Lab 5 – Diluting Tetrahymena and Experimental Design
Nathan McCoy
September 20, 2018
Lab 5: Diluting Tetrahymena
& Experimental Design
Rationale:
Today’s lab was dedicated to understanding and mastering the p-1000 micropipettors. Then also, we worked on diluting stock samples of tetrahymena in 24 well plates into differing diluted solutions. We then used the diluted solutions to work out the amount of cells per mL, using the diluted solutions and compound microscopes. Then we also worked with our new lab teams to discuss questions surround micro plastics in the environment and how they would affect tetrahymena.
Methods:
- Take out all needed materials, such as the micropipettors, 24 well plates, stock samples of tetrahymena, the compound and disecting microscope, PPT and materials needed for recording data.
- Ensure that all the materials are working properly. Ensure both microscopes are able to focus and the lights are able to turn on and the micropipettors are able to take up samples.
- Place the stock sample of tetrahymena into a center well, ensuring there is space where you have 4 empty slots available to perform the dilution.
- Using the disecting microscope, ensure that the sample of Tetrahymena is alive and swimming in the sample.
- Using the p-1000 micropipettor, take 900 microlitres of PPT and place it in 4 of the wells, ensuring they are in order as to make it easier to keep track of the dilutions performed.
- When the 4 wells are filled with the PPT, then use a p-200 micropipettor to measure out 100 microlitres of the stock solution into the first well.
- Then, using the same well you just placed the first 100 microlitre sample in, take 100 microlitres from there and place it into the second well of PPT. Then from that well, take 100 microlitres and place it in the third well of PPT. Then from the third well that you just placed the 100 microlitres into, take 100 microlitres from there and place it into the final well of PPT. You should have now 4 wells with decreasing concentrations of tetrahymena.
- Following this procedure, use the disecting microscope, determine the diluted solution that looks viable to count the amount of tetrahymena in a 5 microlitre of the diluted sample.
- Take a 5 microlitre sample of the viable sample and place it on a concave slide. Then using the compound microscope on the 4x magnification, determine wether or not you will be able to count the amount of organisms in the drop. If not, wipe the slide clean and try a more or less diluted solution, depending on the amount of tetrahymena found on the slide. Ideally, you would be looking for a sample with 20-40 tetrahymena.
- Record the number of cells in the sample, the dilution factor it was found in, and then using this equation: (# of cells/5microlitres) x dilution factor x 1000) to determine the amount of tetrahymena was in the stock solution per milliliter.
- Repeat steps 9 and 10 2 other times to have multiple times and take the average of the amount of tetrahymena in the stock solution per milliliter to get a more accurate depiction of the amount present.
- After all of the trials and data was collected, we then went to the computer lab where the group members discussed the experimental design on what we wanted to research during our time in CILI-culture lab.
- After looking at our options and what is viable to do in this lab, we completed the Questions that matter question packet. More information on our research question and hypothesis can be found in the “Computer Lab Work/Experiment Creation” section of this lab report.
Findings:
Trial |
Dilution |
Cell count in 5 microlitres |
Cells per microlitre in the drop |
Cells per microliter in the undiluted sample |
Cells per milliliter in the undiluted sample |
1 |
10-1 |
27 |
5.4 |
54 |
54000 |
2 |
10-1 |
11 |
2.2 |
22 |
22000 |
3 |
10-1 |
26 |
5.2 |
52 |
52000 |
Average |
42667 |
Sample working out:
(21.3/5)x10x1000 = 42667 cells per milliliter of undiluted sample
Through the study of the dilutions and the calculations, it was found that per milliliter of the stock solution, there was about 43000 tetrahymena in the stock solution per milliliter. This was calculated by using the 4x magnifications and moving the slide around to ensure that I counted all of the organisms in the sample. Then I did the experiment 3 times to have multiple trials to allow for the source of error and probability of random errors. Some errors that may have occurred in the experiment is the compound microscope only focuses on a small section of the sample and therefore I could have not seen some of the organisms in the sample.
Storage:
The storage of the microscope involves putting the stage down to the lowest it is able to go and leaving the microscope on the lowest magnification (4x) for the next time it is used. Then also wrapping the electrical cord around the microscope and placing the cover on top of it. For the slides, washing them properly and drying them are essential for storage of these fragile pieces of glass. The micropipettors need to have the tips taken off and placed back in their holders. The well plate should be cleaned properly to have all of the samples cleaned out properly for the next use of it. All of the PPT should be given to your instructor and any stock sample that you may have remaining.
Future Steps:
In the future, further trials with the diluted sample could give a more accurate representation of the amount of tetrahymena that would be found in the stock solution. Then also, you could use the technique of diluting solutions and finding the amount of the organism is present in the sample to look at varying conditions and how they affect the number of organisms in the sample. Differing environments such as with the varying depths in the soil, or different temperatures would be interesting to test.
Computer Lab Work/Experiment Creation:
During the time we had in the lab, my team came up with the experiment to test the effects of micro plastics on the reproduction rates of tetrahymena. We plan on doing this by having a control group with no micro plastics and then having other groups with varying concentrations of micro plastics to study the effect micro plastics may have on their reproductive systems. In looking at this experiment, we researched things such as the reproductiveness of tetrahymena, some techniques that would be used and information such as evaporating rates and how that could affect our experiment. Then also, we did determine that we would you PET (Polyethylene terephthalate) would be a viable option for our micro plastic as this is one of the most common types of plastic found in the world. Some next steps would be to look for where we would be able to acquire this plastic, how small we would need the plastics to be and the exact time frames that we would need to leave the samples for in order to see difference. Then also, we would need to look at how we would measure differences quantitatively and how we would show that on a graph.