The purpose of this experiment was to determine the effects of tonicity on a cell membrane using red blood cells, potato strips and three unknown solutions (A, B, C). First three slides were prepared containing RBC’s and unknown solutions A, B and C. A control slide was prepared only using RBC’s. After observing each slide under the microscope it was determined that unknown solution A was hypertonic because the RBC appeared to have shrunk. The RBC in unknown solution B appeared to be swollen, therefor, the tonicity of unknown solution B was hypotonic. Unknown solution C showed no change to the RBC shape, it was suggested that unknown solution C was isotonic.
To confirm the tonicity of unknown solutions A, B and C, a potato strip was placed in 3 separate tubes containing each unknown solution. After each potato strip soaked for twenty minutes it was proven; unknown solution A was hypertonic due to the flaccidity of the potato strip. Unknown solution B proved to be hypotonic because the potato felt extremely rigid. Lastly, the potato strip soaking in unknown solution C was flexible which proved to be isotonic. From those results each unknown solution was established and allowing the determination of tonicity for unknown solutions A, B and C.
The cell membrane was discovered by Swiss botanist Carl Naegeli and C. Cramer in 1855.2 The cell membrane, also known as the plasma membrane is a phospholipid bilayer. Each phospholipid molecule contains a polar head, composed of a phosphate group and glycerol that is hydrophilic (water-loving) and soluble in water, as well as a nonpolar tail, composed of fatty acids that is hydrophobic (water-fearing) and insoluble in water.3 The polar heads are on the two surfaces of the lipid bilayer facing the extracellular and intracellular environment, while the nonpolar tails are in the interior of the bilayer away from the water. Because the fatty acid tails cling together, phospholipids in the presence of water form a self-sealing bilayer. The most important function of the plasma membrane is to serve as a selective barrier for materials entering and exiting the cell.
Plasma membranes have selective permeability. Gases pass through easily, water passes through via transport channels known as aquaporins, ions penetrate the membrane very slowly, and larger molecules (such as protein) cannot penetrate the plasma membrane without the help of transport proteins. Materials move across plasma membranes in two ways: passive and active transport. In passive transport, substances move across the membrane from an area of high concentration to an area of low concentration (down the concentration gradient) without the use of energy. In active transport the cell must use energy to push substances from areas of low concentration to areas of high concentration (against the concentration gradient). Passive transport includes osmosis, which was discovered by French botanist, Henri Dutrochet in 1826.4 Osmosis is the net movement of solvent molecules across a selectively permeable membrane from an area with high concentration of solvent molecules (low concentration of solute molecules) to an area of low concentration of solvent molecules (high concentration of solute molecules).
5 Osmosis attempts to equalize the solute concentrations on both sides. Tonicity is the amount of solute in a solution. A Solute is any dissolved substance in a solution. An isotonic solutions concentration of solutes is equal to inside the cell. The solvent leaves and enters the cell at the same rate, therefore there is no net change; the cells contents are in equilibrium with the solution outside the cell wall. A hypotonic solution outside the cell has a concentration of solutes that is lower than inside the cell. This tonicity causes the solvent to rush into the cell, forcing the cell to swell and sometimes burst (osmotic lysis). A hypertonic solution has a higher concentration of solutes than inside the cell, causing the solvent to leave the cell. Cells placed in a hypertonic solution will shrink as the solvent leaves the cells.
Plant cells react differently to osmosis than animal cells. When an animal cell is placed in a hypertonic solution, water will leave the cell causing it to shrink, this is known as crenation. When a plant cell is placed in a hypertonic solution the cell membrane will pull away from the cell wall, making the plant flaccid, this is known as plasmolysis. When an animal cell is placed in a hypotonic solution, water will rush in to the cell, causing it to swell and sometimes burst. A plant cell placed in a hypotonic solution will also swell due to water rushing in, but will resist rupturing due to the rigid cell wall. Plant cells become more rigid in a hypotonic solution. In this activity we will be observing the effects of potato slices and red blood cells being placed in varying molar levels of NaCl.
The materials used for the first part of the experiment comprised of the following: a microscope, 4 slides, 4 slide covers, blood samples, lancet, a sheet of paper towel, 3 test tube droppers, Solutions A, Solutions B, and Solution C. Blood samples from a volunteer within the group were used to conduct the experiment. The volunteer’s hands were thoroughly washed and an alcohol swab was applied to further sanitize the hands. To gather the blood samples needed, a lancet was properly placed on the forefinger and a firm pressure was applied, which activated the needle inside to spring forward and pierce through the skin. The pierced through finger was massaged to ensure sufficient amount of blood was extracted. A drop of blood was placed in each of the slides. Immediately after, 1 drop of Solution A was added to Slide 2, 1 drop of Solution B was added to Slide 3, and 1 drop of Solution C was added to Slide 4. Slide 1 served as the control, therefore, no solution drops were added to Slide 1.
All 4 slides were lined up on a paper towel with its corresponding labels: Control, Solution A, Solution B, and Solution C. Once all slides were prepared, the microscope was adjusted appropriately. The slide labeled “Control” was placed under the microscope at the lowest magnification. The microscope was further calibrated and adjusted accordingly to the higher magnification to view best results under the microscope. The team reviewed the tonicity and size of the cells under the microscope and observations were noted. The next 3 slides were viewed under the microscope in the same manner as the control slide. Each slide was examined, evaluated, and analyzed by the individual team members. Observations and conclusions were drawn for each slide and solution.
