11/16/18

Trent McMillan

Lab 13: Ciliate Isolation and Soil Texture

Objective:

The objective of this lab was to further our observations of ciliates within our non-flooded plates and/or soil texture samples as well as to determine the classification of our soil texture.

Procedure:

Soil Texture-

1. Retrieve your soil texture sample from the test tube rack, make sure you are picking up the new
2. Measure the amount of clay, silt and sand in mm using a clear ruler
3. Use the texture classification chart to determine the texture of the soil

Ciliate Isolation-

1. Use a compound microscope to observe drops out of your non-flooded plate or soil texture tube for ciliates
2. When you find a ciliate, use methyl cellulose to slow it down
3. Next, take pictures and videos of the organism and if possible try to classify it.

Data:

Conclusion:

In conclusion, as we prepare our final presentations it is crucial that our groups have plenty of pictures of ciliates to try to classify and present to the class. The soil texture gives us insight into what ciliates thrive where, or what kind of soil they fit in best.

Future steps:

The semester is winding down and it is time for us to put together our final presentations. The skills of which we have collected thus far are sure to help us in whatever research lies ahead in the future and the experiments and observations that we have done/made are sure to push us towards a love for scientific discovery.

Trent McMillan

11/08/18

Lab 12: Classifying Ciliates

Objective:

The objective of this lab was to discover soil biodiversity through characterizing and categorizing ciliates from the soil samples of which we collected earlier this semester. We also learned to apply the core concepts of evolution and natural selection to the diversity of soil ciliates, and learned to perform a soil texture test as well.

Procedure:

Soil Texture Test:

1. Using a falcon tube, put 4 ml of the sample soil into it after removing the leaves and sticks.
2. Add water until it reaches the 8 ml mark on the tube.
3. Shake/vortex until the water is thoroughly mixed with the soil and then go to a lab instructor to add 1 drop of the dispersing agent.
4. But the falcon tube back on the vortex to further mix the soil, water, and dispersing agent.
5. Label the tube and store it in a tube stand until next week, when we will observe and classify the soil texture.

Ciliate Characterization:

1. Observe the non-flooded plate by looking around the edges for ciliates using a dissecting microscope.
2. Once you find a cell, collect it using a micropipettor and place it on a slide in order to observe it under a compound microscope.
3. If the cell is indeed a ciliate, use methyl-cellulose or another stain to slow the ciliate down and examine it properly.
4. Record observations with pictures or drawings.

Data:

Soil Texture-

The results from our soil texture tests will be ready next week once the mixture has had time to settle and divide.

Ciliate Characterization-

Although I did find a couple of ciliates this week, the water drop that they were in was too dirty to be able to take pictures or examine the cell effectively. Once I would stain the drop, due to it slowing the ciliate down, I could no longer find the ciliate because of all the dirt particles that were surrounding. Dr. Adair suggests that we find a way to transfer the observable ciliate into a cleaner, water only drop next time so that we don’t have to work around the drop and will therefor will be able to come up with more results. Based off of what I could see, the ciliates that I found were all average size and oval shaped. They moved quickly and tended to spin every now and then.

Conclusion:

In this lab, we conducted the soil texture test in order to see what kind of coil the ciliates of which we find are surviving in. This is a big deal in terms of ciliate discovery as we get to see the variety of ciliates within different, or the same, types of soil. The continuation of the ciliate characterization provided us more practice with the microscopic, micropipettor, and ciliate discovery skills that we have learned thus far in the semester and allowed us to apply our own thinking and knowledge of ciliates to our own observations.

Future Steps:

Next week we will continue to examine our soil samples and the ciliates that are within them, using more clean samples out of the non-flooded plate to enhance our ability to observe ciliates found appropriately. I look forward to conducting observations further and seeing what all we find in our soil samples.

Trent McMillan

11/01/18

Lab 11: Soil Ciliate Discovery

Objective:

The objective of this lab was to investigate our soil samples taken earlier this semester by: calculating the percent of water lost from the soil, finding the pH of the water that the soil was in, and observing the soil in search for ciliates within it.

