The Effects of Physical and Chemical Stress on Cellular Membranes
Essential Questions: What are the effects of temperature stress on cellular membrane?
What are the effects of organic solvents on cellular membranes?
Background Information: Beet tissues will be the model to investigate membrane integrity. Roots of beet contain large amounts of a reddish pigment called betacyanin that is located almost entirely in the large central vacuoles, which are surrounded by a vacuolar membrane called the tonoplast. The entire cell is surrounded by a cell membrane and a cell wall.
In the two procedures you will subject beet cells to a range of temperatures and organic solvents, and determine which treatments stress and damage the membranes the most. If stress damages the membranes, betacyanin will leak through the tonoplast and plasma membrane. This leakage will produce a red color in the water surround the stressed beet. Thus, you can measure membrane damage by measuring the intensity of color resulting from a particular treatment.
Membranes are sensitive to extreme temperatures. High temperatures cause violent molecular collisions that destroy a membrane as a physical barrier to diffusion. Conversely, freezing temperatures cause water to crystallize as ice and expand because of hydrogen bond alignment. This expansion and formation of ice often ruptures membranes.
Organic solvents dissolve a membrane’s lipids. Acetone and methanol are common solvents for various organic molecules, but acetone has the greater ability to dissolve lipids.
Hypothesis: If more temperature is added to the cellular membrane, then the color of each solution will be more intense. If a higher percentage of solution is added to a cellular membrane, then the color of each solution will be more intense.
- Tap water
- Seven dry test tubes
- Distilled Water
- Hot Plate
- Isotonic Saline
We first cut six uniform cubes of beet using a scalpel. We made sure that all of the cubes were the same size. Next, we placed the cylinders of beet tissue inn a beaker and rinsed them with tap water for two minutes in order to wash the betacyanin from the injured cells on the surface. All of the cubes were washed in the same way, and afterwards we discarded of the colored rinse water. Gently, we placed each of the beet cubes into different, dry test tubes. When moving the beets we were careful to try not to stab, crush, or otherwise damage the cubes. Finally we labeled the test tubes 1-6. We used cold and hot treatments for different test tubes. For the cold treatment, we placed tube 5 in a refrigerator (5 degrees Celsius) and test tube 6 in a freezer (-5 degrees Celsius). Test tubes 5 and 6 were left in the cold for thirty minutes. After the 30 minutes we removed the beets from the freezer and refrigerator and added 10.0 mL of distilled water at room temperature to each of the two tubes. The cold treated beets soaked in the distilled water for 20 minutes. While waiting for test tubes 5 and 6 to cool, we began the hot treatments. We took the beet section out of tube 1 and immersed it in a beaker of hot water (70 degrees Celsius). After 1 minute in the hot water bath, the beat was returned to test tube 1 and 10.0 mL of distilled water was added. Afterwards we cooled down the hot water bath to 55 degrees Celsius and immersed the beet from the tube 2 for 1 minute. We returned the beet from test tube 2 and added 10 mL of distilled water at room temperature. The hot water bath was then cooled down to 40 degrees Celsius and test tube three was immersed in the bath for one minute. When we finished we returned the beet to test tube three and added 10 mL of distilled water. Finally, we cooled the hot water bath to 20 degrees Celsius and added test tube four for one minute and then added 10 mL of distilled water to the test tube. The treated beets in tube 1-4 were allowed to soak in the distilled water at room temperature for 20 minutes. Then the beets were discarded and the extent of the membrane injury was measured according to the amount of betacyanin that diffused into the water. For all of the six temperature treatments the relative color of each solution between 0 (colorless) and 10 (darkest red) was recorded in the data table 9.1. Also, the absorbance level was measured for all of the test tubes by placing it in an absorbance reader. The closer the number is to 0, the less that is absorbed.
Seven uniform cubes of beet were cut using a scalpel. They were trimmed so that they were all around the same size. The cubes of beet tissue were placed in a beaker and rinsed with tap water for two imiutes to wash the betacyanin from the injured cells on the surface. Then, the colored rinse water was discarded. Each of the seven sections was placed in dry test tubes. We tried not to crush, stab, or otherwise injure the beets. We labeled the test tubes 1-7. 10 mL of the appropriate solvent listed in the table were added to each of the seven test tubes. All of the beets were kept at room temperature for 20 minutes and shaken occasionally. Then we removed and discarded the beet cubes and measured the extent of the membrane damage according to the amount of betacyanin that diffused into the water. We quantified the relative color of each solution between 0 (colorless) and 10 (darkest red). The absorbance was also recorded by an absorbance recording machine. The data was recorded in table 9.2.
This table represents the measures of color intensity of betacyanin leaked from damaged cells treated at six different temperatures.
This table measures the color intensity of betacyanin leaked from damaged cells that were treated with various concentrations of two organic solvents.
Conclusion: We accept the hypothesis due to the data that was collected throughout the experiment. When viewing the data that was collected in the tables throughout the experiment, we noticed that as the temperature of the beaker was cooled by a number of degrees, the color intensity of the solution dropped. Eventually, at 20 degrees celcius, the color intensity reached zero and stayed in that position for the remaining temperatures that we tested (20, 5, and -5 degrees celcius). This information can be found in Table 9.1 above. The next portion of our hypothesis came from adding 10 mL of different concentrations of two different solutes (acetone and methanol), to seven separate test tubes each holding their own cube of beet. After waiting twenty minutes for the solutions to settle, we found the data placed in Table 9.2. This data supported the portion of our hypothesis that states, “If a higher percentage of solution is added to a cellular membrane, then the color of solution will be stronger”. One example of how this data supports our hypothesis is found in the relationship between the acetone 25% solution and the acetone 50% solution. Whereas the 25% acetone solution absorbed .05% of the light, the 50% acetone solution absorbed .23% of the light. This makes the difference between the solutions .18%. Meaning that the 50% acetone solution absorbed more than twice the amount of light than that of the 25% acetone solution.
During this experiment we learned how organic solvents and temperature changes affected the beet membrane and it’s connection and similarity of a cell membrane. As the temperature increases around a cell, the cell membrane becomes more permeable, which allows for more materials to pass though the membrane. As the temperature lowers, the cell membrane becomes less permeable and restricts the amount of materials passing through. In the beet experiment, there was more color absorbed at 70 degrees celcius than there was at -5 degrees celcius. This relates to the cell membrane because as temperatures increase, the membrane is more permeable and allows more materials in and out of the cell. When temperatures decrease, the membrane is less permeable and restricts the amount of materials passing in and out of the cell.
When adding solutions to the beet cubes, we learned that the high-percentage solutions caused the beet to bleed more betacyanin than the low-percentage solutions did. Cell membranes are sensitive and once high percentages of a solution are added to the cell, like acetone or methanol, the lipids in the membrane dissolve and the cell becomes more permeable because of the loss of lipids. Low percentages of a solution are not as strong as high percentages and don’t break as many lipids as higher percentages do. This allows for the cell to be somewhat permeable, but does not allow as many materials in or out of the cell as much as high percentage solutions allow. In the experiment, there was .23nm of color absorbed at 50% acetone when there was .01nm of color absorbed at 1% of acetone in Table 9.2 above.
During this experiment, there was possible human error and technological error. When cutting the beets for the experiment, each beet cube was close to size with other beet cubes, but the measurements were not perfect measurements. Also, measuring the amount of distilled water added to the beets could have been possibly off by 1mL and not exact. Technological error would be the spectrometer and the amount of color absorbed each beet cube, temperature, and percentage solution added to the cube. The spectrometer calibration could have been off before we measured the color absorbed or the cap might have not covered the test tube to accurately measure the color absorbency.