Although my phage ended up dying at the last minute, this has been one interesting semester of lab. I have never learned so much about research and biology than I have in this class. The different methods that we would do every day and be able to interact with each other whenever we needed assistance or an opinion made me want to come to lab everyday. I made a lot of new friends in this class and we still have one more semester together. I can’t wait to come back next semester and finish what we have started!
So on Monday I was able to check the concentration of my DNA using the spectrophotometer. The concentration of my DNA was surprisingly low. I don’t know what the next step will be, I think I’ll be making a gel, but today I will be making a grid because I was not able to make a grid last time as it was breaking apart. Let’s hope I can find a phage today.
On Wednesday I completed my 10 plate procedure after I got positive results. I did not have any difficulty doing the procedure as it was basically the same thing as before however with 10 plates. I felt a little behind on my work so I came into the lab with Katelyn and Gabi so I could flood my plates today. The 10 plates were all webbed and looked great so I got a head start and flooded my plates today.
So last Wednesday I was able to finish my calculations and actually plate my 5 samples on the same day so I could get on track. However, there was an issue with the 1x top agar and all of the plates ended up being contaminated. Now obviously that is a problem, so today was kind of a make-up session for that mistake. I made a fresh batch of 1x TA and used new phage buffer just to make sure that there would not be any more issues with this procedure. When I get back to the lab on Wednesday, hopefully I can get started on my 10 plate procedure.
After I filtered my plate last Wednesday, I came back to do purify my plaque once more but this time after diluting my sample to 10^-10. It didn’t take me too long to finish on Monday as I have really gotten used to the whole purifying step and can do it with my eyes closed. Tomorrow, I will start titering (I think that’s what it’s called) and catch up to everyone else.
On Wednesday, I got the go ahead to start flooding my plates as my -1 sample’s plaques seemed similar and web-like. I added phage buffer to that plate and put it in the cold room so I will start working with that on Monday.
After being MIA for about a week (I actually missed not being in lab!), I finally got to check out my plates from the week before. Everything seemed to look fine, I got really nice looking plaques and yesterday I was able to do my third and last plaque assay (hopefully) for my purification process. I’m going to check out my plates tomorrow and hopefully I can go onto the next step.
On an unrelated note, I attended a lecture by Dr. Robert N. McClelland tonight and he told us his story of how he operated on both John F. Kennedy and Lee Harvey Oswald back in 1963. Dr. McClelland explained every detail of what occurred in the operating room on November 22, 1963 in Parkland Memorial Hospital. The riveting story of how a calm Jackie Kennedy walked in to the room of her husband and say goodbye and how a couple days later he was doing surgery on the man who shot the president of the United States himself. It was an eye-opening experience and he even showed us the shirt that Dr. McClelland wore when operating on the president which still has JFK’s blood stains. Even though it doesn’t have anything to do with our phages research, I thought I would just share my experience with this legend in medicine.
Quick update on my phages, I am almost done with the purification process and am not having any difficulties so far. Yesterday, there was a shortage of top agar and people were unable to move forward. However, I was a little selfish and took up the rest of my group’s leftover top agar so I ended up finishing up pretty early. Come Monday, my plaques will hopefully look the same and I can finish up the purification process. Nitish, out.
Yesterday, I checked to make sure that my plaque assay still had phage in all of its samples. Thankfully, all samples had some plaque in them, even though they were not that many plaques in each agar plate. Things are finally moving along as I started my second set of plaque assays to make sure that I still have phage. I will check my results tomorrow and do my last set of plaque assay. After two months of constant failure, things are finally working in my favor for this project.
I got to check out my plaque assays today and found no phage whatsoever. I can’t really tell if I made a mistake when doing the plaque assays or if I’m cursed. I’m pretty sure it’s the latter. Anyways, I think the issue might have been with the top agar as it was having some issues with solidifying again. This time I picked the plaques from the spot test that I did for these plaque assays instead of the actual “0″ samples so that could make a difference. I made sure I was extra careful while doing the plaque assays and the TA that I made seemed to be working pretty well, especially since it actually solidified. Well…here goes nothing.
So after 2 grueling months of tedious procedures and failures, I finally got some phages. Now like I mentioned before there were some complications with the 7H9 top agar as it didn’t really solidify well enough. Just to make sure that the results that I received are persistent, I did a plaque assay with samples 5 and 6 (which are the samples that had some phage in them) and we will see on Wednesday whether there really are some phages in them. I am actually pretty optimistic on this one so hopefully we can get a move on.
Two days ago, we got to check the result of our plates and see if we got any phages in them. Now remember, I got a couple of new soil samples to do the experiment with last week and I failed the first time. So when I looked at my plate, I was shocked (not really) by the fact that I didn’t get any phages whatsoever, again. Dr. Gibbon and Dr. Adair proposed that since we were so far into the semester that we try using mycobacterium smegmatis (SMEG) instead of Arthrobacter for our samples. Today, I put my samples on an Agar plate so hopefully that will work out when I check it out on Monday. The Agar never really solidified when I placed my samples on the plate but I hope that won’t change too much as I put the plate in the incubator and that should solidify the Agar and samples. Now I just have to wait until Monday…
Ok, that was kind of cheesy. So I know that it’s kind of late for my first blog entry but I guess I’ll give you a quick update of what has happened so far…nothing, nada, nil. The three samples that I dug up before the school year started didn’t get any phages no matter how many adjustments we made to the experiment. So last Wednesday, I went around campus getting new soil samples. I was going to places where the dirt was a little wet, as Dr. Gibbon mentioned something about how bacteriophages prefer wet environments. Once I collected my samples, I started preparing them to be harvested on Monday.
I came back on Monday to harvest the samples in the agar plates with my lab group and everything seemed to be running smoothly. We were implementing the aseptic technique and were very careful to make sure that we didn’t contaminate our plates as we had issues with that in the past. The samples were pipetted onto the plate and we placed them into the incubator for 48 hours. So now it is Wednesday and we were excited as we wanted there to be signs of phages in our samples, but the plates ended up being grainy. So what now? Since the agar plates were a little grainy, Dr. Adair suggested that we incubate the plates a bit longer just to see if there is any change. We also had a hunch that the top agar that we used was a little “outdated” and could be a factor. We developed a new batch of 1x TA and used that for the same samples to see if there would a be difference with fresh TA. We’ll see how the plates end up on tomorrow after biology class and I’m hopeful that we will find phages but again I’m not really that hopeful about it.