Procedure:

Percent Water Lost-

1. After the soil sample was originally collected, we weighed the sample in a petri dish without the lid on it, this is our wet soil mass.
2. After being dried out for a couple of weeks, the soil was weighed in the same petri dish without the lid on, this weight is the dry soil mass.
3. We then calculate the percent water lost using the equation: (wet mass-dry mass)/(wet mass) x 100= percent water lost.
4. Record the percent water lost in your lab notebook.

pH of Soil-

1. Remove about 1 ml of liquid from the top of the soil sample in the petri dish and transfer it into a microfuge tube.
2. Spin the tube in the centrifuge for 1 minute to pellet the soil.
3. Remove a small strip of pH paper (range 5.0-9.0) from the roll and place it in the liquid-filled microfuge tube, submerging the strip in the liquid until the color of the paper changes.
4. Compare the color of the submerged strip to the pH key, determining the pH of the liquid.
5. Record the pH in your lab notebook and on the soil data sheet.

Ciliate Investigation-

1. Create a non-flooded plate using the soil sample in the petri dish (flooding the soil but not to an extent where the water is overflowing).
2. Push the soil to the side of the petri dish and observe the sample under a dissecting microscope in search for ciliates.
3. If you find a possible ciliate, use a micropipette to take up the ciliate and place it on a concave slide in order to observe it under a compound microscope.
4. Record observations and try to identify the unknown ciliates.

Results:

Percent Water-

Wet Mass- 38.0g

Dry Mass- 35.2g

Percent Water Lost= 7.368%

Soil pH-

pH=7

Ciliate Investigation:

Although my partner was able to fine one, I was unable to find a possible ciliate within my soil sample. A cause of this may have been that since the sample wasn’t re-hydrated until the day before, the ciliates may have still been inside their cristae (which allows the ciliates to survive without water present). I will continue to observe the sample, hoping that I can find a few ciliates to investigate.

Conclusion:

As we enter the third part of this semesters CILI-CURE lab, we are now given the opportunity to investigate ciliates and work on making connections in order to find out what kind of ciliates thrive in our own natural surroundings. This lab, with the finding of pH, percent water lost and actual ciliates, is an addition to what we have been working towards this whole semester.

Future Steps:

In the future, we will use the procedures that we learned in this lab as we dive deeper into investigating ciliates and investigate other organisms in future labs.

10/25/18

Trent McMillan

Lab 10: Lab Report Reflection

Objective:

The objective of this lab was to help us in writing our rough draft of our Lab Report and make changes in our final figures that will be used in the rough draft.

Abstract:

The abstract should contain a brief outline of the entire article along with some background, purpose,  supplemental information, and a conclusion or recommendation. The abstract should be no longer than 250 words and should not include details of the methods. The abstract is designed this way to inform a reader of what the report is about and whether it pertains to the information they are searching for or not.

Introduction:

The introduction should give the background of the experiment and all the information needed in order for a reader to understand why the experiment is important. This allows readers to collect before continuing on with the report enough information so that they understand the reasoning behind the methods and results of the experiment.

Materials and Methods:

The methods section should explain everything you did during the experiment in a concise manner. It should be very detailed so that readers are able to replicate the experiment step for step accurately without causing a problem in their replication.

Results:

Each step that has an outcome in the methods section should have a result along with representing figures in this section. This gives readers the ability to see what you got out of your experiment and possibly see where they can go from in making their own experiment to continue the research of your subject. Basically, this allows the scientific community an opportunity to further our understanding of the experimental question.

Discussion:

The discussion should be the conclusions of which you take from your results. Asking important questions that recommend what should be addressed now that we know certain information plays a major role in this section by allowing readers to attack a new angle or go deeper into an angle of which you have already taken. Without the discussion section there would be no communication between the author and reader about where we think is the correct direction to go in terms of new experiments.

Citations:

Just like any other paper, any time you use someone else’s work you must give them credit via references. Plagiarism is stealing someone else’s work and is never accepted within the scientific community.

Future Steps:

In the future, I will use what I have learned about properly writing a lab report as I continue to perform research (hopefully on a bigger stage someday) and conduct scientific writing.

