Lab #12: Ciliate Classification

11/9/18

Haiden Jordal

Objective:

The purpose of this lab was to learn and practice the ability to classify observed ciliates using morphological characters to find the correct phylum and class. We also are brainstorming the possible ways diversity comes into play and how evolution and natural selection amplifies diversity and connects all branches of life with a common ancestor.

Procedure:

Preliminary Review:

First in lab, we reviewed the different branches of life and how all life can be classified under seven supergroups: Excavata, Stramenopile, Alveolata, Rhizaria, Archaeplastida, Amoebozoa, and Opisthokonta. We looked at the different phylums under each supergroup, and then the different classes of species under the phylum ciliophora.

Soil Sedimentation Test:

1. Scoop up around 4 mL in volume of dry soil that was obtained September 20th into a falcon tube.
2. Add 6 mL of water to the soil for a total of 10 mL.
3. Add a drop of soil dispersion solution into the tube.
4. Place the tube on the vortex for a few seconds to allow the mixture to mix.
5. To allow for the different sediments to settle place the tube on the class test tube rack and wait until next week to obtain the results of the soil composition.

Ciliate Observation

1. Observe the non flooded plates under the dissecting microscope.
2. Look specifically at the soil-water interface for live ciliates.
3. If the the soil sample is too dry than it would be useful to add some additional water.
4. `Swirl the soil and water around to collect any isolated ciliates.
5. Transfer 5 ul of liquid sample to a concavity slide.
6. View the slide underneath the compound microscope with the scanning, 10x and 40x lenses.
7. Record the number and diversity of ciliates found on the slide. Take pictures and videos of the observed ciliates.
8. Attempt to either isolate or culture a ciliate by removing it from the soil contaminated drop into a drop of clean water in order to better observe its features.
9. Try to classify any observed ciliates.

Observations:

Soil Sedimentation Test:

The results for this test will be observed next week when the sediment has settled and the sizes of the sedimentation deposits are clear and measurable.

Ciliate Observation:

In comparison to our last soil ciliate observation, this week saw more ciliates. Our group was able to find multiple ciliates in each of our own slides and Madelyn was able to find a Rotifer in her sample. My first slide, I used a smaller 2.5 ul drop of water to try and limit the amount of soil particles picked up by the micro pipette. This was successful, as around 5 different ciliates were visible moving: one long and pointed on either end, the others different sizes of oval and circles. A picture of the side was not taken due to the speed of the ciliates and the camouflage of the soil particles. Before isolation of one of the ciliates could take place, I noticed that my small drop had dried up, making it nearly impossible to locate the ciliates as they had stopped moving and were either blending in with soil particles or were hidden underneath larger deposits. A second drop was procured, this time a larger 5 ul drop to prevent evaporation. The second trial only produced one evident ciliate that looked similar to the long pointed one observed on the original drop. The ciliate was still too quick and the drop still too full of soil to take a picture of the ciliate.

Storage:

The concavity slides were cleaned and stored on the counter to dry. The micro pipettes were put back on the racks and the used tips were disposed into the tip cups. Both microscopes were turned off, covered and moved back to the center of the lab table. The petri dishes of soil were covered with the lid and stored away again. The falcon tubes are placed in test tube racks to allow for settling and are left their until next lab. The bags of dry soil were collected and stored in a class container.

Conclusions:

Based on observations made both this week and last, it is evident that, even in just our small samples of soil, there are hundreds of ciliates and most of different sizes, shapes, and structures. The soil ecosystem is thriving with life all of which we are so far from understanding completely. Ciliates and other microscopic soil organisms all play a huge role in the biodiversity of the soil and can all be affected by different human caused interactions like pollution and climate change. Being able to identify and classify different ciliates allows us to understand their diversity and their relationship with the soil food web.

Future Steps:

Using a number of keys for ciliate classification, we should be able to isolate and characterize the different soil ciliates we observed in lab. We will have to isolate ciliates in clean water and slow them down with methyl cellulose to better observe them. In addition, we will continue the soil sedimentation test by measuring the amount of sand, sediment, and clay that has settled inside the falcon tube. From these measurements we will them be able to calculate the soil composition percentages.

Lab #10 Lab Report Reflection

Haiden Jordal

10/25/18

Objective: Begin the process of writing a clear, concise, and informative lab report over the experiment we set up and completed over the last few weeks in lab. Learn the different parts of a scientific report and use the data we analyzed in lab to effectively communicate our results to the rest of the science world.

