09/28/2018

Objectives:

The objectives for this lab were to obtain a basis data to inform future labs, and to test new assays on the Tetrahymena. Other objectives of the lab included utilizing micropipettes and completing serial dilutions, as well as calculating the dilution factors of these certain solutions, such as in the Tetrahymena and Iodine solution. Students will also reinforce the skills of properly performing wet mounts and stains, as well as micro-pipetting techniques.

Purpose:

The purpose of this lab was for students to get hands on with their experiments, and to start the process of testing micro-plastics on Tetrahymena. Students learned how to perform basic skills in prior labs so that they could utilize them starting today. This lab allowed for students to demonstrate their mastery of the scientific method and basic labs skills, whilst adding on top of it the art of data collection and testing possible assays never tested before. The data collected today will be useful to students in the future, as they can use this data as a base for other experiments and variables. Lastly, students were able to practice and master performing and calculating serial dilutions, as well as utilizing new technology, such as the iPhone camera adaptor, to see the Tetrahymena.

Procedures:

1. Obtain a clump of BLACK bailing twine and chop it into very fine pieces. Note that only CILI-CURE 33 is using black twine, so stick with this color.
2. Weigh the pieces of twine on the scale out to be 5 grams, and place into a sterile jar.
3. Add 50mL of PPT treatment into the jar. Boil and vortex the solution for the entirety of the lab.
4. Hand off this solution to Dr. Adair, where she will autoclave and extract the “twine juice” in open lab on Friday.
5. Next, we will practice serial dilutions and testing out new assays. Obtain a compound microscope, concavity slides, a P20 and P200 micropipettes and tips, Tetrahymena culture, Iodine, and a Petri dish.
6. Using the P20 micropipette, gather 5uL of Iodine and place on the Petri dish. Be sure to eject the tips into a proper disposal place after use.
7. Next, take a P200 micropipette and add 20 uL of Tetrahymena sample to the 5 uL of Iodine, mixing with the same techniques utilized in last lab, ejecting the tip afterwards.
8. We observed that with this solution, there was an abundance of Tetrahymena. There were too many to count, so dilutions had to take place.
9. To counter this effect, using a P200 micropipette, we added 45 uL of PPT to dilute the 5 uL solution of Tetrahymena and Iodine, creating a 50 uL solution.With this, we created a 1:50 dilution, or a 10^-5 dilution.
10. Using the P20 micropipette, obtain three 5 uL samples from the 5o uL sample and place them on a concavity slide in different places.
11. Observe at 4x and 10x, and decide which magnification is better. Create a graph labeled with the trial number, dilution factor, cells per 5uL, cells per uL, cells per uL in the undiluted sample, and cells per mL in the undiluted sample.
12. Observe and record data in the chart.
13. Next, we move on to testing various assays. In this lab, I chose to test the vacuole formation assay with India ink.
14. Obtain India ink, concavity slides, Tetrahymena culture, a P20 micropipette, a cover slide, and a compound microscope with a black disc.
15. Pick up 20 uL of Tetrahymena culture and place it onto the center of the concavity slide.
16. Next, obtain 5 uL of India ink, and mix it in with the Tetrahymena on the concavity slide, using the pumping motion from last lab.
17. Quickly place the cover slide over the solution, and observe at 400x magnification.
18. Using the phone adaptor, record the microscope’s FOV, waiting for 10 cells to pass by. After this, set a timer for 10 minutes.
19. Observe the cells from the video, pausing and rewinding when needed. Count the black dots you see inside the cells. These are the vacuoles, and they are black because of the India ink dye.
20. Record this number in a chart labeled with the time, vacuole count of 10 cells, magnification, and the total number of cells.
21. Due to lack of time and difficulty locating the cells at first, this experiment only proceeded to the 10 minute mark. If time allows, test until the 30 minute mark, observing the Tetrahymena every 10 minutes and recording the data in the chart.
22. Return all supplies and clean up lab area, and finish your QTM of the day.

Data and Observations:

Below is a table for the cell counts for the Ciliate Count Challenge.

Below is a table for the vacuole counts observed in Tetrahymena with India ink.

Time	Vacuole count	Magnification	Total # of Vacuoles	Photos of vacuoles (the black dots)
0 minutes (base)	6,1,1,3,4,4,1,2,3,3	400x	28 vacuoles
10 minutes	7,3,2,2,5,7,1,1,3,4	400x	35 vacuoles
Average	X	X	31.5 vacuoles	X

Storage:

We returned the micropipettes and eject their tips, re-covered the compound scopes, and returned the Tetrahymena sample, India ink, and iodine to their proper places. We washed the concavity slides and coverslips with 20% bleach solution and laid them out to dry on a paper towel.

Conclusions:

In conclusion, we observed our average cell count in the undiluted sample to be 103,334 cells per mL. This was tricky to procure, because we had to dilute the sample with 45 uL of PPT, resulting in a 10^-5 dilution factor. Utilizing the India ink assay, we were able to count and record the number of stained vacuoles, resulting in an average of 31.5 or ≈ 32 vacuoles per 10 cells. However, it was observed that the number of vacuoles increased over the 10 minute span. This data lead us to hypothesize that over time, the vacuoles of Tetrahymena will ingest more of the dye through phagocytosis. From this, we deduced that if there was a high concentration of micro-plastics in the soil with the Tetrahymena, the cells could take in more of this pollutant over time, which in turn could result in mutations, changes in behavior and swimming patterns, and overall cell counts.

Future Steps:

In the futures I would give myself more time to observe the Tetrahymena in specific assays. Future labs could possibly utilize a solution that doesn’t require waiting for the Tetrahymena to swim by, and everyone could utilize an iPhone camera adaptor to video record samples.