Objectives: Identify a ciliate using a non-flooded plate and determine the soil texture.

Objective: Continue to look for ciliates in soil samples and determine the soil texture.

Purpose: To find and identify a certain ciliate that will presented to the class and add more metadata.

Procedure for Soil Texture:

1. Remove sticks and leaves from the soil sample.
2. Add 4 mL of soil in a Falcon tube.
3. Add 6 mL of water and mix vigorously.
4. Add 1 drop of diapering agent and re-mix.
5. Let the tube sit undisturbed till the next lap.
6. Take a picture of the tube next to a ruler and measure the % sand, silt, and clay.
7. Use the USDA triangle and soil type calculator to determine the texture of the soil.

Results for Soil Texture:

The soil was prepared and will be examined at the next lab.

Objective: Determine the soil ciliate biodiversity within the soil samples collected earlier this semester using a non-flooded plate. Also, find the % water content and pH of the wet soil.

Purpose: To find and identify a certain ciliate that will be presented to the class.

Procedure for % Water Content:

1. Add ~8 mL of deionized water to the wet soil and record the mass (water was added on Tuesday).
2. Subtract the mass of the petri dish when it was empty from both the wet and dry soil mass.
3. Use the equation below to find the % water content:

[(wet soil mass – dry soil mass)/wet soil mass] X 100=% water content

Results for % Water Content:

[(22.6g-19.7g)/22.6g] X 100=12.8%

Procedure for pH Protocol:

1. Using a micropipette remove 1 mL of the water from the dry soil and transfer it to a microfuge tube.
2. Spin the tube in the centrifuge for 1 minute.
3. Place a piece of pH paper on a clean area of the petri dish lid.
4. Transfer the liquid from the microfuge tub onto the pH paper and wait for 1 minute.
5. Use the chart and the color of the pH paper to determine the pH.

Results for pH Protocol:

The pH was 7.0

Procedure for Identifying Ciliates using a Non-Flooded Plate:

1. Using the dry soil, push the soil around the perimeter of the petri dish.
2. Use a micropipette and transfer 100 ul of the liquid in the middle of the petri dish to a clean petri dish.
3. Use a dissecting microscope and look for any ciliates.
4. If there are ciliates in the sample, transfer the 100 ul drop to a concavity slide and place a cover slide over the sample.
   • Use methyl cellulose if the ciliates are moving too quickly.
5. Using a compound microscope, record the number of ciliates observed.
6. Transfer a ciliate into 500 ul of media in a 24 well plate.
7. Put the lid on the petri dish with the soil to decrease evaporation.

Results for Identifying Ciliates using a Non-Flooded Plate:

I did not find any ciliates in this lab.

Future Steps:

I will continue to look for ciliates in my soil sample, then identify and capture one of them for my group presentation.

Purpose: To prepare the figures that will be used for the results section in the research paper. Also, to practice presentation skills for future research presentations.

Title and Authors: Describes what the research is about and who contributed to the research. Also, the title, class, and section are included. This section is important because authors should be acknowledged for their work.

Abstract: The abstract is a single paragraph composed of 200-250 words and outlines the entire research project. Some background information, the purpose, information about the general methods used, the most important results, and a conclusion or recommendation are included in the abstract.  Details from the methods section and reference citations are excluded. This section is important because it summarizes the entire research paper.

Introduction: Provides background information about the research. Also, includes why the research is important. This section is important because it contains the information that supports the hypothesis and rational for conducting the research.

Methods: This section describes the protocols done in the research. The reader should be able to recreate the experiments from the information in the methods section. It should be written in the past tense. A statistics section should be included. The methods section is important because it describes how the research was conducted and how it can be recreated.

Results: This section includes visual representations of the data (table, graphs) which are labeled and with captions that summarizes the results. Each step in the procedure that has an outcome is mentioned in the text of the results. The results section is important because it provides evidence for the discussion section.

Discussion and Conclusion: This section includes the conclusions about the significance of the results in regards to the problem that the research was concern about. Comparisons of the results to other research is included. Important questions about what should be addressed next is also included. This section is important because it decides if the research was relevant or not and what should be done with this information.