Abstract:

An abstract is the first piece of writing that a reader sees when viewing your scientific paper. It should be a concise and clear summary over the rest of the paper so that the reader can identify whether or not the information within the paper is of value to their own research. It should include background information, purpose and the methods that we used to come to our concluding results. Within this information a reader can determine whether the research paper is credible and significant or not.

Introduction:

The introduction provides as a preliminary experimental design/ set up that introduces the reader to the topic being researched and the necessary background information that was read to incite inquiry. This is where the references first get used and the prior research done in the particular field of focus is given necessary credit. The intro can establish the importance of the results of the experiment and elaborate on the overall importance that can stem from the knowledge gained upon completion of the experiment.

Materials and Methods:

The materials and methods section lists and describes all of the steps taken throughout the conduction of the experiment. It is an important section because it is what determines the reproducibility of the experiment. If the materials and methods page is thorough and detailed then the reader will be able to reproduce similar results and further confirm or deny the observation gathered. It is essential to any scientific research paper as it establishes wether or not the methods used were credible enough to accept the conclusions.

Results:

The results are quite possibly the most important aspect of each scientific paper as it is what every reader and fellow scientist are reading the paper for. The results are a collection of specific tables, graphs, and statements that explain exactly what was found upon completion of the experiment. It should include the data from each individual procedure that was completed. Figures should be conjured by analyzing the data and compiling it into charts and graphs that give a clear and evident message as to what the results of the experiment say. No interpretation of the results should be explained in this section but only a relay of information.

Conclusion/Discussion:

In the discussion, the full scope of the results is interpreted and recited to convey possible meaning and underlying implications. It is an opportunity for the writer to give specific thoughts on the procedure and data and discuss possible sites of error and flaw in experimental design. This section will allow the reader to make their own conclusions from the results and lead to calls to action or reformation. The significance of the experiment and the results should be fully realized and a plan for future research can be established.

References:

The reference section allows for the writer to give credit to other pieces of research that may have assisted in the experimental process. In APA format, the references should be easy to read and helpful to readers by giving additional background and other scientific literature that is necessary to go into more depth.

Future: Take what I learned in the process of writing my scientific paper to further my skill of being able to communicate my findings to the scientific community. The skill of scientific communication, is a necessary skill to allow my research and my ideas to be heard, believed, and considered credible. Without this ability, any information obtained by experiment or other scientific analysis is practically useless as it would not be read or interpreted by anyone else.

Lab #7 Toxicity Assay; Controls and Treatment

10/04/18

Haiden Jordal

Objectives: 

Today’s objective is to finally run through our experiment. Using the results we get from this experiment we should be able to answer or scientific question and either accept or reject our original hypothesis. The question we are testing in lab is: Will the introduction of micro plastics to a solution of T. pyriformis cells have a significant effect on the ciliates’ growth or mobility? Our hypothesis inferred that it will have a significant negative effect on the Tetrahymena’s reproductivity and ability to move.

Procedures:

Sampling

1. Find the lab counterpart that performed the same behavioral assay as me in the last lab.
2. With the new partner, label two test tubes, one Treated and one Control.
3. Obtain the flasks of samples and be sure to swirl before pipetting.
4. Use the serological pipette to transfer 4 ml of the solution of tetrahymena treated with the twine juice into the tube labled “treated”. Transfer 4 ml of the solution of tetrahymena mixed with the PPT media into the tube labeled “control”.
5. Place tubes in test tube rack to carry.

Spectotrophometers

1. Measure the optical density at wavelength 600 nm of each solution of: PPT, PPT + Twine Juice, PPT + TH, and PPT + Twine Juice + TH.

Cell Counts

1. Working quickly, so as to prevent the drops from evaporating, Transfer 2 ul of each of the treated and control Tetrahymena onto a flat slide.
2. Add 1 ul of Iodine to the drops.
3. Observe the drops under the compound microscope and locate the most appropriate magnification for counting cells.
4. Take a picture of the slide to count later for accuracy.
5. Repeat the process with two more drops each of the treated and control samples.
6. Take the average of the cell counts for each sample and calculate the average concentration of TH cells/ml.
7. Use the equation (avg of cells/ 3ul) x (D.F. 10) x (1000 ul/ml) x 1.5 = concentration (cells/ml)

Behavioral Assay (swim speed)

1. Place 20 ul of each culture on a clean flat slide
2. Set the slide on top of a metric ruler over the mm side.
3. Observe the cells under a dissecting microscope.
4. Choose one specific cell and use a timer to record the time it takes the cell to move from the inside of one mark the inside of the next.
5. Write down the time and repeat the process for at least 10 cells.
6. Calculate the average time and speed using mm/s.
7. Repeat that whole process for the other drop of culture.