Trent McMillan

10/18/2018

Lab 9: Results and Data

Objective:

The objective of this lab was to work more with Excel and create more figures in order to help us with our coming research paper and presentation. With our groups, we made our own charts, tables, etc., that will be used as we continue to learn about and execute our own scientific writing.

Procedure:

1. Open the class’ data Excel sheet, which contains both control and treatment data for cell counts, optical density and individual assays.
2. Copy the data into a new Excel sheet in order to create your own figures after finding the averages and descriptive statistics for each set of data.
3.  Make sure to add figure titles, axis labels, standard error bars, and asterisks to significant data for each figure.
4. Do this for Cell Counts, Optical Density, and whichever assay you performed individually.
5. Save each figure as a JPEG so that you can upload it and use it in your paper/online notebook.

Data:

Storage:

Since we were in the computer lab, all figures and data were stored on our computers.

Conclusion:

This lab taught me a lot about the advantages of using Excel to create figures for any type of data and our research data more specifically. The ability to use Excel effectively will play a major role in the writing of our research papers as well as our final presentations. In addition to Excel, learning more about how to properly write scientific literature and results sections will increase the professionalism within our papers and lead us on the correct path towards writing our first very own properly constructed research paper.

Future Steps:

As the semester is getting closer to an end, we are putting all the research and data that we have collected in use towards writing our research papers and constructing our final presentations. What we have learned thus far will go on to help us after this semester, though, as we continue our paths of science and scientific discovery. Our new-found knowledge of scientific literature and skills within programs like Excel will continue to help us throughout our scientific careers.

Trent McMillan

Lab 8: Data Analysis

10/11/18

Objective:

The objective of this lab was to further our skills in using Excel for descriptive statistics, calculations for large quantities of data, and to use Word to present and describe our procedure/data.

Procedure:

1. Open a new Excel workbook and insert the data from our control and treatment cell counts; also insert data from the control and treatment directional change assays.
2. Place data into separate columns based on their categories.
3. Perform the descriptive statistics function on the data and insert the chart in the spreadsheet.
4. Create a histogram for the data and insert it into the spreadsheet.
5. Perform an F-test using the results from the descriptive analysis.
6. Perform a T-test for Unequal Variances using the results from the descriptive analysis.

Data:

Descriptive Analysis:

Histogram:

F-Test:

T-Test:

Conclusion:

Lab 8 enhanced our skills within Excel that will be critical to know as we continue with our experiments and research. Knowing how to use the different functions, table and graphs will be crucial to correctly organizing and observing our data as we continue to learn more and more about the effect of polypropylene microplastics on tetrahymena.

Future Steps:

In the future, we will continue to properly analyze the data that we collect using these functions to be as precise as possible in order to create the most accurate and replicable experiments that we can. Knowing how to use and observe these different functions will help us is many ways and even further in our scientific careers than this lab alone.

10/04/18

Trent McMillan

Lab 7: Materials and Methods-Performing the Experiment-10/04/18

Objective:

The objectives of this lab were: to introduce us to the writing of the materials and methods section of our scientific research paper, apply the skills we learned last week to our own hands-on experiment with the treatment solution to test the effects of polypropylene on Tetrahymena using behavioral assay’s, learn how to use serological pipettors with the bulb, plastic pump, and electronic pump, and to strengthen our dilution/standard deviation skills within the lab.

Procedure:

Absorbance-

1. Use the spectrometer to measure the optical density(OD) at wavelength 600nm of each solution.

Solutions-

PPT (clean media), PPT + “twine juice” treated media (clean treated media), PPT + TH (media + tetrahymena), PPT + TJ + TH (treatment + tetrahymena)

Sampling-

-Flask of 50 ml of culture

1. Transfer 5 ml of treatment and control into sterile glass tubes using a serological pipette.
2. Swirl flask before pipetting.
3. Pipette from top of culture

Cell Count Measurements-

1. Everyone count three drops (control & treatment) using 2 μl of Tetrahymena and 1 μl of Iodine; take a picture of each and count later for accuracy.
2. Work quickly so the drops don’t evaporate.