Citations and References: This section includes the sources that provided the information that was used in the research project. APA style was used to cite the sources. This section is important because it prevents plagiarism.

Future Steps: Finish the rough draft of the research paper. Then make changes to it and create the final draft.

Objectives: Create figures for the data on cell counts, optical density, and the vacuole assay.

Purpose: These figures will be used for the results section of our research paper.

Procedure:

1. Enter the mean data in separate columns.
2. Select the columns and in the Insert tab, select Recommended Charts.
3. Choose the bar graph (for vacuole assay choose line graph).
4. Click inside the chart and select Chart Design.
5. Click on Add Chart Element and select Axis Title (both vertical and horizontal).
6. Also, go to Error Bars and then more Error Bars Options.
7. Select custom on error amount and enter the standard error for both positive and negative (do not do this step for vacuole count).
8. Add a descriptive title for the chart and change the axis titles to what each element represents.

Results:

Future Steps: The results section will be finished and the figures will be presented to the class by my group.

Objective: Create descriptive analysis, histograms, F-tests, and t-tests for the cell count and vacuole assay data taken by the class.

Purpose: To create the statistical section in our research papers and see if the treatment effected the Tetrahymena.

Procedure for Descriptive Analysis:

1. Using excel, organize all of the control data in one column and all of the treatment data in another column.
   • For the vacuole count make four separate columns: 5 minutes control, 15 minutes control, 5 minutes treatment, and 15 minutes treatment.
2. On the data tab, go to data analysis tool. Select descriptive statistics.
3. Select the range of the data for the control, then select a location for the output.
4. The summary statistics should be checked and then click OK.
5. Select the range of data for the treatment and select another location for the output range, then repeat step 4.
   • For cell count you should have two descriptive analysis charts and for the vacuole assay you should have four descriptive analysis charts.

Results:

Cell Count:

Vacuole Count:

Procedure for Histogram:

1. In a column, input numbers you want for the bin numbers of the histogram.
2. Using the data analysis tool, select Histogram.
3. Select the data of the control as the input range and select the cells that contain the bin numbers for the bin range. Don’t select labels. 
4. Select a location for the output and check chart output. Click OK.
5. Repeat steps 1-4, but use the treatment data.
   • For cell count you should have two histograms and for the vacuole assay you should have four histograms.

Results:

Cell Count:

Vacuole Count:

Procedure for F-Test:

1. Using the data analysis tool, select F-Test Two Sample for Variances.
2. Input the control data for variable range 1 and treatment data for variable 2 range.
3. Select a location for the output, then click OK.
   • If the variance for variable 2 is higher than the variance for variable 1, swap the data for the variables.
4. If F>F critical one-tail, then the null hypothesis that the variances of the two samples are equal is rejected. This means that the variances of the two populations are not equal.
   • For cell count you should have one F-Test and for the vacuole assay you should have two F-Tests.

Results:

Cell Count:

• The variances are not equal.

Vacuole Count:

• Variances are equal.

• Variances are not equal.

Procedure for t-Test:

1. Using the data analysis tool, select t-Test: Two-Sample Assuming Unequal Variances if according to the F-Test the variances are not equal. Select t-Test: Two-Sample Assuming Equal Variances if according to the F-Test the variances are equal.
2. Input the control data for variable 1 range and input the treatment data for variable 2 range.
3. Select a location for the output and then click OK.
4. If the t Stat is greater than the t Critical two-tail, the null hypothesis is rejected. This means that there is a significant difference between the 2 means.
   • For cell count you should have one t-Test and for the vacuole assay you should have two t-Tests.

Results:

Cell Count:

• The two means do not have a significant difference.

Vacuole Count:

• There is a significant difference between the two means.

• There is a significant difference between the two means.

Future Steps: I will grow my experience with statistics and use this information for making the statistics section in the Methods & Materials section of my research paper.

