Lab #7 Toxicity Assay; Controls and Treatment

10/04/18

Haiden Jordal

Objectives: 

Today’s objective is to finally run through our experiment. Using the results we get from this experiment we should be able to answer or scientific question and either accept or reject our original hypothesis. The question we are testing in lab is: Will the introduction of micro plastics to a solution of T. pyriformis cells have a significant effect on the ciliates’ growth or mobility? Our hypothesis inferred that it will have a significant negative effect on the Tetrahymena’s reproductivity and ability to move.

Procedures:

Sampling

1. Find the lab counterpart that performed the same behavioral assay as me in the last lab.
2. With the new partner, label two test tubes, one Treated and one Control.
3. Obtain the flasks of samples and be sure to swirl before pipetting.
4. Use the serological pipette to transfer 4 ml of the solution of tetrahymena treated with the twine juice into the tube labled “treated”. Transfer 4 ml of the solution of tetrahymena mixed with the PPT media into the tube labeled “control”.
5. Place tubes in test tube rack to carry.

Spectotrophometers

1. Measure the optical density at wavelength 600 nm of each solution of: PPT, PPT + Twine Juice, PPT + TH, and PPT + Twine Juice + TH.

Cell Counts

1. Working quickly, so as to prevent the drops from evaporating, Transfer 2 ul of each of the treated and control Tetrahymena onto a flat slide.
2. Add 1 ul of Iodine to the drops.
3. Observe the drops under the compound microscope and locate the most appropriate magnification for counting cells.
4. Take a picture of the slide to count later for accuracy.
5. Repeat the process with two more drops each of the treated and control samples.
6. Take the average of the cell counts for each sample and calculate the average concentration of TH cells/ml.
7. Use the equation (avg of cells/ 3ul) x (D.F. 10) x (1000 ul/ml) x 1.5 = concentration (cells/ml)

Behavioral Assay (swim speed)

1. Place 20 ul of each culture on a clean flat slide
2. Set the slide on top of a metric ruler over the mm side.
3. Observe the cells under a dissecting microscope.
4. Choose one specific cell and use a timer to record the time it takes the cell to move from the inside of one mark the inside of the next.
5. Write down the time and repeat the process for at least 10 cells.
6. Calculate the average time and speed using mm/s.
7. Repeat that whole process for the other drop of culture.

Alternative Assays

1. Madalyn performed the directional change assay and Kelsi performed the vacuole formation assay.

Observations:

Cell Count

Concentration of treated cells: 326,500 cells/ml

Concentration of control cells: 88,000 cells/ml

Optical Density

Behavioral Assay

Treated cells moved at a pace around .21 mm/s faster than the control cells.

Storage:

At the aseptic area, the serological pipette was left for other groups to use. The slides used for cell counts and behavioral assay were cleaned and stored on the counter to dry. The micro pipettes were put back on the racks and the used tips were disposed into the tip cups. Both microscopes were turned off, covered and moved back to the center of the lab table. The tubes of both the treated and control samples were left in their racks on the table alongside the rack containing the iodine.

Conclusion:

The results of this experiment show both an increase in cell count for the Tetrahymena in the treated culture and an increase in mobility for those same cells observed in the behavioral assay. The increase in cell concentration was quite significant going from 88,000 cells/ml in the control group to 326,500 cells/ml in the treatment group. This change suggests that the cells interacting with the micro plastics in the treatment group were able to reproduce and stay alive at a much higher rate that the control, contradicting our hypothesis. This contradiction is continued with the observation of the behavioral assay and the fact that the treated cells moved approximately .21 mm/s faster than the control cells. So not only were they more abundant in the treatment, something about the micro plastics enhanced the mobility of the Tetrahymena.

Future Steps:

In the near future, our individual results will be shared on a spreadsheet to the entire class. The observations can then be compared and a pattern or common result may be identified. Upon this realization, a better conclusion can be made as to whether or not the treatment negatively or positively affected the Tetrahymena. According to my results alone, it would appear t be positive, but that does not rule out any experimental random error. It is also important to start thinking systematically and brainstorm how our results can be used to understand further relationships that micro plastics an playa part in in the real world. If in fact the micro plastics have an immediate positive effect on Tetrahymen, the long term effects are still unknown and can be of equal if not more importance. We should be sure to look into the procedure of heating and filtering and determine if the process of obtaining the micro plastics could be flawed in the fact that it is not completely sterile.