Mackenzie Singer

1/24/19

Purpose:

The purpose of this lab was to present information on metabarcoding to the class because we will be using the procedure throughout the semester. We also created non-flooded plates from the soil we collected that we can use later in the lab and tested the pH levels of our non-flooded plates.

Procedure:

Soil Collection:

1. Designate a tree on campus to collect soil from.
2. Take pictures of the full tree, the leaves, and the collection site.
3. Use a spoon and dig about 1-2 inches deep into the soil and place it in a plastic bag.
4. Fill out the data sheet with the tree’s information and submit it to canvas.

Non-Flooded Plate:

1. Obtain a large petri dish and weigh it without a lid. Record mass in grams.
2. Pour enough soil into the petri dish so that the bottom is fully covered.
3. Add DI water using a pipette, making sure that the water covers all of the soil but isn’t over-flooded.
4. Weigh the petri dis containing soil. Record in grams.
5. Use a dissecting or compound microscope to observe and take note of any organisms in the soil.

Soil pH:

1. Fill a test tube with 3mL of soil.
2. Fill the test tube with water up to the 8mL line.
3. Shake the tube for at least 3 minutes.
4. Pipette about 1mL of water from the falcon tube and move it to a falcon tube
5. Centrifuge the falcon tube for a minute.
6. Take a pH strip and place it in the falcon tube. Let sit for one minute.
7. Compare the color of the strip to the colors on the package. Record the pH value.

Data and observations:

Storage:

Petri dishes were labeled and placed at the back of the lab. The soil test tubes were placed into a storage rack. All microscopes were unplugged and covered. Used falcon tubes and pipettes were thrown away.

Conclusion and future steps:

I am very excited to observe the DNA of ciliates and trees around campus throughout this semester. I was very interested in the presentations and ways to observe DNA. I could not find any organisms in my soil, but I am hoping that a closer look in next week’s lab will allow me to find ciliates.

Mackenzie Singer

Lab 6

9/28/18

Objectives:

The purpose of this lab is to familiarize students with procedures that will be used in future labs involving tetrahymena and microplastics. We divided up the different testing procedures so that one student in each group understands how to do the lab. It also helped us get more comfortable with dilutions.

Procedure:

PP Microplastic Production

1. Cut polypropylene (PP) into small pieces.
2. Weigh out .5 g of PP and put it in a glass jar.
3. Add 50 mL of protease-peptone-tryptone (PPT) into the jar.
4. Boil and stir/vortex for the entire lab.
5. Filter using 50 µm filter paper into sterile 50 mL tube.
6. Autoclave “twine juice” and store for the next week.

Ciliate Count Challenge

1. Add 20 µl of tetrahymena to 5 µl drop of iodine in a petri dish.
2. Mix by pipetting up and down.
3. Place three 5 µl drops of the iodine solution onto a clean slide.
4. Under a compound microscope at 4x magnification, count the number of Tetrahymena in each droplet.
5. If cell count is over 50, dilute it 1:2 to make it easier to count by adding 25 µl of PPT to 25 µl of tetrahymena. (my group’s dilution factor)
6. Count the cells and record in lab notebook.

Direction Change Assay

1. Add a 5 µl drop of tetrahymena culture to a clean flat slide.
2. Observe the slide under a dissecting microscope. Use the slide over the black plate and under the reflecting light.
3. Choose a cell to follow for 10 seconds. Use a timer to keep track of time.
4. Count how many times the cell changes direction in the 10 second time span. Record in a chart in your lab notebook.
5. Repeat steps 3-5 for 10 cells.
6. Find standard deviation.

Simple Swim Speed Assay

1. Place a 20 µl drop of tetrahymena culture on a clean flat slide.
2. Set a metric ruler in the field of view on the microscope at 4x. Adjust the slide so that you can see the cells over marks on the ruler.
3. Choose one cell to watch and line it up with the inside of a mark.
4. Start a stop watch when a cell hits the mark and stop the time when it reaches a distance of 10 marks away. Only cells swimming straight should be included.
5. Record 10 cells.
6. Swim speed can be expressed as mm/s.

Vacuole Assay

1. Place 20 µl of tetrahymena culture on a clean concavity slide.
2. Add 1-5 µl of India ink to the drop of cells and pipette up and down to mix the cells and ink.
3. Place the coverslip over the drop and use 400x magnification on a compound microscope. Start a timer. Scan the slide for 10 cells and count any lysosomes. This is time=0.
4. After 10 minutes, count the number of lysosomes for at least 10 cells.
5. Repeat the procedure for 20 and 30 more minutes.

Data:

Cell Count: 34 cells in 2 µl 1:2 diluted drop.

Directional Assay:

Speed Assay

Vacuole Assay

(My group member did not have enough time to complete the entire procedure, and only recorded 2 of the cells.)

Storage:

The “twine juice” was stored in jars for next week’s lab. The used tetrahymena solutions were disposed of and slides were washed and set out to dry. Microscopes were put away properly on lab table.

Conclusion/Future Steps:

This lab was useful to help us understand the procedures we will be doing in a future lab to test the tetrahymena. I became much more comfortable with the dilutions and using the micropipettes. The steps used in this lab will be extremely important when we use them to test the effect of the microplastics on tetrahymena. One thing that would be essential to fix is timing because not everyone was able to complete their full procedure.

Mackenzie Singer

Meet Tetrahymena-Lab 4

9/13/18

Objective:

The purpose of this lab was to introduce students to the tetrahymena that will be used for future research and experiments during the semester. We also practiced using the micropipettes so that we can get comfortable using them for future procedures.

Procedure:

1. Obtain a 24-well plate and transfer 100 ul of tetrahymena stock into a single well using a micropipette.
2. Observe the tetrahymena using a dissecting microscope.
3. Using a P-10 micropipette, pick up 5 ul of tetrahymena.
4. Transfer the 5 ul of tetrahymena into a concave slide.
5. Observe the tetrahymena under a compound microscope under 4x and 10x objective lenses. If 40x is needed, use a cover slip over the well.
6. Count the number of tetrahymena in the 5 ul solution. Record in lab notebook.
7. Using field of view measurement, find the relative size of the cells. Record in notebook.
8. Record any other observations in notebook along with a sketch.

Observations:

Trials	/Number of cells in 5 ul	Approximate diameter of cell	Picture
1	200	5um

Storage:

For this lab, we did not label or store any of the used samples. The used solution was kept in a 24-well plate and the micropipettes and microscopes were kept on the lab tables.

Conclusion:

The dissecting and compound microscopes give very different views of the tetrahymena. It is much easier to observe them close up with the compound microscope and on a large scale with the dissecting microscope. Future steps I would take with tetrahymena is to use the organism to perform more advanced experiments in the future, as it will be our model organism.

Computer Lab:

In the computer lab, my group read articles on tetrahymena and thought of a hypothesis to test for soil tetrahymena. In marine environments, microplastics pose a threat to the growth rate of organisms by reducing the rate. We want to test if the microplastics reduce growth rate in soil environments also. We will use microplastics containing BPA placed in soil with tetrahymena versus a control tetrahymena in soil with no microplastics to decide whether growth rate is reduced in soil.