9/20/2018

By: Marci Jordan

Bio Lab 1105-31

Pre-Lab and Lecture

Learning Theory: Understanding Deeply

As we learned before a fixed mindset means we cant change. The brain is able to do much more than we ask, therefore the brain can grow. Understanding deeply means asking a lot of questions to better understand the subject. Through questions you re-emphasize what you know and clarify things you don’t. After completing CILI-CURE, we will recognize and experience the diversity of life, process of experimental design, connections between living things, cell structure and function, microscopy, and the joy of learning and discovery. Through the process of completing the units, we must ask questions to deeply understand the purpose of why we do this to apply it in our scientific future. To understand deeply I’ve been asking questions like “How does this equation apply?” and “Specifically what are ciliates?” “Why is there such a wide variety of types of ciliates?”

Scientific Method and Experimental Design:

The process of the scientific method include asking a question -> Research -> Forming a hypothesis -> Designing an experiment -> recording data -> and coming to a conclusion, then repeat. It is necessary that the research question that is posted is testable. For this experiment, we are designing in very classical scientific method to form an experiment that has a single variable and control group.Epistemology is the branch of philosophy concerned with theory of knowledge. Epistemology studies nature of knowledge, justification, and rationality of belief. With our experiment with Tetrahymena and microplastics, how does this affect the overall soil environment? How long has it been a problem? When did microplastics peak to where it became a negative impact?

Deductive and Inductive Reasoning

Scientific research is described as “The process of determining some property y about something, to a degree of accuracy sufficient for another person to confirm this property y” (Glass). Experiments can use both types of reasoning; experiments are designed and performed using deductive but applied using inductive. Inductive reasoning involves evolving from a series of specific cases into a overall general statement or prediction. Therefore, the conclusion in inductive arguments is never guaranteed because the experiment is yet to be performed. In our course we want to design an experiment with some kind of statistical probability for the effect of x on y. Literature will be used to help make a good design.

Hypothesis: Falsification Framework and Experimental Design: 24 well plate

Philosophy of sciences varies depending on area of study and researcher perspective. Not all science is hypothesis driven, but for our experiment it’s okay to try to falsify a hypothesis based on results. The purpose of a 24 well plate is to test the effect of substance x against Tetrahymena growth rate in a culture. As seen later in this lab report after performing serial dilutions we went to the computer lab to complete experimental designs with our lab group. My group already had an idea to it was just a mater of getting it on paper. Our detailed experiment idea is listed below but I had a few questions when thinking about the 24 well plate. When forming our experiment I didn’t know what measurements to use. How much Tetreahymena should I put in each well? How much mncroplastics should I put in each well? How will I make the mass of microplastics precise?

BioSkills: Serial Dilutions and Calculating the Concentration of Cells in a Culture

To count the number of cells in a culture, we learned to preform a dilution. A dilution involves using micropipettors and a stock culture. When reading the pre-lab I was very confused as there were detailed steps about how to dilute but I was unfamiliar with the process. Once in class we performed a skill performance task of diluting our own stock solutions in a 24 well plate . You start with one well with the full amount of stock solution given. Then you take a smaller portion of that mixture and mix it with the next well. Then you apply the same process going down the chain. I wonder if this is the only order to dilute a solution? Are there other method? Once we begin our experiment will i need to dilute anything? The normal format for recording the concentration of a culture is in cell/mL. No one wants to count the entire mL or even a small dense volume as there could be a high concentration of cells. So instead we count a small volume of a diluted sample and compute the number of cells/ml.

Soil Collection

This is where we merge pre-lab to in class lecture. Prior to the pre-lab we were instructed that this week we would need to bring soil samples into lab. During pre-lab were we instructed why we need the sample, how and where to get the sample, the location of sample gathering based on lab section, specifically where on the tree to gather the sample, instruction to upload pictures and more information about the soil gathering on another website, and how to properly label our samples. Then in class the first thing we did was soil collections. We labeled the top and bottom of a petri dish with our full name and soil identifier. Next we measured the mass in grams of the empty dish (bottom only) and then the bottom dish plus enough soil to cover the bottom. My data is shown below. Then we covered it and stored them in the fume hood. They will stay here for about a week to dry them out so the Tetrahymena form spores.

Purpose

The purpose of this lab was to get us familiar with serial dilutions. As I mentioned earlier reading about serial dilutions on the pre-lab was confusing so having hands on experience would clear up a lot of questions. We also gained more experience properly using I micropipettor which is very valuable knowledge for when working in a lab in the future. Lastly, to construct our experiment for the rest of the semester. Now we have a solid question that we can apply and research.