Objective: Count the number of Tetrahymena in the treatment and control groups. Measure the OD600 of proteose-peptone-tryptone (PPT); PPT and “twine juice” together; PPT and Tetrahymena together; and PPT, Tetrahymena, and “twine juice” together. Also, record the number of vacuoles in Tetrahymena over time in both the treatment and control group.

Purpose: To get the data needed for our research paper.

• I was responsible for the cell count and vacuole assay for my group.

Procedure for Making the Sampling Groups:

1. Use the flasks with 50 ml of the treatment and control group.
2. Swirl the flask.
3. Transfer 5 ml of the treatment and the control into sterile glass tubes using a serological pipette.
4. Pipette from around the top of the culture.
5. Keep the tubes labeled and covered.
6. Place the tubes in a test tube rack.
7. Each table will use the same tubes for their assays, OD600, and cell counts.

Data for the OD600:

Procedure for Cell Count:

1. Create three drops using 2 ul of Tetrahymena and 1 ul of iodine.
2. Do this for both the treatment and control groups.
3. Record the number of cells.

Data from Cell Count: 

Procedure for Vacuole Assay:

1. Place 10 ul of Tetrahymena culture on a flat slide.
2. Add 2 ul of India Ink to the drop of cells and pipette up and down to mix the cells and the ink.
3. Place 2 ul of methyl cellulose in the center of the drop.
4. Place a cover slide on the drop and focus the slide on 100-400x magnification on a compound microscope.
5. After 5 minutes, record the number of vacuoles for 10 cells.
6. Repeat step five after 15 and 30 minutes.
7. Do this for both the control and treatment groups.

Data for Vacuole Assay:

•  I did not have enough time to record the number of vacuoles after 30 minutes for the treatment group.

Future Steps: I will be using this information for writing my methods and materials section and my introduction for my research paper.

Objective: Preforming serial dilutions and cell count, a lysosomal assay, and produce Polypropylene microplastic.

Purpose: To prepare for the research report and study Tetrahymena behavior and lysosomes.

Procedure for Producing Polypropylene Microplastic:

1. Shed/cut the polypropylene twine into small pieces with scissors.
2. Measure 0.5g of (PP) into a sterile glass jar/breaker.
3. Add 50 ml of sterile proteose-peptone-tryptone (PPT) media.
4. Boil and stir/vortex for the entire lab.
5. We will autoclave your “twine juice” and store for next week.
6. Open lab: Filter using a 5 um filter paper into a sterile 50 ml tube.
7. Aliquot so each group is using the same microplastic.

Data:

Empty jar: 125.3 g

Jar with polypropylene: 125.8 g

Procedure for Serial Dilution and Cell Count:

1. Add 20 ul of cells to 5 ul Iodine.
2. Add 3 ul drops to a slide and count the stained cells; record your average.

Data:

Average: 35,000 cells/ml

Procedure for Lysosomal Assay:

1. Place 20 ul of Tetrahymena culture on a concavity slide.
2. Add 3 ul of India Ink to the drop of cells and pipette up and down to mix the cells and the ink.
3. Quickly place a cover slide on the drop and focus the slide on 400x magnification on the compound microscope and start your stop watch.
4. Scan the slide for 10 cells and count any vacuoles. This is your Time 0.
5. After 10 minutes, count the number of vacuoles for 10 more cells.
6. Repeat the procedure for 20 minutes and 30 minutes.

Data:

My teammates  did the behavioral assays: swim speed and percent directional charge.

Future Steps: This data will be useful for constructing the research project. The micro-plastic production will be put in a microwave for an hour, then into a water bath at 55°C overnight. In open lab, it will be filtered and autoclaved.

Objectives: Practice micropipetting, learn serial dilutions and concentration calculations, and prepare our soil samples. Also, determine the research question that the class wants to address and learn experimental design methods.

Purpose: To grow our skills in micropipetting and learn techniques that will be used in designing our research paper. Also, preparing soil samples for future labs.

Procedure for Preparing Soil Samples:

1. Write your name and soil identifier on the top and bottom of a Petri dish.
2. Measure the mass of the bottom of the Petri dish and record the mass.
3. Add the soil sample and then measure the mass of the bottom of the Petri dish and record the mass again .
4. Place the top of the Petri dish on and let the soil dry out.

