If At First You Don’t Succeed…

Try, try, try again.

A polluted river. What a perfect place to get a virus living in the dirt, one might think. The person who thought that was me. Because I had gone to Hampden previously and had seen the sign near the river warning people to not go into the river because it contains harmful runoff, I thought that this area would be crawling with phages. I collected my dirt, making sure not to step into the river for fear of contaminating myself with whatever lied within. I went back to my dorm, confident that there were millions of little phages ready to lyse some M. smeg the following Monday.

I was wrong.

My first attempt at direct isolation ended up in plates that looked just like the control with no plaques on it and I sure was bummed. Maybe I just got unlucky? Maybe the phages were just tired and didn’t feel like lysing the bacteria? I knew the latter was clearly not true, but maybe I had just been unlucky. So then I tried direct isolation with the same filtrate, thinking that this filtrate that I had inside my microcentrifuge tube must be home to many phages.

I was wrong. Again.

I held back my anger as I looked at my second round of plates and saw exactly what I had seen last time; nothing. Just more plates that looked like the control. I felt like I had wasted so much time, plating and filtering and shaking these tubes, and for what? For nothing! It was with a heavy heart that I took another sterile tube and went back behind the UTL’s, begrudgingly scooping up dirt, knowing that I had to start all over again. All I needed was one plaque.

I returned to class with this new dirt sample and was told to do a different procedure; enriched isolation. Direct isolation, the procedure I had done the two times previous, had a smaller phage yield. Enriched isolation would increase my chances of getting a phage by a large margin. For the first time in a while, I had a real feeling of hope as I mixed some enrichment broth and 7H9 with my dirt. I extracted and filtered the supernatant and did serial dilutions up to 10^-5, plated them, and left class with a smile on my face. I knew that next class I would have plaques on my plate ripe for the picking.

I was wrong. Again. For the third time.

IMG_3749.jpg

This time, however, it wasn’t that I had no phages. The issue was the I had too many phages. All of my plates, even the 10^-5 plate, were basically all cleared, indicating a very high phage concentration. I scratched my head because I didn’t know how to feel. I guess this was a good thing? I mean, at least I had plaques. I decided that maybe streaking some of these plates would help dilute these phages. Maybe, next class, I would have some plaques!

I think you’re probably catching onto the theme here…

If this class gave credit for having plates that are as clear as they were before putting them into the incubator, I would have an A+. The streaked plates I had been so excited about were clear and one was contaminated. After yet another defeat, the amount I wanted to hang up my lab coat and leave forever was at an all time high, but not because I had not succeeded. It was because I had to do serial dilutions again. But this time, I had to do ten rounds. The thought of that made my stomach turn. There were so many ways I could mess up and contaminate my filtrate and have to restart, especially because I was still not very good at the procedure. Facing my fears and all of the odds favoring me messing up aseptic technique, I began the process of doing ten serial dilutions. After completing the serial dilutions, I plated the following plates: 10^0, 10^-1, and 10^-6 through 10^-10. As sweat dripped down my face from the stress and from the bunsen burner’s proximity, I placed my plates into the incubator.

Oh, what a feeling.

Plaques on 10^-6.jpg

Plaques. Plaques everywhere. On my 10^-6 plate, I had a beautiful array of clear dots, followed by fewer on my 10^-7 plate and only 2 on my 10^-8 plate. My dilutions had actually worked. All of my failure, all of my problems, were all worth it. These lessons that imposed themselves on me are definitely some that I can apply to my future in research, and, in life.

Sometimes, things don’t work. A hypothesis is refuted, a plaque doesn’t show up. The reason we experiment is to learn and to try new things and to fail. In my eyes, a failure in the scientific community may be just as valuable as success. Failure allows us to step back and view what we have done and see what could have gone wrong or what we can do to improve our procedures. Failure is a blessing in disguise, and it’s important to not fall victim of the nasty appearance that we perceive failure as having. Sometimes, following up on an unexpected finding or something that went wrong can be more beneficial than what one was looking for in the first place. I am happy to say that my failures have helped me learn more about laboratory procedures, and I have definitely learned that patience, in the scientific community, is definitely a virtue.

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