Lab 7 10/4/18
Date of Experiment: October 4, 2018
Objectives:
- Determine the optimal density of various medias using a spectrophotometer
- Calculate the cell count of the control and treatments cultures
- Carry out speed assays on the control and treatment cultures
Purpose: To determine whether the microplastic concentration in the media can affect Tetrahymena
Materials:
- PPT and “twine juice” media
- PPT and Tetrahymena (control culture)
- PPT and “twine juice” and Tetrahymena (treatment culture)
- Spectrophotometer
- Flat slide
- Iodine
- Ruler
- Stopwatch
- Micropipette
- Serological pipette
- Sterile glass tubes
Methods:
Sampling
- Swirl each flask containing 50 ml of culture
- Using a serological pipette, collect 5 mL of the control and treatment group from each of the flasks
- Place each 5mL of culture in a sterile glass tube
- Use the cultures from these tubes to conduct optimal density, cell count, and swim speed assays
Cell Count
- To determine the average cell count of the Tetrahymena, use a micropipette to add three drop of 2uL of Tetrahymena and 1uL of iodine on a flat slide
- Placing the slide under a compound microscope, observe the cells of each drop under 4x magnification
- Record the amount of cells observed in microliters to be able to better see the cells, converting these into milliliters to get a better estimation of the amount of cells in a culture.
- Calculate the average of cells per milliliters
- Repeat steps 1-4 for both the control group and treatment group
Swim Speed Assay
- Divide the work with a lab partner, with one person being responsible for the swim speed assay of the control group whereas the other is accountable for the swim speed assay of the treatment group.
- To observe the swim speed assay of the Tetrahymena, use a micropipette and place 20 microliters of Tetrahymena onto on a flat slide and place these under a compound microscope
- Placing a ruler under the slide, align the millimeter mark with the cell you’ve chosen to observe
- Using a stopwatch, record the time it takes for the cell to swim 1 millimeter (making sure to only record time for cells swimming straight the entirety of the trial)
- Repeat the previous step for ten trials, calculating the speed of each cell
- Find the average and standard deviation of the cell speed
- Repeat steps 2-6 for both the control group and the treatment group, recording the results of both experiments
Observations:
Optimal Density
Transmittance=0
| Media | Absorbency |
| Twine Juice | .056 |
| Control | .068 |
| Treatment | .075 |
Cell Count
| Drop 1 (cells/2uL) | Drop 2 (cells/2uL) | Drop 3 (cells/2uL) | Average cells/mL | |
| Control Group | 24 | 37 | 16 | 12666 |
| Treatment Group | 40 | 25 | 50 | 19166 |
Control Culture Swim Speed Assay
Average: .30 mm/s
Standard Deviation: .03
| Cell | Time (seconds) | Speed (mm/s) |
| 1 | 5.86 | .17 |
| 2 | 2.50 | .4 |
| 3 | 3.25 | .31 |
| 4 | 5.05 | .20 |
| 5 | 3.60 | .28 |
| 6 | 2.65 | .38 |
| 7 | 2.16 | .46 |
| 8 | 3.38 | .30 |
| 9 | 4.00 | .25 |
| 10 | 4.06 | .25 |
Treatment Culture Speed Assay
Average: .25 mm/s
Standard Deviation: .03
| Cell | Time (seconds) | Speed (mm/s) |
| 1 | 3.56 | .28 |
| 2 | 5.33 | .18 |
| 3 | 5.94 | .16 |
| 4 | 3.61 | .28 |
| 5 | 2.05 | .48 |
| 6 | 4.55 | .24 |
| 7 | 4.24 | .24 |
| 8 | 4.65 | .22 |
| 9 | 4.95 | .20 |
| 10 | 4.66 | .21 |
Results:
Upon setting the transmittance to zero, it was determined the absorbency of the twine juice was .056, the control group was .068, and .075 was the treatment. These results are used to determine the optimal density as each increasing absorbency indicates the ability of light to pass through molecules. The average cell count of the control group was about 12,666 cells whereas the average for the treatment group was about 19166 cells. The swim speed assay conducted for the control has an average speed of .30 mm/s whereas the treatment group displayed an average speed of .25 mm/s.
Storage:
Upon finding the optimal density, cell count, swim speed assays, the micropipette tips were disposed of, the microscope slides were rinsed with 10% bleach solution and water and dried on paper towels. Test tubes containing the various cultures and medias were stored in a test tube rack, and stored in the lab.
Conclusion/Next Steps:
The average cell count of the control culture in comparison to the treatment culture posed as a surprise since it was assumed that microplastic concentration in the Tetrahymena media would significantly decrease the cell count. These results differ from the swim speed assays as the speed of the control group was higher than that of the treatment group. These findings suggest that microplastics concentration may increase the rate of Tetrahymena, but such toxins affects it functions, as seen in the cells decreased speed. Using these results, I would further this experiment to show how such change in speed affects how Tetrahymena’s fulfill its role in the environment, in spite of the increase in the amount of cells.