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Estephania Campa BIO-181L R945 February 07, 2019 Professor Francis Cell Membrane Structure and Permeability Lab Report Introduction The cell membrane is used to help hold the organelles in place and give the cell a shape, but one of its most important features and function is its semi-permeability and how it allows certain molecules to com in and out. (Reece,Wasserman , Minorsky, Urry , Jackson, Campbell,& Cain, 2011). Many of the cells functions such as protein synthesis, photosynthesis, absorption, and ingestion all involve different transports through the membrane. [ CITATION Fra191 \l 1033 ]. The random movement of particles is called Brownian movement [ CITATION Are161 \l 1033 ] and be further classified to diffusion and osmosis. Diffusion is the movement of particles from an area of high concentration to an area of low concentration. [ CITATION Ree11 \l 1033 ]. The particles move down the concentration gradient, thus involves no energy and it moves passively. [ CITATION Are161 \l 1033 ]. Facilitated diffusion is the same as diffusion, except it does not pass through the membrane randomly, it goes through specific “roads.” [ CITATION Fra191 \l 1033 ]. Osmosis is also the passive movement of particles, but it is specific to water. Water moves from low solute to high solute, in efforts to “dilute” the solute and made it equal. [ CITATION Fra191 \l 1033 ]. Different factors that affect passive movement are the size, concentration, lipid solubility and space. [ CITATION Fra191 \l 1033 ]. Active movement needs energy to pass larger molecules and often uses ATP. An example of active movement is the

sodium-potassium ion pump. It carried both molecules in and out of the cell with ATP. [ CITATION Ree11 \l 1033 ]. As each particle is moving in and out of the cell, because the cell one of three things: hypertonic, hypotonic, or isotonic. If the cell is hypertonic, then the outside of the cell is higher in concentration. The cell would lose water and shrink, because water moves from low to high concentration. [ CITATION Fra191 \l 1033 ]. If the cell is isotonic, the concentration is the same inside and outside of the cell, and there is little to no movement, in either direction. [ CITATION Fra191 \l 1033 ]. If the cell is hypotonic, then the concentration is lower on the outside of the cell and would gain water and expand. [ CITATION Fra191 \l 1033 ]. At hypertonic, an animal cell would shrivel up and a plant would plasmolyze with the lose of water. Isotonic animal cells are happy, because there is little to no movement. A plant cell would be wilted, because plant cells like to be turgid, slightly swollen. Hypotonic plant cells like this best, because the cell wall is puffed out with water and it holds the plant upright. Hypotonic animal cells would burst with all that excess water. In this lab experiment there will be four different experiments to test cell membrane permeability. A deshelled egg placed in differing solutions of water, a dialysis bag submerged in sodium sulfate, an elodea leaf mounted on 0.5% sodium chloride, and a beetroot placed in water, boiling water, and alcohol, will all be tested to identify how the membranes react to each solution. [ CITATION Are161 \l 1033 ]. Hypothesis 1. A deshelled egg placed in 10% salt will lose weight because the egg is hypertonic compared to the water and the water will move out of the egg.

2. In the dialysis bag, the chlorine ions and sulfate ions will cross the membrane by diffusion because the molecules are small enough. 3. A plasmolyzed elodea cell will become turgid after being placed in a hypotonic enviroment, by process of osmosis, the water will move into the cell. 4. Beets placed in heat will release more water because the molecules in the vacuole will move faster and burst, releasing more pigment and water. Objectives

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Identify if a substance is hypertonic, isotonic, or hypotonic based on how the solvent diffused.

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Practice making a wet mount and using it to identify plant cells.

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Test solutions for starch with iodine, chloride ions with silver nitrate, sulfate ions with barium nitrate, and glucose with Clinistix strips.

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Describe how both diffusion and osmosis move molecules through membranes.

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Compare different samples of beetroot to one another and describe the difference in pigmentation in each one.