Objective

The objective is to successfully deeply understand how to dilute a solution. To understand what is happening each step and correctly calculate the cell concentration of the sample. Also, to properly use micropiprttors of various volumes, with different tips. Lastly, to form a proper experimental design dealing with Tetrahymena and microplastic pollution. It must be able to falsify and very detail that it is clear what is the question and can anyone replicate the experiment.

Hypothesis

I believe that students will mess up diluting the solutions are many wont read all the way through first. However, I think everyone will perfect and master using the pipettors correctly. I believe forming proper experimental designs will be hard as some groups were changed and I know some individuals already had ideas that there group might not want to do. Also, it is hard to find information or research on Tetrahymena and microplastics so difficulty of research success will impact forming a experimental design.

Procedures

In Lab

Goal: To determine the concentration of Tetrahymena in the “stock” solution.

1. Begin by observing the Tetrahymena in the “stock” well of your 24-well plate using the discecting microscope. This is your undiluted sample or 10^0
2. Record your observation in lab notebook: living, dead, concentrated….

Performing a 10-fold series dilution:

1. Label 4 wells of the 24 well plate: 10^-1, 10^-2, 10^-3, 10^-4
2. Add 900 µl of Tetrahymena culture media to the 4 wells
3. Add 100 µl of 10^0(undiluted) stock culture to the “-1” tube, mix briefly by slowly pipetting up and down. Change tips

When pipetting from the Tetrahymena stock cultures, avoid the bottom of the well where dead cells and debris have accumulated. Living Tetrahymena cells are located near the surface. Develop a consistent method for removing cells from the same vertical level. You may pipette while observing your cultures under the dissecting microscope while learning this technique. Learning to be consistent reduces sampling error.

1. Add 100 µl of 10^-1 sample to “-2” well, mix briefly by slowly pipetting up and down. Change tips.
2. Add 100 µl of 10^-2 sample to “-3” well, mix briefly by slowly pipetting up and down. Change tips.
3. Add 100 µl of 10^-3 sample to “-4” well, mix briefly by slowly pipetting up and down. Change tips.

In Computer Lab

Get together with group and create a experimental design with Tetrahymena and microplastic pollution. Use the QTM as an outline to aid in this process. The goals of today is to determine the research question that we want to address and create experimental design/method. Meet with members of your assigned team and discuss the experimental design that you would like to propose.

Data

In lab

In Computer Lab

My group decided to see how microplastics affect Tetrahymena’s behavior. A more detailed methods description is on the QTM and not recorded in my lab notebook. We will be watching 4 different enivorments within a 24 well plate. Group 1 will be the control, group 2 will have .005g of microplastic pollution, group 3 will have .1g of microplastic pollution, and group 4 will have .15g of microplastic pollution. All wells will have 1000µl  of Tetrahymena extracted from our soil samples. We will observe them for 1 week and see how the different various concentrations of microplastic pollution affects them compared to the control group (group 1) with no microplastic pollution.

What Went Wrong

In the in lab procedures above I only listed the procedures that we did. Felicia announced at the beginning that we would not be doing it all as we needed to go to the computer lab. After all we just needed to learn about dilution so it is the only part we did. The above is not the accurate data that I got from what I saw in my microscope in the lab. The whole class was having problems seeing the Tetrahymena at different dilutions; dilution D1 being the hardest. Although I couldn’t see ciliates I still learned about dilutions which was the goal. I understood that as you went down the chain the solution became more diluted which means less concentration of cells which means less cells. Therefore, I inferred my own numbers per well and had them decrease as the dilution increased. I showed Felicia my numbers and she said they were acceptable. I used the numbers I created and used them to work through the equations to find the columns in the table. Therefore, I understand what effects dilutions have on the concentration of cells and how to use the equations to find various methods of measurements.

Conclusion

After lab today I learned a lot of valuable information on a wide variety.I now deeply understand what serial dilution is and how it is performed. I consider myself a professional at micropiettes and feel more confident in a lab. I’m very excited that my group has our experimental design set up and Felicia said it was great and will be a good idea to test. I’m very excited for the rest of the semester as I can now apply what we have been learning in class.

Next Step

My next step in this process is all about application. The past weeks have been learning about skills needed for our research project. But now the time has come and I cant wait to get my hands dirty. My own first lab experiment!! Skills such as dissecting microscopes, compound microscopes, micropipettes, making wet mount slides, staining, and now series dilutions are coming together to see the effect that microplastic pollution has on Tetrehymena, the best model organism.