The Third and Fourth Week

Week Three

This last span has been filled with results. First, I received the DNA sequences for the samples. I organized the results, and ran them against a library of known bacteria 16s rRNA genomes. For a brief summary of this process, the samples are sent out to a company which runs the sample DNA through something like a PCR reaction.The gene is mapped when a computer reads the light wave lengths emitted from dyes which bind to their base pair respectably. (Red binds to T for example). A graph is sent back with the wave length of light emitted, with letters corresponding to each light emission. I went through all the bacteria samples graphs and made sure they were accurate, because the beginning and the end of the sequence are often not clearly defined. So I checked manually to make sure that letters corresponded to the graph properly, and I left gaps for parts that I could not discern. I then blasted the sequence to an only program called BLAST, and compiled the results of the type of bacteria and how closely the sample DNA matched the sequence in the library. Only one of the samples did not have accurate sequences; that one might have been contaminated or perhaps the DNA was not pure enough.

With the samples now matched with a species, I designed an experiment to test growth in the presence of a antibiotic called Gentamicin. I would compare the rate and extent of growth of bacteria in a small well. The plate I used for this had 96 wells, so there was ample room for some controls with no bacteria, and three trials for each variable. The first test was just to see if the bacteria would grow in the wells with a general media broth (environment with nutrients to feed the bacteria). The ones we tested did, and from there I moved forward. The next trial would only fit half our samples, because for each sample there are four varying levels of antibiotic to test, and three trials for each. I made the media solutions with the proper amount to antibiotic, and filled the well with it using a special automatic pipette. From there I put 1µl of bacteria in each well, and ran them on an optical density machine. The optical density shows how much light can bass through the well. The more bacteria, the less light passes through.  After the bacteria is given time to grow, data is collected. That was why I took so long to post; I was waiting for the results of this experiment before I posted this post. Unfortunately, my wait was in vain. I used too much antibiotic (about thirty times too much) and so nothing grew in those wells. I will be repeating this experiment again next week, and then trying using a different media.