De-Shelled Egg Experiment Materials

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3 De-shelled eggs 3 Beakers (600 mL) 10% sodium chloride (NaCl) solution 20% sugar solution

    Procedures

Scale Gloves Timer 3 Weigh boats

1. Every lab bench will get one de-shelled chicken egg, to be used by that entire group. Lab professor will assign each group a specific solution. 2. Carefully, weight the egg on a scale, using a weigh boat. Record this number in Table 1, under pertaining solution. 3. In a beaker, place the egg, and cover the egg with the solution assigned to that lab bench. 4. For 1.5 hours, let the egg sit in the solution, at room temperature. 5. Once the egg had been in the solution for 1.5 hours, remove the egg from solution using gloves. Place the egg back on the weigh boat and weigh it. Record new weight in Table 1. Share the results with the class. Dialysis Bag Experiment Materials

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Dialysis tubing Clips Funnel Dialysis solution (5% glucose, 1% starch, and 1% sugar) Culture dish Timer

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6 Small test tubes (100 mL) Iodine Silver nitrate 1% barium chloride Clinistix test strip Sodium sulfate (0.5%)

Procedures

1. Tie one end of a piece of dialysis tubing, with clips. Open other end of tubing. 2. With a funnel, fill the tubing with dialysis solution (5% glucose, 1% starch, 1% NaCl), until nearly full. The NaCl molecules will dissociate and be present as Na+ and Cl-. 3. Secure the tubing with a clip, eliminating as much air as possible. 4. Thoroughly rinse the bag by running it under some tap water.

5. Record the tautness and weight of the bag in Table 3 6. Put the tubing in a culture dish and note the tautness. Fill out the first column of Table 3. 7. Pour 0.5% sodium sulfate (Na2SO4) into the dish, until the tubing is half submerged in the solution. Sodium Sulfate will dissociate to Na+ ions and SO4- ions. Finish the “before” section of the “Outside Dialysis Tube” column in Table 2. 8. Leave the dialysis tube in the culture dish for 40 minutes. 9. Remove the tube after 40 minutes, note the tautness of the bag, and weigh it. Record data and observations in Table 3. 10. Rinse the tube under running water and empty the solution into a clean 250 mL beaker. 11. Obtain 6 test tubes (2 groups of 3 tubes) and set them up as follows: a. Put 10 drops of the tube contents in 3 tubes. b. Put 10 drops of the solution in the culture dish in the other 3 tubes. 12. Test for starch, chloride ions, and sulfate ions in the test tubes as follows: a. Starch Test: Add 3 drops of iodine to the first tube in each set. If starch is present, it will cause a color change. b. Chloride Test: Add one drop of silver nitrate to the second tube of each set. If chloride is present, then a white precipitate will form. c. Sulfate Test: Add three drops of 1% barium chloride to the third tube in each set. If sulfate is present a white precipitate will form. 13. Test for glucose inside the bag and outside as follows: a. Get three Clinistix strips

b. Using a pipette add two drops of water, two drops of solution outside of the tube in the dish, and two drops of solution in the tube on the three different strips of Clinistix. 1. Green is a positive, compared to the negative control of water. c. Record the results in Table 2

Elodea Leaf Experiment Materials

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Elodea leaf Compound light microscope 5% NaCl

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Dropper Glass microscope slide Cover slip

Procedures

1. Place an elodea leaf on a slide, add a drop of water and place a cover slip over the leaf, thus preparing a wet mount. 2. Use a light microscope on low and high power to examine the cells of the leaf. 3. Draw the cells in Figure 1.A and label it along with the correct magnification. 4. Remove slide from microscope, take off the cover slip, and add three drops of 5% NaCl. Put cover slip back on and let it sit for 5 minutes. 5. After 5 minutes, examine the cells under high power. Observe the cell and draw them in Figure 1.B 6. Draw one cell labeling and showing the chloroplasts and central vacuoles in the plasmolyzed state. 7. Take the slide off mount and rinse the leaf with water. Remount the leaf with regular tap water.