Direction Change Assay-

(One each for control and treatment solutions)

1. Two per table
2. 20 μl drop of either the control or treatment.
3. Record the # of direction changes in 10 seconds 10 times (use a different tetrahymena each time)
4. Record the data average as : [# of changes-time spent spinning]

Data:

Cell Counts of 2μl Tetrahymena Drops-

Control: Average- 17 cells

Treatment: Average- 70.66 cells

Spectrophotometers-

-Optical Density @ 600

Directional Change Assay:

Control- Average(changes): 4, Average(time spiraling): 4.6

Treatment-Average(changes): 3.5, Average(spiraling): 4.75sec

Conclusion:

In conclusion, the lab served as, at least for me, a confidence-boosting lab. I say that because we are getting the opportunity to put to use the skills and information that we have been learning throughout the semester. Like the article that we read in the pre-lab says, science can be discouraging at times, making you feel “stupid” because you don’t know the answer. Labs like this, where we get to use our new-found skills and observations to come up with our own results brings the enjoyment and thrill of scientific research back into the “classroom”. Labs like this are what we look forward to when putting in the work to prepare for it early on, and actually conducting it just reassures students like us that the not-so-fun preparation is well worth it.

Future Steps:

We will use the information collected in this lab to continue our research experiments and include in our Scientific Research Paper. Being that the whole class essentially is doing the same experiment, our future results should be fairly accurate considering the repetition of it throughout the class. Also, by learning more about, and by writing our own Materials and Methods section for our research paper, I now have a further understanding of what a Scientific Research Paper requires.  I am excited to continue learning and putting new skills to work in the lab as well as writing my own research paper based on my own research!

09/27/18

Trent McMillan

Lab 6: Experimental Design and Preparation

Objective:

The objectives of this lab were to: create “twine juice”- a polypropylene solution that we will use for our upcoming experiments in researching the effect of microplastics on Tetrahymena, determine ciliate concentration using  a shortcut dilution method, and to develop an understanding of and carry out a specific Behavioral Assay-which lets us visually interpret how cells are behaving.

Procedure:

PP “Twine Juice” Production-

1. Weigh out 0.5 g of polypropylene twine using an aluminum foil tray, zero out the tray and weigh the twine on the scale.
2. Cut the twine into as small of pieces as possible, and put the pieces into a sterile jar.
3. Add 50 ml of sterile PPT media using a graduated cylinder.
4. Boil and stir/vortex for entire lab.
5. Autoclave “twine juice” and store for next week.

Serial Dilutions and Cell Counts-

1. Using a micropipettor, transfer 20 μl of Tetrahymena stock solution to a petri dish and add 5 μl of iodine.
2. Observe under a compound microscope using the 4x magnification lens.
3. Record the number of tetrahymena in the in the sample.
4. Repeat steps 1-3 two more times and then calculate the average cells/ml.

Direction Change Assay-

1. Using a micropipettor, transfer 18 μl of media to a concave or flat slide, then add 2 μl of the tetrahymena stock solution and mix with the micropipettor to create a 1:10 dilution.
2. Using the black light-covering plate for the bottom light, place the slide on the black plate and observe the dilution through a dissecting microscope.
3. Watch a specific ciliate for 10 seconds and keep track of how many times it changes direction during the time.
4. Repeat step 3 ten times, examining a separate ciliate each time around.
5. Calculate the average amount of direction changes.

Data:

Serial Dilution Cell Counts:

Direction Change Assay-

Storage:

PP Microplastic Production-

We gave the “twine juice” to our instructor after producing it. The instructors will autoclave the twine juice and store it properly for our next lab.

Serial Dilution and Cell Counts-

The solutions of which we examined were left in the tube racks for the next class to use, we turned off and put up all of the microscopes and micropipettors properly. We then took all of the slides and petri dishes that were used and cleaned them with bleach water and left them to dry.

Direction Assay:

The storage for this procedure was the same as the Serial Dilution and Cell Counts procedure.

