Blog Post Week 11

Its close to the end of the semester so we are trying to wrap everything up. This week we just nanodropped our samples and took a trip down to the  ASU Tempe DNA Sequencing Lab. ( Lots of walking and trying not to get lost). We hope that next semester we will get our sequencing results and confirm the samples we found as positives.

Blog Post Week 10

I was not able to make it to the lab earlier this week so I do not have much to say. There is no update on the microplastics, there is no money to buy new equipment to try to make this project work. For Pseudomonas the plan is to make gels today (December 1, 2016) to use for tomorrows group meeting. We need a total of 8 gels since we are screening multiple samples. we started to make gels the day before to make it a little easier for the team, there are days were we have to screen many samples and it takes a long time to set the gels and actually run them and not everyone can stay to the end.

Blog Post Week 9

It was a slow week in the lab. For microplastics there is nothing I can really do at this point. I spent this week trying to catch up on all the work that I need for Stem and my classes; I am currently really behind in almost all of my classes. For the Pseudomonas project we ran PCR on the samples that were collected from the microbiology classes, something happened to the thermocycler where our samples were not held at the right temperature so we have to redo PCR on some of the samples. Since the campus was closed on Thursday and Friday we did not have our regular Friday meeting. The plan is to screen the samples through gel electrophoresis next week.  

Blog Post Week 8

For microplastics this week I tried the beaker method separation (figure 1) since I finally had shaved down enough plastic to attempt it. I was supposed to filter my sample once I was finished separating to see what percent of microplastic we could get back but the microbiology lab needed all the filtering equipment for the class. So I decided to leave the sample sitting and filter another day. When I came back to the lab to filter Matt had made a discovery! The sample had sat for two days. Our though was that all the plastics would float because they had a lower density than the 5M NaCl solution, but this did not happen (figure 2). Instead some of the plastics sank to the bottom. We know that the filed plastic is a microplastic so we had to find a solution with a higher density. This means that we have to find a cost effective solution. Matt suggested we attempt to make a 5M NaCl and 10% sucrose solution. This still did not have a high enough density, the highest density that we had was 1.301 but it was with a 5M NaCl and 60% sucrose solution (figure 3), which is not the best. We are once again stuck until we can find a good funnel to allow to do the separation and a solution with a greater density.

Figure 1. Beaker method Seperation 

Figure 2. Sample that was left out
Microplastic settled 

Figure 3. Attempt to make solution
with greater density

Pseudomonas Update: The microbiology labs did DNA extraction of their samples and we were in charge of nanodropping. The microbiology classes also ran a PCR and we were in charge of nanodropping that as well and screening them through gel electrophoresis.

Blog Post Week 7

This was a slow week in lab. I am behind on my background and method so I spent my time trying to catch up. I have also spent time filing down plastic. It takes me a long time to file down one gram of plastic to attempt the beaker method. Matt would like me to try the beaker method at the least five times. I have only down it once since I have been trying to file down as much plastic as possible.

Pseudomonas Update: The microbiology classes have started collecting water and biofilms sample. Our team is now in charge of using the class samples and taking the date. This week we took pictures of the plates and transferred some sample onto broth. We also began to organize a file that will include the classes data.

Blog Post Week 6

Figure 1. Beaker Method

Figure 2. Unsuccessful
Turkey Baster

We are still trying to find new a good protocol to follow for the microplastics. Since we could not get a funnel or any separation apparatus to work Matt suggested we tried the beaker method once more. This time the beaker method would be different in a way. Matt wanted me to get a known amount of plastic and mix it with our soil sample to see what percentage of the microplastics we could get back. I started with filing down plastic 

(I used a PVC Pipe as the plastic and a file to file the pipe) once I filed down enough plastic I weighed 1gram of it and 50 grams of soil and combined them together. I continued to add 200mL of NaCl solution to the sample and continue with the beaker method (figure 1). With the beaker method it states we transfer using a turkey baster but all the microplastics would get stuck on the side of the turkey baster making it unsuccessful (figure 2). I attempted to transfer the micropipette using a sterile transfer pipette but the microplastics clumped on the tip not allowing a proper transfer (figure 3). I tried with a micropipette and a regular green pump pipette and it did not work. Matt suggested that I pour the microplastics and the liquid into the other beaker (figure 4), He thought this would be the best way to transfer even though in was very inefficient. To completely transfer I had to add another 200 mL of solution to get the most microplastics, I added another 200mL and repeated my steps (total volume of 600mL of 5.0M NaCl Solution). Once I was done transferring the microplastics, I had to move on too filtering. I had concerns that our sample would not filter because of how dirty the sample was. Filtering did not go well either since the hose collapsed (figure 5). Everything that we have tried so far has not worked, we are going to keep trying. 

Pseudomonas update: We recently did gel electrophoresis and moved on to DNA extraction from the gel (figure 6).    

Figure 3. Clogged
Transfer Pipette 

                                                                                              

Figure 4. Transfer through pouring

Figure 5. Filtration/
 Collapsed hose

Figure 6. DNA extraction from gel

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.