The following materials were prepared for the second part of the experiment: four pieces of potato sliced in identical proportions, Solution A, Solution B, and Solution C in its respective containers with corresponding labels. One potato was placed on a clean piece of paper towel and was labeled the control. The three remaining slices of potato were each placed in a Solutions container and submerged for twenty minutes. After twenty minutes, potatoes were taken out of the solutions and placed on the paper towel. Each potato was evaluated and analyzed by the individual team members. Observations were noted and conclusions were drawn for each potato and solution.
Image I. A drop of blood is smeared onto a glass slide, without any added solution, and then examined under a microscope. This is the “Control” slide, which will facilitate comparison and contrast of red blood cells in different unknown solutions.
Image II. A drop of blood is smeared onto a glass slide with an “Unknown Solution A” and examined under the microscope. Compared to the Control, shrinkage of red blood cells is evident, which suggests crenation.
Image III. Solution B is added to a drop of blood on a glass slide, which is then evaluated under a microscope. In comparison to the Control slide, the red blood cells are swollen.
Image IV. This image is displaying a drop of blood that is mixed with “Unknown Solution C”. Upon observation, the red blood cells maintained the same shape as our control sample. The solution equally moved in and out of each cell.
Cells placed in solution A, displayed signs of crenation, indicating the solution was hypertonic. The cells that were placed in solution B showed signs that they were swelling and that hemolysis taking place as well as, indicating the solution was hypotonic. Lastly, cells were placed in solution C, which maintained constant volume and pressure, identical to our control indicating the solution was isotonic. The findings were consistent with the principle behind tonicity. Hypertonic solutions have a higher concentration of solutes than the cell; therefore, the cell displays water flowing out to maintain equilibrium, thus resulting in crenation. On the other hand, in hypotonic solution, the extracellular space has a lower concentration of solutes, thus enabling water to flow in, which results in cell swelling and possibly hemolysis.
In a hypotonic environment, where the water moves into the cell by osmosis and causes its volume to increase to the point where the volume exceeds the membrane’s capacity and the cell bursts.6 In isotonic solution, the solute concentrations are in equilibrium so there is equal movement of water in or out of the cell. Tonicity is the relative concentration of solutions that determine the direction and extent of diffusion. Cells have a certain molarity and when they are placed in a solution of different molarity, a concentration gradient forms and that creates osmotic pressure on the cell’s membrane. In order to maintain equilibrium between the cell and the solution, passive transport occurs.
As mentioned above, there are three levels of tonicity: isotonic, hypotonic and hypertonic. We also observed strips of potatoes in the same solutions A, B and C. When the potato was placed in hypertonic solution, the cells shrunk, allowing more room to bend without breaking. In an isotonic solution, there was equal movement of water so the potato remained at the same rigidity. In a hypotonic solution, the cells became swollen and closer together, making the potato more rigid.
Initially, this experiment was to determine the effects of tonicity (Hypertonic; cells shrink, Hypotonic; cells swell, Isotonic; cells remain the same) on a cell membrane using red blood cells, potato strips and three unknown solutions (A, B, C). The data collected during this experiment supported the determination of the effects of tonicity, the relative concentration of solutions that determine the direction and extent of diffusion.
After the initial prick of the finger a drop of blood was placed on each slide. For slides A, B and C there was one drop of the each unknown solution then the cover was placed over the blood. Immediately, there after the slide was placed under a microscope for a real “naked eye” view of the red blood cells. There were 4 slides in total including the control slide. What was not expected to occur was for the controlled slide to have had too much blood dropped which resulted in the cells not separating at all. It was determined that a second control slide was needed. The three slides with the unknown solution were inspected under the microscope as well.
During this time it was noted whether each unknown solution mixed with the blood sample was Hypertonic, Hypotonic or Isotonic. After, completing this experiment the next step was to do the same with the potato strips. The potatoes were placed in each unknown solution for twenty minutes. It was also noted that each of the potatoes in the unknown solutions had the same reaction as the red blood cells. The potato in unknown solution A was hypertonic due to the flaccidity of the potato strip. The cells within the potato shrunk. Unknown solution B proved to be hypotonic because the potato felt extremely rigid. The cells became swollen. Unknown solution C proved to be isotonic.
The potato was flexible and not too rigid or flaccid. The potato placed in solution C was the most similar to the control potato, which was not placed in any fluid. The purpose of this experiment was to be able to differentiate and recognize what Hypertonic, Hypotonic and Isotonic are and how it looks through the microscope. The members of group one were able to differentiate and recognize this! The initial hypothesis of this experiment is supported.
1. Chamberlain et al. Effects of Tonicity on Cell Membrane . Human Physiology Labratory Manual, 8th Edition, Expt 6 part C and D 2. Chronology of Life. http://www.whatislife.com/education/fact/history_table.html. Accessed November 22nd, 2014. 3. Tortora GJ. Functional Anatomy of Prokaryotic and Eukaryotic Cells. In: Microbiology an Introduction. 9th ed. San Francisco, CA: Pearson Education, Inc; 2007: 77-113. 4. Osmosis Background. http://rheneas.eng.buffalo.edu/wiki/Osmosis:Background. Accessed November 22nd, 2014. 5. Fox SI. Interactions Between Cells and the Extracellular Environment. In: Human Physiology. 12th ed. New York, NY: McGraw-Hill;2011:129-159. 6. Campbell, N., & McClendon, J. (2014, October 24). Cytolysis. Retrieved November 23, 2014, from http://en.wikipedia.org/wiki/cytolysis