8. Observe the cells once again and draw observations in Figure 1.C. Beetroot Experiment Materials

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9 Beetroot cubes Small test tubes (13 x 100 mm) Reagent alcohol (70%)

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Hot plate Glass beaker (250 mL) Tongs

Procedures

1. Rise 9 cubes of beet under tap water. 2. Place three cubes in each test tube. 3. Prepare the tubes for testing, as follows: a. Fill first tube with half water b. Fill second tube with half water and set in a boiling bath until the water inside the tube boils as well. c. In the third tube, add reagent alcohol (1mL) and then add water till it is half full. 4. Shake the tubes and let them sit for 30 minutes. 5. After 30 minutes compare the pigmentation of each tube to one another and record observations in Table 4

Data Table 1 The Effect of Hypertonic/Hypotonic Solutions and Weight Solution

Weight Before Experiment

Weight After Experiment

Weight Change

Tonicity

Pure H20

94.43 g

98.10g

3.67 g

hypotonic

20% Sugar

87.4 g

87.13 g

-0.27 g

Isotonic

10% Salt

92.00g

96.03g

4.03 g

Hypotonic

Table 2 Results of Investigating the Permeability of Dialysis Tubing to Various Substances Substance

Starc h Chloride Ions Sulfate Ions Glucose

Inside Dialysis Tube Before Experiment (+)

After Experiment (+)

Outside Dialysis Tube Before Experiment (-)

After Experiment (-)

(+)

(+)

(-)

(+)

(-)

(+)

(+)

(+)

(+)

(+)

(-)

(+)

Table 3

Documented Tautness (Stiffness) and Weight of Dialysis Bag Before and After Experiment

Change in Characteristic of Dialysis Bag

Before Experiment

After Experiment

Difference

Stiffness (observation)

Squishy

Stiff

Got stiff, with water gain.

Weight (mg)

29.91 g

31.21 g

It gained 1.3 g

Figure 1 02/06/2019 – Estephania Campa

02/06/2019 – Estephania Campa

2/06/2019 – Estephania Campa

Table 4 Permeability of Beetroot Cell Membrane after Treatment with Heat and Reagent Alcohol Tube Control Tube

Color of Solution Pale red / fuchsia

Blood red / dark red

Boiled Tube Tube with Reagent Alcohol

Red

Analysis The solution with 20% sugar was isotonic, with the egg losing little to no weight. The solution with 10% salt was hypotonic, gaining weight. The egg submerged in water was hypotonic as well, gaining weight too. The salt and sugar solution eggs result in errors that will be discussed later. The greatest weight gain came from the salt solution, at 4.03 grams added to the egg. The greatest weight loss was in the sugar solution, losing 0.27 grams. Once again, the salt and sugar solutions resulted in errors, that will be discussed later. The ions Na+ and Clshould have been unable to pass through the membrane because an egg’s membrane would not have allowed it to pass. [ CITATION Ree11 \l 1033 ]. The sugar molecules did not pass through the membrane because the molecules are too big, the egg membrane is semi-permeable to them. In the dialysis bag experiment chloride, sulfate, and glucose were able to pass through the membrane, but starch did not pass through. Each substance crossed the membrane by process of diffusion. The chloride and sulfate moved from an area of high concentration, to an area of low concentration, thus moving outside of the bag. Glucose crossed the membrane into the bag, also by diffusion. Water also inherently moved into the bag, by process of osmosis, water moved from low to high solute. Both the glucose and water moved into the bag, causing it to gain 1.3 grams. Since all these substances moved by process of diffusion or osmosis, which are passive transport,

there is no energy required. The size of molecule does affect membrane transport. As seen in the starch, which was unable to pass.[ CITATION Fra191 \l 1033 ]. It did not pass the iodine test, for the samples of solution outside of the tubing. Those molecules were too big for the membrane to passively move them through. All the molecules that were able to passively diffuse through the membrane did. Glucose, chloride ions, and sulfate ions were all able to passively diffuse. The silver nitrate test showed there was chloride ions, in and outside the bag; the barium chloride test, tested positive for sulfate ions, in and out of the bag; and the Clinistix strips tested positive for glucose, in and outside the bag. There was no test for sodium ions, because sodium only moves through active transport and it would not have passed the membrane. [ CITATION Ree11 \l 1033 ]. Water exited the elodea leaf cell when the salt solution was added. This happened because the water in the cell moved by osmosis, to an area of higher solute. If these cells were allowed to remain in this solution for several hours, the cell would plasmolyze, the cell wall would retract from the sides.[ CITATION Fra191 \l 1033 ]. This process can be reversed as seen in the experiment. Once shriveled cells, that were plasmolyzed, became turgid when regular water was reintroduced to the cell. [ CITATION Fra191 \l 1033 ]. Most of the organelles were still compact in the center of the cell, but if left for a while, the cell would be back to “normal.” Plants do not have skeletons that help keep them upright, but they have a very rigid and strong cell walls. [ CITATION Ree11 \l 1033 ]. When these cells are turgid and hypotonic, it allows plants to hold an upright position, even without help. [ CITATION Fra191 \l 1033 ]. Plants begin to wilt when in an isotonic state. Without the extra water to keep the central vacuole full, the plants begin to wilt.