Alternative Assays

1. Madalyn performed the directional change assay and Kelsi performed the vacuole formation assay.

Observations:

Cell Count

Concentration of treated cells: 326,500 cells/ml

Concentration of control cells: 88,000 cells/ml

Optical Density

Behavioral Assay

Treated cells moved at a pace around .21 mm/s faster than the control cells.

Storage:

At the aseptic area, the serological pipette was left for other groups to use. The slides used for cell counts and behavioral assay were cleaned and stored on the counter to dry. The micro pipettes were put back on the racks and the used tips were disposed into the tip cups. Both microscopes were turned off, covered and moved back to the center of the lab table. The tubes of both the treated and control samples were left in their racks on the table alongside the rack containing the iodine.

Conclusion:

The results of this experiment show both an increase in cell count for the Tetrahymena in the treated culture and an increase in mobility for those same cells observed in the behavioral assay. The increase in cell concentration was quite significant going from 88,000 cells/ml in the control group to 326,500 cells/ml in the treatment group. This change suggests that the cells interacting with the micro plastics in the treatment group were able to reproduce and stay alive at a much higher rate that the control, contradicting our hypothesis. This contradiction is continued with the observation of the behavioral assay and the fact that the treated cells moved approximately .21 mm/s faster than the control cells. So not only were they more abundant in the treatment, something about the micro plastics enhanced the mobility of the Tetrahymena.

Future Steps:

In the near future, our individual results will be shared on a spreadsheet to the entire class. The observations can then be compared and a pattern or common result may be identified. Upon this realization, a better conclusion can be made as to whether or not the treatment negatively or positively affected the Tetrahymena. According to my results alone, it would appear t be positive, but that does not rule out any experimental random error. It is also important to start thinking systematically and brainstorm how our results can be used to understand further relationships that micro plastics an playa part in in the real world. If in fact the micro plastics have an immediate positive effect on Tetrahymen, the long term effects are still unknown and can be of equal if not more importance. We should be sure to look into the procedure of heating and filtering and determine if the process of obtaining the micro plastics could be flawed in the fact that it is not completely sterile.

Lab #5: Experimental Design and Serial Dilutions

09/20/18

Haiden Jordal

Objectives:

There were three parts to this lab. First the purpose of obtaining the soil samples from the soil around the trees is to later be used in research of soil ciliates. The second part, was serial dilutions. The objective of this part was to teach students to use micropipettes to dilute a solution in order to calculate a concentration of cells when there is an overwhelming amount of cells to count in an original solution. The last part in the computer lab allowed students to record their results from in the lab on a class wide spreadsheet. It was also necessary to brainstorm and identify an experimental design to go along with an experimental question and hypothesis. All of which should be testable and ready to replicate in lab.

Procedure:

-Soil Collection

1. Choose an area of soil within the rhizosphere of a bald cypress tree by the creek to collect a soil sample.
2. Bring the soil sample to lab for labeling and record the mass of the soil.
3. Place sample inside a petri dish under the vent hood for later use.

-Practicing with Pipettes

• Use water to practice picking up and dispersing different amounts with the newly introduced 1000 ul pipette.

-Serial Dilutions

1. Identify the stock solution of the 24-well plate and observe the activity of the Tetrahymena under the dissecting microscope.
2. Transfer 900 ul of media to each of four wells in a single column using the 1000 ul micropipette.
3. Transfer 100 ul of the Tetrahymena stock to the first well and label it 10^-1.
4. Mix around the solution by pipetting it up and down. Then change tips.
5. Transfer 100 ul of the solution of the first well into the second well and mix. Change tips.
6. Repeat this process for the remaining wells.
7. Determine the well that has a countable concentration and transfer 5 ul of it to a concavity slide.
8. Observe the slide under the compound microscope at 4x magnification and then at 10x and record the number of cells transferred.
9. Repeat this twice with two more wells.
10. Calculate the concentration of cells in cells/ml.
11. Formula-    cells/ml = (# of cells/ volume of drop ul)x(1000 ul/ml)x(dilution factor)
12. In the computer lab, transcribe the results from lab onto the spreadsheet provided by Dr. Adair for class data.

-Experimental Design

1. Utilize the computer lab to find research and brainstorm an experimental question.
2. Based on the question, create a falsifiable hypothesis.
3. Design the experiment, explain the methods including the set up making sure to list the control and tested variable.