Conclusion:

In conclusion, creating the “twine juice” was an important step in kicking off our research experiments. The twine juice will allow us to properly experiment the effect of microplastics on Tetrahymena since it is a controlled substance that everyone in the class will be using, this will also enhance the results of different experiments throughout the class as we will all be using the same microplastic. The serial dilutions and cell counts will be very helpful in coming labs as we experiment on Tetrahymena, this procedure enhanced our knowledge toward conducting experiments of this sort and made us more familiar with the procedure. The directional assay was also a great way to practice and get familiar with procedures we will be using while conducting our research experiments. Knowing how to examine the behaviors of our cells before and after they are effected by microplastics will play a huge role in our experiments success.

Future steps:

In future labs, we will use all of these procedures to benefit our upcoming experiment.  The twine juice will used to introduce our Tetrahymena to microplastics and the other procedures will enhance our results. I am looking forward to putting these new-found skills to use as we conduct our research, knowing that they will benefit both my knowledge of scientific research as well as our experiment.

09/20/28

Trent McMillan

Lab 5: Experimental Design and Serial Dilutions:

Objective:

There were three different objectives pertaining to the lab this week. The first being the collection of soil from around trees along the creek near the BSB. We collected this soil to use in later labs as we conduct experiments and research on the ciliates that live right here on campus. The second objective was to learn about and perform serial dilutions: using micropipettors to dilute a solution in order to find the concentration of cells within a solution and break down the overwhelming amount of cells in the original solution to make it easier to observe a single cell. Performing serial dilutions is an important skill that will be used heavily as we experiment our ciliates as well as in labs to come. The last of the objectives for this lab was experimental design, where we, in our new research groups, got together and set up our procedure and basis for the research and experiment we will be conducting on ciliates. This pertains to making a falsifiable hypothesis that can be tested and replicated by other scientists. Experimental design is important throughout all of science, as it is the first step to every experiment (and eventually solution) of any scientific question.

Procedure:

-Soil Collection

1. Go to your section’s designated area along the creek near the BSB and collect soil (half of a sandwich bag) from the rhizosphere of a Bald Cypress tree.
2. Bring the soil to the lab to label, weigh, and store your sample in the fume hood for use in a later lab.

-Serial Dilutions

1. Observe the characteristics and activity of the given Tetrahymena solution in a 24-well plate through a dissecting microscope.
2. Using the 1000 μl micropipette, put 900 μl of media in each of 4 wells in the same 24-well plate as the given solution.
3. Label each well, starting with 10^-1, ending with 10^-4, putting 100 μl of the original solution directly into the 10^-1 dilute well first.
4. Mix the solution around in the well, then take 100 μl from the first dilution and put it into the second, then from second to third, then from the third to fourth.
5. Decide which diluent has the most countable cell concentration and transfer 5 μl of it onto a concave slide.
6. Observe the slide through a 4x and 10x magnification compound microscope and record the number of cells transferred in the diluted  solution.
7. Calculate the concentration of cells in cells/ml: cells/ml=(#of cells/ volume of drop μl)x(1000 μl/ml)x(dilution factor)

-Experimental design

1. Go to the computer lab and get in your new research groups, look for other experiments/research that can help you come up with a hypothesis and experiment of your own.
2. Based on the question your group comes up with, make a falsifiable hypothesis (that can be tested and proven wrong or right).
3. Design the experiment of which your group will be conducting, include your control and testing variables.

Data:

-Soil

-Serial Dilutions

Due to time being short, I only got the chance to observe the 10^-2 dilution through the compound microscope. I chose this dilutions because it was the most observable after looking at all of them through the dissecting microscope.

-Experimental Design:

• Experimental question: my group and I came up with the question: How do microplastics effect the metabolism/function-ability of Tetrahymena?
• Hypothesis: When introduced to microplastics, the rate of growth/metabolism in Tetrahymena is slowed or stunted, causing the death or lack of production of  Tetrahymena.
• Experimental Design: Using a 24-well plate, we divide the plate into three sections of 8. In the first section, the control group, we will put only Tetrahymena and food for them to live on. The second section, the first testing variable, we will put colored microplastics and Tetrahymena without food for 24 hours to observe how the Tetrahymena reacts/intakes the microplastics. And in the third section, we will transfer the food deprived, microplastic effected Tetrahymena from the second section into it with food for them to eat. We will then be able to observe how the Tetrahymena eat and digest the food with microplastics in their system. We will record the development of each section weekly for as long as time permits to see if our hypothesis is correct.