The beetroot experiment allowed for anthocyanin to pass through the cell membrane, because the membrane was burst when the beetroot was cut. [ CITATION Fra191 \l 1033 ]. This allowed for the initial release of the pigment, in the control, as well as the other samples. The control turned a pale red. More pigment was released in the boiling water, because the molecules begin to move faster, eventually bursting through the vacuole and membrane and allowing for more release of pigment in the beetroot, than the control. Boiling proteins causes them to denaturize and unravel. [ CITATION Ree11 \l 1033 ]. The boiling water pigment turned dark red. In the isopropyl alcohol, the beetroot did release pigment, but not as quickly, and not as much, compared to the boiling water, but more than the control. The isopropyl alcohol pigment turned an average red color. Lipid solubility in alcohol is high and this is likely why the alcohol beetroot was darker than the control. Once the dye is out of the membrane, if diffuses throughout the entire solution, because if goes from a high to low concentration. With a higher temperature, pigment diffuses faster, because the molecules are moving more rapidly. Conclusion The purpose of this lab was to test cell membrane permeability in a de-shelled egg, dialysis tubing bag, an elodea leaf cell, and a cube of beetroot. Each test showed one facet of membrane permeability and how Brownian movement occurs. [ CITATION Are161 \l 1033 ]. The hypothesis for the de-shelled egg - a deshelled egg placed in 10% salt will lose weight because the egg is hypertonic compared to the water and the water will move out of the egg, was not supported by the data in this experiment. The hypothesis for the dialysis bag experiment - in the dialysis bag, the chlorine ions and sulfate ions will cross the membrane by diffusion because the molecules are small enough, was supported by the data. The hypothesis for the elodea cell experiment - a plasmolyzed elodea cell will become turgid after being placed in a hypotonic

enviroment, by process of osmosis, the water will move into the cell, was supported by the data. The hypothesis for the beetroot experiment - beets placed in heat will release more water because the molecules in the vacuole will move faster and burst, releasing more pigment and water, was supported by the data. Errors arose in the de-shelled egg experiment. The sugar solution should have made the egg lose weight as it is hypertonic, but it ended up being isotonic. In addition, the salt solution should have been isotonic, but it ended up being hypotonic and gained 4.03 gram. In both eggs, what could have occurred is that they both were not as “concentrated” as an egg may normally be. The eggs could have been sitting in the de-shelling solution for too long or the solution itself could have been contaminated. There was also a small error in the Dialysis Bag experiment. The starch should have turned color after adding the iodine, but it did not. What happened was that the solution was not mixed well and there was not enough starch present in the tube. The professor added more starch to the tube and then the solution turned the correct color. This lab was an excellent way for the students to learn about cell membrane permeability and how different molecules move across cell membranes, cell walls, and membranes in general.

References Areda, D., Boyles, R., Francis, G., & Hite, A. (2016). Laboratory manual for General Biology I. Retrieved 05 February 2019, from http://lc.gcumedia.com/bio181l/laboratory-manualfor-general-biology-i/v1.1/#/chapter/6 Francis, G. (2019). Week 4: Membrane & Cell Permeability. BIO-181L: General Biology 1 – Lab. Phoenix, AZ: Grand Canyon University. Reece, J. B., Urry, L. A., Cain, M. L., Wasserman, S. A., Minorsky, P. V., Jackson, R., & Campbell, N. A. (2011). Campbell Biology. Retrieved 05 February 2019, from https://viewer.gcu.edu/24WWXP...