Observations:

Soil:

Serial Dilutions:

Experimental Design:

• Experimental question: How will the introduction of propylene microplastics effect the population of living Tetrahymena ciliates?
• Hypothesis: The propylene will decrease the survival rate of tetrahymena in the contaminated populations.
• Experimental design: Using 24 well-plates, there will be two rows of observation: one with six control groups and the other with six treated groups. There will be media placed in the control groups to maintain the single variable of the microplastics. This is done in order to reduce error. The original population size will be recorded and then recorded again once a week for three weeks to observe the continual growth or decay of populations of all twelve wells.

Storage:

The micropipettes were put back on the rack after use while all of the pipette tips were disposed into the tip waste cups. The compound microscope was put back in the center of the lab table and covered after being set to 4x magnification and lowered to lowest stage setting. The dissecting microscope was turned off and placed back in the middle as well. The 24 well-plate and the media used in the dilution were both stored towards the side of the table, while the concavity slide was washed and placed on the counter to dry. The unused tips were put back in the lab drawer to the side, and the lab instructions went back in the drawer in the center of the table. In the computer lab the computers were properly logged off of.

Conclusion: 

In lab, I learned the valuable skill of serial diluting. It is essential in determine the concentration of microscopic cells when there are far too many cells to count in a single sample. Diluting the sample to keeps the concentration constant while the number of visible cells reduces. This makes counting them and calculating the average number of cells per much easier and accurate. In addition, we began our first steps to creating our own scientific literature by using the framework of other sources and a guide to come up with our experimental design and method. It is evident that continuing to use scientific literature in our research will be key to effectively producing our own publishable experiment. In lab today, we came up with the idea to use propylene as our microplastic toxin as it is one of the most abundant microplastics in the world.

Future Steps:

In the future with our new groups, we will be starting our development of our experiment. Further research will be needed to understand the habits of Tetraahymena over time. Also while we decided upon propylene, it could be useful to research other types of potential microplastics that can be hazardous to soil ciliates like tetrahymena and whether or not we are using the best one for observation.

09/13/18

Haiden Jordal

Objective: Students will observe tetrahymena under different magnifications and microscopes. With further observations, students will be able to learn more about the model organism and how it functions. Students will also learn how to use micropipettes and use their primary source evaluating skills to find articles relevant to the research of tetrahymena.

Procedure and Materials:

1. Practice using the micropipettes with disposable pipette tips using water and varying measurements.
2. Obtain a tetrehymena sample in the 24-well plate.
3. Observe the Tetrahymena in the well plate under the dissecting microscope.
4. While looking through the microscope, pick up 5 ul of cells using a P-10 micropipette.
5. Place the 5 ul of cells on a concavity slide and observe them under the compound microscope.
6. Approximate the diameter of a cell with the knowledge of the FOV measurements.
7. Record observations in lab notebook.
8. Utilize the computer lab to brainstorm and research articles relating to possible experiments to be conducted between tetrahymena and microplastics.

Observations:

Dissecting Microscope
Compound Microscope 40x
Compound 100x

Equipment Storage: Slides were cleaned and left to dry on the counter. Microscopes were covered after being set on lowest magnitude and stage brought up, and placed back in the middle of the lab table. The three ringed activity binder was placed back in the middle drawer. And all of the micropipette tips were disposed and the micropipettes were hung back on the rack in the center of the table.

Conclusion: Tetrahymena play an important role in biology and the study of genetics and many other cell functions. It is evident that the cells are easy to observe based on the amount of cells we could study under the microscope at one time. Also, since tetrahymena are easy to acquire, it makes replicating experiments and results quick and cost effective. With the different levels of magnification under the microscope and the FOV measurment calculated last week, I could approximate the diameter of one average tetrahymena cell to be around 30 micrometers(um).

Future Steps: With what we started in the computer lab, it will be important to study the tetrahymena more closely and accurately to be prepared to use them in our own experiments to understand the relationship between them and microplastics. Scientific literature will be a big resource to gaining the information necessary to understand their functions. In our experiment, we will be conducting trials of survival rates of tetrahymena that are exposed to soil contaminated with microplastics versus tetrahymena that are living in pure soil. The significance of this research is to find potential solutions to the looming threat of microplastic pollution to our biological ecosystem. With effective experiments we will be able to focus our area of study to wether or not microplastics will have a profound effect on soil organisms and wether or not that effect should be detrimental to the environment as a whole.