, I was determined to find my very own phage baby.
Many other students in the lab were having success with the enrichment method so after streaking out a plaque from Eve’s plate, Suzie and I ventured out in torrential rain to the Beach to get dirt samples. We returned to lab completely drenched but hopeful that in our 15 mL conical tubes resided phage. I came to lab one day to discover that the plate I was streaking to purify my adopted phage was contaminated! On the bright side, my enrichment plating worked! I had an awesome dilution with some plates completely clear and others covered in plaques. From there my journey was fairly smooth sailing. I completed three streaks with only one contamination, but that situation was quickly averted by going back to the previous plate to re-streak.
Next, it was time to do a titer assay in order to calculate my titer. I had to do three titers total. The first round of plates was contaminated, the second round was not incubated so there wasn’t any growth, but the third round worked perfectly. My calculated titer was 1.04×107 pfu/ml. From here I attempted to find my maxweb. The first time I tried to do so my plates were all strangely clear. Either I didn’t add M. smegmatis to the TA when plating or my titer was so high that it ate all of the bacteria on the plates. I tried for a second round plating a 1x, 2x, 1/2x, 1/4x as well as a 1/5x and 1/25x. This attempt was much more successful. Both the 1/4x and 1/5x were perfect webs! I decided to continue on with the 1/4x and used this to do the 10 max web plates. I came in on a Saturday to see gorgeous web plates. I flooded these with phage buffer and after a couple of hours (so the phage would go into the PB) Katie helped me by filter-sterilizing the phage buffer, resulting in my high titer lysate.
On October 31st I was fortunate enough to see my phage face-to-face!! It was an incredible experience using the electron microscope to take pictures of my own phage that I hunted and purified by myself (with the assistance of my awesome professors and TA’s of course). My phage, Minion (named after the little yellow characters in the movie Despicable Me), is approximately 233 nanometers and yields a plaque size of about 3 mm. The plaques are clear in the center and turbid around the edge. My high titer lysate titer is 4.10×1010 pfu/ml. Next I’ll be purifying my phage DNA and competing in the Phage Olympics!!
JHU Phage Hunters 
Minion is beautiful, Gillian! Congratulations on your gold medal in the phage olympics.
Out of curiosity, was the contamination that occured during streaking apparently from another phage or another bacteria? What would you guess is the source of this contamination?
Thanks! Sadly, I actually had to rename Minion because the name was taken by another phage hunter 😦 So Minion is now Manatee!
I had two contaminations throughout the whole process of isolating and purifying Manatee and both were from different bacteria than M. smeg. I haven’t had a contamination from another phage luckily! I’m not really sure what the source of the contamination was…maybe a contaminated wooden stick?? Thanks for commenting! 🙂
I have been doing this for a while, but I have gotten into trouble! My plaque assay have had contamination for the fourth consecutive time now!!! Despite trying to maintain high standards of sterility during the work. What can I do to avoid this? While sometimes the plaques do not form?