Using the plasmolysis threshold to estimate the concentration of solutes and water inside living cells
Under your compound microscope, the cell membrane of a turgid cell is invisible because it is pressed up against the inner surface of the cell wall. But in a plasmolyzed cell, the cell membrane is visible because it has pulled back away from the cell wall. We will use this fact to estimate the concentration of water and solutes inside living cells. In Exercise 2 you will be placing cells into a series of “environments”; i.e., test solutions with known solute concentrations. Your objective is to find the “isotonic points” for each cell type– the test solution whose solute concentration is equal to the solute concentration inside the cell. At the “isotonic point” point cells are close to the balance point between turgid and plasmolyzed. Viewed under a microscope, an isotonic environment is one in which about half the cells appear turgid and half are plasmolyzed.
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Table 2: Using plasmolysis and turgor to determine the solute concentration inside plant cells.
When the cell membrane is …
The environment is said to be …
The concentration of solute in the environment is…
plasmolyzed (shrunken & visible) in all cells
hypertonic greater than that inside the cell
turgid (invisible) against the cell wall in all cells.
hypotonic less than that inside the cell
turgid in about half the cells, plasmo- lyzed in about half
isotonic equal to that inside the cell
Procedure 1. Continue to work with 1-2 partners. View the videotape on plasmolysis and how plasmolysis is used to determine the isotonic points of cells. In the videotape, the solute used is sucrose (sugar). We will be using sodium chloride (table salt) as the solute. 2. Obtain a wedge of red onion and peel off a few thin sheets of outer epidermal tissue. To see the difference between plasmolyzed and unplasmolyzed cells, place a sample of onion epidermis in small beakers containing10 ml pure water (0% salt solution) and one with 10% salt solution. Wait for 10 minutes. The cells in pure water (0% salt) will be unplasmolyzed, so can be used as a standard for comparison. 3. To make sure you have actually seen the difference between plasmolyzed and unplasmolyzed red onion cells, sketch some actual cells in the circles below. Unplasmolyzed onion cells ( x) Plasmolyzed onion cells ( x) Being able to see the difference between plasmolyzed from unplasmolyzed cells is crucial for your experiment in Part III. If you are having trouble, be sure to ask for help from one of the learning center instructors.
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4. While waiting for the samples in step 2, put additional samples of onion tissue into a series of 4-5 beakers containing concentrations of salt between 0 and 10%. Label a series of microscope slides with 0%, 10%, and your other testing solutions. 5. Allow the samples in step 4 to remain in solution for 10 minutes. Then prepare a wet mount of each specimen. One by one, examine each specimen under the microscope. Work quickly so the specimens do not begin to dry out under the microscope. 6. For each specimen, look only at the cells that contain red pigment. Count 50 cells and record in Table 3 how many of the 50 are plasmolyzed and unplasmolyzed. The natural pigmentation in red onion makes it easier to see whether the cell membrane has pulled away from the cell wall. 7. Calculate the percentage of cells plasmolyzed = Number plasmolyzed_ x 100% Number of cells counted and enter the data in Table 3 on the next page. 8. Repeat steps 5 and 6 (above) for several salt solutions. HINT: You are looking for the concentration at which about half (40-60%) of the cells are plasmolyzed, half unplasmolyzed. This indicates a concentration close to the boundary between plasmolyzed and turgid. If you plan ahead, you won’t need to test all of the concentrations! Look at the results of 0%, 5% and 10%, and then decide whethr you need to test 2 – 4%, or 6- 9%. You may then need to run only one or two additional concentrations to “zero in” on the solute concentration isotonic for onion epidermal tissue. 9. For each of the solutions you test, the percentage of cells plasmolyzed = Number plasmolyzed_ x 100% Number of cells counted Enter these results in Table 3. 10. From Table 3, the isotonic point for red onion appears to be about _____ %.