Blog Post Week 5

Figure 2. Funnel Separation Apparatus

Figure 1. Funnel Separation Apparatus

My micro plastics project is still in the very early stages. We continue to have problems with the actual separation of the microplastics from the soil. We have tried a different technique using a funnel, tube, and a binder clip to separate the micro plastics from the sediment. It does not work either because it still gets clogged. I have attempted to use different size funnels and different tubes without any success. Matt suggested that we attempt to do the separation in a beaker. For the beaker method I was supposed to add our 50 grams of soil sample and 200mL of NaCl solution stir and let it to settle. Once the sediment had settled I was to use a turkey baster to transfer all the liquid from that beaker into a clean one (figure 3) and only transferring a small amount of the soil into the other beaker. This approach did not work well either when I was transferring floating objects would get stuck to the sides of the beaker (figure 4). The floating parts are what I want. The idea was to attempt the beaker method and continue using a separation apparatus. It did not go this way. It did not work. I have to continue trying to see what method will work. At one point the funnel with the tube setup might work, I think it will give it another try. I have to find a better clamp. The clamp I used was very good for holding everything inside the funnel but it is kind of difficult to but it back on quickly. The soil seemed to be going through slowly but it sped up very quickly and I could not stop it in time due to the clamp. I am starting to run out of idea, I am doing more research to find a better protocol.                         

Figure 3. Start of Beaker Method

Figure 4. End of Beaker method, Failed 

    

Pseudomonas Update: This week the plan is to run gels for our positive controls. We ran PCR on our positive controls last Friday. We will screen the positive control and choose the best ones to create more positive controls. We plan to also do DNA extraction from the gels as well.

Blog Post Week 4

Figure 1. Sodium Chloride Solution in the making

This week for the microplastics I started with making a five molarity sodium solutions that is necessary for the separation process. This took a very long time. I started with adding the appropriate amount of salt need to have three moles in a liter, once that was dissolved I would continue adding one mole at a time. It took a really long time to dissolve the fifth mole of salt. To help dissolve the salt I used heat. I came in on Monday to actually try to do the separation. This was fail, I started with weighing out 50grams of soil that had been previously sieved by Matt. Once I weighed out my sample I added 50 grams into a 500mL separation apparatus and 50 grams into a 1000mL separation apparatus. I then continued to add 200mL of the sodium chloride solution to each of the apparatus. Very carefully I took each apparatus and shook them to mix the soil and the solution. Once the mixture settled I attempted to let the soil run through the apparatus to only keep the microplastics which would be floating. This process did not work, the apparatus got clogged so nothing went through. I got that cleaned and I moved on to do hydrogen peroxide digestion that Matt had. The sample was supposed to stop bubbling and we were supposed to dry it. It did not go this way; the sample would not stop bubbling. We are going to attempt to use a different percentage of hydrogen peroxide. Another thing I did was go out and collect three soil sample, these sample had to be dried before I could do anything with them. When I came back the samples were dried so I continued to pulverize the samples. Once the samples were pulverized I continued to weigh out 100 grams and pass the samples through the sieve. I also tried to do the hydrogen peroxide digestion with these samples, they continued to bubble. This stage of the project is really trying just to get everything working. For the Legionella project we ran PCR on Pseudomonas aeruginosa DNA to create more positive controls for ourselves and the microbiology labs.  

Figure 2. Clogged seperation apparatus 

Figure 3. Hydrogen Peroxide Digestion

Blog Post Week 3

Sample gel positive on far right
(lane 8)

 So far for the pseudomonas project we have done gel electrophoresis (This is where we check for banding of our PCR product that tells us if we have positives if we have banding or negatives which shows no banding). We ran our gels and there was some banding but there were troubles with the PCR, while preparing some samples did not contain the full 25 microliter needed for the PCR. We came in again to run PCR on all of the samples. The whole preparation for PCR went much smoother this time around, the appropriate amount of DNA, PCR water, and forward/reverse primers were added. All that’s left to do for those samples is to do gel electrophoresis. If we have positives we hope to do DNA gel extraction to send our samples to DNA sequencing at Arizona State University. This week I have also began to look at protocols for separating microplastics from soil samples. This is still in the early stages; we are still trying to see which protocol is the best to use. This is really new to me but I am excited to begin learning something new. I hope to learn new skills with this project. I will still be working with Legionella and Pseudomonas in addition to the new project.

PCR preparation

Microplastics Protocol

Gel electrophoresis

Blog Post Week 2

This week for the pseudomonas project we will be doing gel electrophoresis on our samples. We have done water and biofilm collection. We moved on to do filtration, using a filtration apparatus we trapped any bacteria onto a filter. That filter was then transferred onto a plate to allow for us to culture the bacteria. The growth was then transferred into broth to allow to proceed into DNA extraction. For the DNA extraction we follow a protocol. I found this protocol interesting because we can see the chromosomal DNA threads being formed. We moved on to Nano drop to see the DNA concentration. Once we finished Nano dropping we moved on to PCR. What PCR does is amplifies our DNA using primers that are specific to the pseudomonas we are looking for. Next week we hope to have our gel electrophoresis results and maybe move on to DNA sequencing.

Blog Post Week 1

This week was spent working on my powerpoint presentation for the National Role Models Conference in Washington DC. The conference allowed us to go an present our undergraduate research to fellow undergraduate researchers and judges. I had to give an oral presentation which was completely nerve wrecking for me. I have always struggled with oral presentations but the only way that I will get better is by actually going to these conferences and presenting. My presentation was on our findings of Legionella pneumophila. Even though I did not win an actual prize, I won a great experience, the chance to network, and the chance to see other great undergraduate work. At the actual conference I had a chance to attend other undergraduate presentations, which were really interesting. Some of the presentation I got to attend were on lipedema, which is a fat loss resistant condition that affects women. There was also session on statistical methodologies that is being applied to Dravet syndrome dataset that I had the chance to attend. The presentations were divided into three categories. The categories were Biology and Chemistry, math and engineering, and public health and diseases. My presentation was placed in health and infectious diseases. The conference had panels where they spoke of the importance to apply for internships, and to find good role models to further our STEM careers. They gave a lot of helpful information into how to start your own independent research projects. Being able to attend this conference was great, it really motivated to keep trying in my career.
**I have attached a picture of the Lincoln Memorial in Washington. There was so much algae growth in the water! (science related). It is kind of hard to tell in the picture, but it was there. All I wanted to do was collect water and swab for biofilms to test what I could find in it, sadly I could not which was very disappointing. There could have been Legionella or other Pseudomonas present in that water!