Storage:

We stored our soil samples under a fume hood, we will leave them there to dry out and cyst until we re-hydrate them for our experiment. All of the pipette tips were disposed of properly in the trash and the micropipettes were put back onto their holders. We put the covers back on our dissecting and compound microscopes and put them back into the center of the table where they belong, and we cleaned off the slides of which we used to observe our dilutions. We put the 24-well plate to the side of the table for our instructors to depose of/store properly. We put the unused tools (such as pipette tips) back where they belong in the side drawer and we made sure our table was clear and clean. Once finished in the computer lab we logged off of the computer properly and made sure to pick up all of our materials that we had brought in there with us.

Conclusion:

In conclusion, we finally got to start on our way to creating our own experiment and conducting our own research just like real scientists do everyday. We are building a great basis of skills (such as serial dilutions, micropipetting, etc.) that will help us with these experiments as well as in the future, skills of which are a necessity to conducting scientific research. Collecting our soil and observing it is just the start to our researching lives that will be full of ciliates that we are beginning to research now. Learning about experimental design and getting to design our own experiment has been a fun, motivating, aspect of this lab so far, and I can’t wait to see the results we each get out of our personal experiments.

Future steps:

In the future, with our research groups, we will get to conduct our own research with our own experiments, which is something many of us have been dreaming about doing for a while now. Although we aren’t researching something like cancer itself, we are getting to research an aspect of science that could lead to discoveries of solutions for many world problems like cancer.  We are literally going to the root of all living cells, and experimenting how some of today’s decisions will impact the world that future generations will grow up in. We may only be doing a small part, but we are getting the opportunity to contribute to scientific studies that could impact the whole world. Once we get this experiment going, we will continue to rebuild and re-test our hypothesis, either proving our hypothesis to be false or correct.

Objective:

The objective of Lab 4: Meeting Tetrahymena was to gain familiarity with micropipetting techniques and Tetrahymenas. Also, we worked on accessing and researching primary scientific literature in order to investigate microplatics and their effects on soil ciliates such as Tetrahymenas.

Procedure:

1. Make sure the table is clear and clean.
2. Place 24 well plate given by the instructors containing a Tetrahymena solution under the dissecting microscope and fine the Tetrahymenas.
3. Put a sterile tip onto the end of a 5 μl micropipette.
4. Take 5 μl out of the Tetrahymena solution and put it on a concave slide.
5. Put the concave slide under a compound microscope then observe and record observations/data.
6. Clean area and put up equipment properly.
7. Once the lab is clean, go to the computer lab, and in groups of three search through primary scientific literature to find ideas for possible research ideas/experiments.

Results at 10x Magnification:

Sketch of Tetrahymena: 

Conclusion:

In lab 4: Meeting Tetrahymena, we practiced micropipetting (Tetrahymena) solutions, first onto a dish and then onto concave slides. This will be helpful in future labs as we conduct research and experiment ciliates in tiny proportions. We also worked with dissecting and compound microscopes while observing the Tetrahymenas, observing that they are small ciliates that move in quick, spiraling motions while improving our microscopic skills at the same time. At the end of the lab when we were searching for primary scientific literature to help us with our research, we discovered and read over several articles that can potentially help with the research/research experiments we will be conduction later on. The ability to properly find a relevant, trustworthy source will improve our researching knowledge and results as we further investigate ciliates throughout the remainder of the lab.

Future goals:

In the future, I would like to further my knowledge on, and research, the effect of microplastics in our soil, the ciliates inside it, and more specifically Tetrahymenas. I believe that microplastics are going to be a huge focus for science in our generation and I look forward to doing as much research on them, and their effects on our life, as possible.