BIological Membrane Integrity

Lab 3 – Homework
1. Purpose of today’s experiment:
To determine the effect osmotic pressure might have on cellular membranes, specifically when beet slices are placed in NaCl solutions of varying concentrations.
2. Hypothesis:
The osmolarity will directly increase with increasing NaCl concentrations.
3. Control = Distilled Water – this was present in all solutions
4. The independent variable – salinity of the 6 solutions; while predetermined, the NaCl concentrations varied from 0% to 15%.
The dependent variable – beet osmolarity; this lab’s focus was measuring the amount of damage inflicted upon a beet sample.
5. Wavelength used: 470nm
6. We used this wavelength because we were measuring the amount of lycopene in each solution; 470nm is the peak of lycopene’s absorbance.
7. Results table:

NaCl Solution
Absorbance Reading
Class Averages for Absorbance Reading
1
0 %
0.098
0.124
2
3 %
0.127
0.117
3
6 %
0.112
0.126
4
9 %
0.091
0.090
5
12 %
0.108
0.121
6
15 %
0.068
0.117

8. Currently, this is a very rough set of data, as the individual groups’ measurements widely varied from each other, and didn’t show a uniform trend. Therefore, I would advise the running of more tests to potentially create a more evenly spread set of data.
9. See attached data.
10. See attached graphs.
11. Damage in lower concentrations:
A few sources of damage incurred on the actual beet slices may come from the handling of the beet during prepping, stirring and removal, as well as from the effects of hypotension in the actual beet cells. Handling would be a potentially major cause, as there was inevitable damage done when slicing the sample, rinsing the sample, as well as from picking the sample up with tweezers and placing it into the solutions. Even though we were careful to limit actual touching, and rinsed prior secretions off, we still had to touch the sample.
Additional damage might have occurred through the stirring done every minute. Again, we were careful, and tried to avoid touching the sample when stirring the solutions, but we were not ‘perfect’ and therefore probably did cause some extra damage through handling, when stirring each solution. Similarly, the actual removal of the beet sample probably caused additional damage (i.e. more secretions of colorant) which we were unable to prevent from mixing with the rest of our solution, as we had to use tweezers to remove each slice from the solutions.
Another form of damage caused in lower concentration solutions, might be that of hypotension in the beet cells. This would be especially true for those samples in 0% NaCl solutions, as the actual salinity of the beet’s internal cellular system would be higher than that of the surrounding solution; water would seep into the cells, which would cause damage via making the cell turgid.
12. Damage in higher concentrations:
As in the case of lower concentrations, handling during prepping, stirring and removal would be a definite source of error. An additional source could be the effects of hypertension in the actual beet cells. Hypertension in the cells would be in effect in these solutions, as the NaCl concentrations of the surrounding solutions are most likely higher than the salinity of the internal cellular system; water inside the cell would leave to dilute the surrounding solution.
13. Materials/Methods:
Materials:
-1 slice beet
-1 frosting tip (small, circular object for cutting the beet into even samples)
-1 dissection pan
-1 graduated cylinder
-1 pair tweezers
-Spectrophotometer, with 1 cuvette
-Paper towels, kimwipes
-Appropriate safety wear (gloves, glasses, lab coat, and leather, closed-toe shoes)
-NaCl solutions at 0%, 3%, 6%, 9%, 12%, 15% concentration
-6 pipettes (1 for each solution, do not intermix)
-6 test wells
Methods:
Using the frosting tip and dissection pan, cut 6 samples of beet of similar-thickness from the original slice, with limited handling and place on a kimwipe. Rinse each with distilled water to remove any secretions, but do not pat dry. Meanwhile, fill each test well with 2.5mL of the NaCl solutions, use a different pipette for each solution to prevent the concentrations from mixing; if needed, label each test well so that the solution’s concentration is clear. Place one beet sample into each test well and let soak for 15minutes total, but be sure to stir every minute, limiting contact with the beet sample as much as possible. When the 15 minutes are over, remove the beet samples and discard.
Your spectrophotometer should be warmed up and blanked such that a cuvette of distilled water is the ‘zero’. Fill the cuvette with the solution from test well 0% and measure the absorbance reading. After recording the measurement, carefully rinse the cuvette out with distilled water and dry with a kimwipe. Repeat this process for each of the remaining 5 solutions until you have filled your data table. Once done, write down found values on the whiteboard to compare data with the rest of the class. Then clean up the lab area by rinsing the cuvette, frosting tip, and dissection pan with distilled water and drying, as well as throwing away any used pipettes. Lastly, return all items to their original locations, and wipe down the lab bench with ethanol.