Preparing for qPCR

Running the new protocol proved to be incredibly challenging. A constant worry during a lab procedure is that no matter how well you may have been doing for the past seven hours, if you mess up during the last few minutes, you will most likely have to repeat everything from the start. While this encourages you to be careful and take your time with a procedure, you also have to keep in mind that you can’t be in the lab forever–we do have classes and other responsibilities. ¬†With that being said, I think the first experiment I ran using this protocol went well, but I did learn a lot of new skills that I’ll need to apply for the following times I perform this experiment.

Working in a lab can be tiring. Sometimes, you feel so inadequate to be there. Who are we to know how to run these experiments? We are students who are still in the process of learning. There are many times when I’ve sat in my science classes and I think to myself “That would have been useful to know when I was conducting x or y experiment”, but the thing is: everyone feels that way. No matter how hard we work, how prepared we are, or how well our results turn out, we will always learn something new that will improve how we perform these tasks. I guess that is the essence of research.

As someone aspiring to be a physician, I know that my time in the lab may help bring a cure for an awful opportunistic disease to some unfortunate people, and even if we don’t manage to accomplish this during my time as an undergraduate student, these skills will be useful when I am conducting my own projects a physician scientist. It’s very nice to think about the great things that we can do in lab, or even the confidence that we get from performing a job well done; however, I think that nothing of that can compare to feeling one must feel when being able to provide health to those who are in the most need. I know that this is why I like going into the lab, because despite many of drawbacks that one has from sitting in front of a bench for many hours, or meticulously timing procedures and weighing materials, this type of work helps us advance as people, help create a better society, and improve the quality of life for those who are in need.

Fluorescent staining and future testing

As mentioned in my last blog, fluorescent staining can have a variety of drawbacks. One of them is the possibility to show that a drug is 100% efficient in treating a disease.

The way that MIC fluorescent testing works allows for a blank control (non-infected cells with drug component), to be compared with an experimental group (infected cells + drug component). This can be problematic since fluorescent readings could show the same values for a control and an experimental group. This comparison would show a hundred percent efficiency, which albeit intuitively, it’s also a red flag for much needed skepticism.

Dr. Yarlett mentions that a new way of analyzing our results may come along since the lab will be getting a brand new toy: a qPCR! the qPCR will be a great addition to the research since it will allow us to look for parts of the parasite that are only present in living protozoan, and it may also reduce the amount of parasite that is needed for infection.

One of the drugs that we are using shows promise. The literature published by Dr. Yarlett in the past shows that the drug we are currently testing may be a great candidate for treatment of crypto. It will be interesting to see what results we will receive once we start the new protocol.

Drug treatment for cryptosporidiosis

Following from the research that Dr. Yarlett, Mary Morada, and I performed over the summer, we continue to test different drugs for treating crypto. From this project, I expect to expand my knowledge on this terrible disease, learn new laboratory techniques, as well as hopefully be able to move forward in finding a treatment for crypto.

Our main questions rise from quantifying the effectivity of a specific drug. We are currently using fluorescent staining to perform our procedures; however, this type of technique has its limitations. Fluorescent staining requires a relatively large amount of the parasite in the cells in order to be able to quantify our results with certainty. The large amount of parasite can have adverse effects on the cell, which may affect the quality of the results. Another problem that can arise from fluorescent staining is that dead parasites may be counted as alive, affecting the quality of the results.

During the summer, we tested different ways of staining our parasite and cells, and we’ve been using a technique which helps minimize these setbacks. The amount of parasite is within a safe range, and the fluorescent antibody is selective for live parasite.