Procedure for Performing a 10-fold Serial Dilution:

1. Label 4 wells of the 24-well plate that are in the same vertical column: 10^-1, 10^-2, 10^-3, and 10^-4.
2. Add 900 ul of Tetrahymena culture media to the 4 wells.
3. Add 100 ul of 10E0 stock culture to the “-1” tube, mix briefly by slowly pipetting up and down. Change tips.
4. Add 100 ul of 10^-1 sample to “-2” well, mix briefly by slowly pipetting up and down. Change tips.
5. Add 100 ul of 10^-2 sample to “-3” well, mix briefly by slowly pipetting up and down. Change tips.
6. Add 100 ul of 10^-3 sample to “-4” well, mix briefly by slowly pipetting up and down. Change tips.
7. Observe the wells using a dissecting microscope and estimate which well has a “countable concentration”.
8. Record the optimal dilution for cell counting and transfer 5 ul to a clean concavity slide.
9. Use a compound microscope with the 10x objective to count the number of cells.

Data:

Future Steps: I will be using this skill for my research paper.

Procedure for the Experimental Design:

1. Discuss with lab groups about the experimental design that we would like to purpose.
2. Think about: relevance, availability, cost, and safety.
3. Come up with a question and hypothesis (make sure its falsifiable).
4. Describe the methods for “Treatments” in a way that another student could set up our experiment. This should include: concentration, timing, and methods of measuring.
5. Describe how and when we will measure the effects of the treatment.

Question:

Does microplastics effect Tetrahymenas’ lysosomes, which results in the Tetrahymenas’ death?

Hypothesis:

The increase of microplastics concentration with Tetrahymenas will decrease the number of lysosomes in Tetrahymenas, causing the Tetrahymenas’ death.

Method:

There will be three different concentrations of microplastics. The control group will contain the same number of Tetrahymenas as in the treatment groups, but with no microplastics. Each table will be given a concentration and then each student will record the number of lysosomes in each Tetrahymena in both the treatment and control group. The average of the number of lysosomes at each table will be taken. This experiment will last for 24-hours, after which the students will count the number of lysosomes and the average number of  lysosomes at each table will be taken again.

How and When:

Each well contains the same number of Tetrahymenas and the same amount of LysoBrite Blue (stain for lysosomes) . The treatment wells will vary in the amount of red polyethylene microplastic. We will measure the number lysosomes of both the control and treatment wells, before and after 24-hours.

Future Steps:

Be more detailed in our Methods section and start working on our research experiments once we find out which experiment we will be doing.

Objective: Observe Tetrahymenas, count the number of cells found, find Tetrahymena’s approximate diameter, and learn the basics of using a micropipettor.

Purpose: Introduce Tetrahymenas and know how to use a micropipettors safely and effectively.

Procedure:

1. Transfer 100 ul of Tetrahymena stock culture to a clean well using the correct micropipettor.
2. Observe the Tetrahymena in the 24-well plate using the dissecting microscope.
3. While looking through the dissecting scope, pick 5 ul of cells from the well using a P-10 micropipettor.
4. Transfer 5 ul to a concavity slide and observe on 4x and 10x using the compound scope. No 40x without a coverslip!
5. Count the number of cells found in each trail.
6. Using your FOV measurements, approximate the diameter of Tetrahymena.
7. Should have 3-4 trails done.

Data:

Trails	Number of cells in 5 ul	Approx. diameter of the cell	Sketch
1 (at 4x)	200 cells	14.3 ul
2 (at 10x)	52 cells	12.5 um

• I ran out of time to do three trials

Pictures:

At 4x

At 10x

Conclusion: I managed to complete the objectives for two out of the three trails. I learned how to use a micropipettor safely and effectively and found the diameter and number of Tetrahymenas in a sample.

Future Steps: I will use the knowledge I have learned about miropipettors in this lab for future labs. Also, I will be using Tetrahymenas for my future research paper.