9/27/18

Purpose

We will prepare the twine to be broken into microplastics by cutting it and mixing it with sterilizing solution. This will be performed in order to conduct the next experiment on the effects of microplastics on Tetrahymena. Then, we will count the Tetrahymena cells per mL using Iodine and concavity slides. This is a simple cell count that teaches basic laboratory and calculating skills. Finally, we will conduct one of three assays to test the lysosomes or behavior of the Tetrahymena. One will do the vacuole formation assay, simple swim speed assay, or direction change assay. This will help us find more basic details of Tetrahymena before conducting our class experiment.

Procedure

Part 1: Twine

1. Cut the twine into many, small parts for absorption.
2. Weigh a weighing boat on a scale. Add 0.5g of twine to the boat.
3. Transfer the twine to a glass jar.
4. Use a graduated cylinder to measure 50mL of solution and mix.
5. Place the jar near the microwave to be heated.

Part 2: Cell Count

1. Micropipette 20uL of Tetrahymena on a petri dish lid.
2. Micropipette 5uL of Iodine and add the droplet to the 20uL of Tetrahymena.
3. Micropipette up and down to mix the solutions.
4. Obtain 3 5uL drops and place them on concavity slides without a cover.
5. Use a compound microscope at 4x to view the Tetrahymena. Count the number in each 5uL drop.
6. Calculate the average number and record the cells per mL.

Part 3: Simple Swim Speed Assay

1. Place a 20 µl drop of culture on a clean flat slide.
2. Set the slide on top of a metric ruler and adjust the slide so that you can see the cells on top of the mm marks on the ruler.
3. Pick a cell to watch and line it up with the inside of one mark.
4. Start the timer and watch the cell until it touches the inside of the next mark. Stop the timer.
5. A stopwatch can be started whenever a cell passes over one of the marks and stopped when the cell touches the next mark, but this is easier if you have one person watching and saying “Go” and “stop” to a partner. Only cells swimming straight during the entire length of the mark should be included.
6. Record the time and repeat for at least 10 cells.
7. Swim speed can be expressed mm/s.
8. Calculate the Average and the Standard Deviation.

Data/ Observations

Part 2:

Part 3:

Average= 3.07 sec

Standard deviation= 0.79 sec

Conclusion

For part 1, the twine juice will be boiled and vortexed. The autoclave will sterilize the juice. Next week, the solution will be filtered for 5uL, so that the materials will qualify as microplastics as well as be small enough to be digested by the Tetrahymena. This lab taught me many basic laboratory procedures and calculations which will be helpful with future procedures. It prepares for the upcoming lab, but it also teaches about the basic Tetrahymena characteristics in the assays. The experiment was divided into separate parts which also helped me work on my time management.