Ok, now I’m finally moving again. Last week, I adopted Aparna’s phage. My phage had spent too much time in the lysate and apparently it all degraded, because none of the lysates I had remaining were able to revive it. Therefore, I had two options: get more soil samples, or adopt. Given that it is currently December, I chose to adopt. Since adopting, I have gone through the DNA isolation processes and I performed the photospectrometry this Wednesday. I got 1458 ng/microliter, which is apparently pretty good. In all, I am finally moving again. Nice work on your phage, Aparna, it is serving me well!

Ok, so there are two problems here.

1. After having calculated the titer I should have needed to completely lyse the plate, there was very little success (that was through this week). In response to that, I went up one dilution (from 10^-4 to 10^-3) and used 40, 60, and 80 microliters of lysate instead of 10, 20, and 30. So based on that, there should have been some sort of lysis. Instead, I went into the lab today and found that I didn’t have a single plaque. Issues. The only response I can have is to let the plates incubate over the weekend and hope. To confirm whether or not I have a phage at all, I also created a spot test of a 10^0 dilution with 10, 20, 30, 40, and 50 microliters. If nothing shows up, I’m really just not sure.

2. Is my phage (pre-emptively, admittedly) named “Patrick” or “Ya Boy Kenny”? It would probably be shameful to name a failing phage after Kenny, so I think it’ll be Patrick: it’s got to have a name if I want to cheer it on.

Whatever it may be, I’m afraid that my phage may have actually died, even though that’s technically impossible.

So when I went and checked my plates on monday, I ended up finding that either 1. my phage had begun to present itself differently or 2. my lysate had gotten contaminated somehow (I had a similar presentation of something very unlike my phage that increased in concentration with my phage and my control- phage buffer plate had no contamination). Based on Dr. Adair’s advice, I just refiltered the lysate. However, upon replating, I was warned that the only arthrobacter we had was likely contaminated (it was no lie! very contaminated). On wednesday, I redid the plating with the new Arthrobacter colony and now I am going to be going into lab on friday to do my ten-plate, assuming that my phage isn’t dead (as I fear).

#phageworldproblems

Okay, I take this all as good news even though it is rooted in EVEN MORE of my mistakes. So apparently when I went back to fix my bad plating which I did to fix my bad sample, I used the wrong lysate concentrate. So 1. I fixed it, 2. because my phage isolated so early on, my Titer was high enough to actually be able to set up my grid for the Electron Microscopy stage. (Just saying, it is amazing all that this entails!!) We get to actually look at a single bacteriophage!! I think that is pretty cool, especially the whole process. It just absolutely blows my mind. But yeah I’m all excited now and I can’t wait to see if my phage came back with an image!

Turns out, it was too weak and I lost it again. Going back a few more weeks to try to revive this poor little phage of mine.

My phage is just too opaque. It can’t put a dent in the agar and can only present itself on the surface. Therefore, it is invisible in something as small as the “0”-“-10” spot testing. Because of this, I had to do seperate plaque assays for dilutions 0,-1,-2,-4,-6,-8 to try and find it. At this point, I have been stalled at isolation for an extremely long time all because of my weak phage and I think I may have to start considering adoption if progress is not made, today. We will see!!

Hopefully, I got caught back up. This time I actually saved the whole syringe full of sample! Looking around the room seeing the people that are ahead of me, it looks like they’ve got some pretty cool stuff going on. I’m excited to get there and I’m happy that pretty much everyone has gotten a phage by now.

I made a pretty severe mistake by not saving my flood solution and I was set back about two weeks (four lab days) on monday. I replated the third round of purifications and I will have to reflood tomorrow. I’m learning never to throw anything away; I’ll be a hoarder by the end of this, I swear it.

On Monday, I attempted to re-plate my concentrated phage solution that I removed from the flooded PB  plate. However, I ended up using the wrong TA solution so the bacterial agar never actually solidified. If there was any plaque development, it is not visible because of my mistake. Today I made a new batch of 1X Arthro Top Agar and used that instead (it solidified within minutes) so now we should be moving forward again!

Yesterday, I got back my plates after two days of incubation and my third round of purifications all came back very positive. Although the colonies produced from my phage are very opaque (honestly, just weak), they are consistent and I believe that I have isolated a single strain of the bacteriophage. Therefore, I started the next step of the process, yesterday, which involves a process called “flooding” where you fill the agar plate with phage buffer and wait for a few days, then use that phage buffer (with phages in it) to do another round of plating in plaque assays. I am going in tomorrow to do that plating so that I do not lose an entire week if my phage isn’t strong enough to survive the flooding. I’m really excited to be moving forward like this.