Tetrahymena are single cell, free living protists found in freshwater. Tetrahymena are interesting to study when it comes to how they ingest their food. They have cilia, which are hair like projections that act as locomotion for an organism, which sweep food close to the cell and then Tetrahymena ingest the food particles through phagocytosis. Phagocytosis is the process of a cell surrounding a particle, creating a vacuole, and eventually completely enclosing around the particle creating a phagosome (Department of Molecular, Cellular, and Biomedical Sciences, 2018). Then, the phagosome merges with a lysosome so that it can be broken down inside the cell and the particle can be used in the cell. In the first experiment, the students observed the process of phagocytosis in Tetrahymena by providing the solution of Tetrahymena with polystyrene microbeads. The students produced one tube of Tetrahymena without the microbeads and one with microbeads and Tetrahymena to show that when microbeads were available to the Tetrahymena they would phagocytize the microbeads. Samples of each tube were put on slides to view under the microscope to count how many microbeads would be phagocytized by each cell. After learning the characteristics of Tetrahymena, doing experiment one, and doing a little bit of research, the students came up with their own experiment to test phagocytosis in Tetrahymena. The students decided to test the rate of phagocytosis of Tetrahymena in different salt concentrations. The hypothesis was, if phagocytosis of microbeads by Tetrahymena are experimented with in different salt concentrations, then less beads will be consumed in higher salt concentration, because Tetrahymena are mostly found in freshwater so they will most likely have a higher phagocytosis rate in lower salt concentrations. Also, the students found an experiment that tested the growth rate of Tetrahymena in different salt concentrations and in higher salt concentrations the population growth rate was lower than in lower salt concentrations.
Experimental Design and Methods:
To start this experiment, the students had to create differing tubes for each control and experimental group. First the students made the tube for the positive control, which was mixing 50 microliters of Tetrahymena and 50 microliters of 3 microliter microbeads together. This is the positive control, because in the first experiment the Tetrahymena from the tube containing this solution phagocytized the microbeads and the salt concentration of this group was 0. The next tube that was made was one of the negative controls, which had 50 microliters of Tetrahymena and 50 microliters of nutrient media. This is the negative control, because in the first experiment there was no phagocytosis since there were no food particles (microbeads) to phagocytize. Once these two test tubes were made, the students let them sit for 10 minutes so that phagocytosis could take place, and then they added 100 microliters of 0.2% glutaraldehyde, which would kill the cells so that they could be looked at under the microscope easily. A third test tube was made, which was the second negative control. The steps to make this tube was to add 50 microliters of Tetrahymena and let it sit for 10 minutes. Then, 100 microliters of 0.2% glutaraldehyde was added and finally after letting it sit for five more minutes, 50 microliters of 3 microliter microbeads were added. This is the negative control, because the cells would be killed by the glutaraldehyde before the Tetrahymena could phagocytize the microbeads. This tube was, also, made to make sure the glutaraldehyde was working. Next, the students started making the experimental test tubes. The first test tube was filled with 50 microliters of Tetrahymena and 50 microliters of 0.22% NaCl, creating a total salt concentration of 0.11%. The second experimental test tube was filled with 50 microliters of Tetrahymena and 50 microliters of 3.5% NaCl, creating a total salt concentration of 1.75%. The students then let the tubes sit for 15 minutes, so that the Tetrahymena could get used to the salt concentrations. Once the 15 minutes was up, the students added 50 microliters of 3 microliter microbeads to both test tubes. Then, the test tubes sat for 10 minutes before 100 microliters of 0.2% glutaraldehyde was added to each. Now that all of the test tubes were prepared, the students could pipette 20 microliters of each test tube onto a slide. So, one slide contained 20 microliters of the positive control, another slide had 20 microliters of the first negative control, and another slide had 20 microliters of the second negative control. Then, 2 slides were made for each experimental test tube, 0.11% and 1.75% salt concentration, because they were the experimental groups. So, there was a total of 7 slides. When put under the microscope, the students counted how many microbeads were found in 5 different cells on each slide. So, a total of 10 cells were counted for each experimental group since each one had two slides. An Olympus CH-2 bright field compound microscope, with a magnification of 40, was used to view the cells. The students made Histograms for the positive control and 2 experimental groups to help determine which concentration led to a higher rate of phagocytosis. A histogram is like a bar graph, but it graphs the frequency of occurrences in a data set (bin). In this experiment, the independent variable was the salt concentration and the dependent variable was the number of microbeads consumed, because the students were testing if salt concentration changes the rate of phagocytosis. In order to keep the experiment focused on the relationship between salt concentration and phagocytosis, the students had many control variables. A few of the control variables were temperature, lighting, and microbead size. These were all kept constant, because if they weren't, another relationship of phagocytosis of Tetrahymena would be tested and would affect the results.
Results:
The objective of this experiment was to find the relationship between salt concentration and the rate of phagocytosis in Tetrahymena. So, the students experimented with three different salt concentrations, which were 0%, 0.11%, and 1.75%. By looking at each histogram, the students were able to see a relationship between salt concentration and rate of phagocytosis. But to help determine the relationship better the average of how many microbeads were ingested for each salt concentration was found. For the positive control the average number of beads consumed was 7.2, for the 0.11% concentration the average number of beads consumed was 9, and for the 1.75% concentration an average of 2.1 microbeads were consumed. Using the averages and the histograms, the students were able to see that the relationship between salt concentration and rate of phagocytosis was that the lower the salt concentration the higher the rate of phagocytosis in Tetrahymena. The average helped figure that relationship out, but the relationship was able to be seen through the histograms, also. Comparing the bins of each histogram helped see the relationship. For example, in figure 1 and 2 most of the cells consumed a number between 0 and 14 beads. But there were a few cells in figure 1 and 2 that consumed between 14 and 28 microbeads. In figure 3, the cells in the highest salt concentration, most of the cells consumed between 0 and 2 microbeads, which is significantly lower than in figure 1 and 2. The highest bin in figure 3 is 4 to 6 microbeads, while the highest bin in figure 1 is 14 to 21, and for figure 2 is 21 to 28. No histograms were made for the two negative controls, because the number of beads consumed by the cells in the negative control groups were 0. This is what should have happened, since in the first control group no microbeads were given to the Tetrahymena and in the second control group the microbeads were added after the cells were killed. In the second control group, a student did count that a cell had consumed two microbeads, but after considering that the cells died before they were given microbeads the students determined the microbeads were probably on top or below the cell, but it just looked like they were in the cell on the microscope.