Cell Membrane Structure and Permeability Lab Report PDF

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Salvador Ramirez Tinoco BIO-181L T 1230 February 04, 2020 John McCulley

Cell Membrane Structure and Permeability Lab Introduction The cell membrane primarily consists of lipids and proteins, with carbohydrates also playing a role. Phospholipids are the most plentiful of the lipids present with unique physical properties. Phospholipids are amphipathic, which presents both a hydrophilic heads and hydrophobic tails. Most membrane proteins are also amphipathic, with the hydrophilic region on the outside of the membrane (Campbell Biology, 2017). The cell membrane serves to facilitate required molecules in and out of the cell, so it can handle absorption, photosynthesis, respiration, ingestion, protein synthesis, and elimination of waste (GCU, 2016). The permeability of the phospholipid bilayer is selective, meaning only certain molecules and ions can pass through active or passive transport. Non-polar and hydrophobic molecules can pass through the cell membranes easier and quicker than polar and hydrophilic molecules. Transport proteins inside of the cell membrane allow charged ions and polar molecules to pass through. Channel proteins, a type of transport protein, contain a hydrophilic tail which serve as a channel for certain molecules and ion. Aquaporins, a type of channel protein, allow the passage of water molecules to easily pass in and out of the cell (Campbell Biology, 2017).

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The Brownian motion of particles is the random motion of molecules colliding, causing a change of direction. The motion of the particles is affected by concentration, temperature, and pressure. The particles colliding move from areas of high collision to an area with lower concentration of collisions. The shift of concentrations is known as diffusion. Diffusion ceases to exist when a consistent concentration is reached, the particles are then considered to be at equilibrium (GCU, 2016). Since the motion of molecules in diffusion is random, no energy is required. Each substance has its’ own concentration gradient, which is the diffusion from high to low concentration. For the non-permeable membranes existing in the phospholipid bilayer, the transport proteins present in the cell membranes allow the diffusion of particles across the concentration gradient. The diffusion of water across cell membranes is known as osmosis. The determining factor for the diffusion of water is the osmotic concentration which water diffuses from an area of high solute concentration (hypotonic) to low solute concentration (hypertonic) until solutes are near equilibrium (Campbell Biology, 2017). The cell wall of a plant can expand or contract. The tonicity of a plant cell directly effects the ability to do so. When a plant cell is placed in a hypotonic solution, water will diffuse into the cell causing an increase in turgor pressure causing the cell to push against the cell wall, eventually expanding the plant cell expands. When placed in a hypertonic solution, water will diffuse out of the cell leading to the cell wall to pull away from membrane and shrink. The shrinking of the cell is known as plasmolysis (GCU, 2016). This experiments in this lab are designed to test the diffusion, function of the cell membrane, and the permeability of specific substances within it.

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Hypothesis 1. A deshelled egg that is placed in a solution containing 30% sugar will see a net decrease in mass because the solutes inside the deshelled egg are lower creating a hypertonic solution for the cell through osmosis. 2. Chloride and sulfite ions will diffuse across the cell membrane because they are small enough to pass the semipermeable membrane. 3. The cell walls of the elodea leaf will undergo plasmolysis through osmosis when placed in a hypertonic solution because the solutes outside the cell is in higher concentration 4. Placing the beetroot in a solution of alcohol will release the most pigment from the cell because the alcohol will damage the cell membrane. Objectives 

Explain the process of diffusion across cell membrane through experiments and explain results.



Show how solute concentrations effect the movement of particles across concentration gradient in diffusion through data collected.



Observe plasmolysis in plant cells when placed in a solution.

De-Shelled Egg Experiment Materials

 

De-shelled eggs 500 milliliter (mL) beaker

 

10% sodium chloride (NaCl) solution 30% sugar solution

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

Scale (Flinn Scientific 410 grams [g]–0.01 g)

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Timer/watch Weigh boats

Procedures Procedures should be listed numerically as shown below. 1. Obtain one de-shelled chicken egg 2. Record the mass of the egg using a weigh boat on the scale and record the mass in Table 1 of the Cell Membrane Structure and Permeability Lab Worksheet. Be mindful not to damage the egg in the process. 3. Using a 600 mL beaker, add enough of the solution to fully cover the egg. 4. Keep the egg submerged in the solution for 90 minutes. 5. After the 90 minutes, remove the egg from the solution using gloves and reweigh using a weigh boat. Record the value in table 1 of the worksheet. 6. Repeat steps 1-5 for the other two solutions. Dialysis Bag Experiment: Materials

    

Dialysis tubing (cellulose) Thread or clips Funnel Dialysis solution (5% glucose, 1% starch, and 1% sugar) Culture dish

      Procedures

Small test tubes (13 x 100 millimeters [mm]) Iodine Silver nitrate 1% barium chloride Clinistix test strip Sodium sulfate (0.5%)

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1. Obtain a piece of dialysis tubing. Then tie a knot at the end of the tubing using a piece of thread. If the dialysis tubing wont open, run it under warm water. 2. Fill the dialysis tubing with a funnel with a solution containing 5% glucose, 1% starch, and 1% NaCl so that the bag is nearly full. The sodium chloride (NaCl) in the solution will dissociate into ions of Na+ and Cl-. 3. Tie another knot at the top of the bag sealing it while eliminating as much air as possible. 4. Clean the outside of the dialysis tube using tap water. 5. Weigh the bag and observe stiffness. Record observations in Table 3 of the Cell Membrane Structure and Permeability Lab Worksheet. 6. Notice the tautness (stiffness) and weight of the bag. Record observations in Table 3 of the Cell Membrane Structure and Permeability Lab Worksheet. 7. Place the dialysis tube into a culture dish and fill the culture dish so that half of the dialysis tube is covered in the solution containing 0.5% sodium sulfate (Na2SO4) which will dissociate into Na+ and S042+ ions. Fill the “before experiment” section for the “Outside Dialysis Tube” in Table 2 of the Cell Membrane Structure and Permeability Lab Worksheet. 8. Leave the dialysis in the solution untouched for 40 minutes. 9. Remove the tube from the culture dish after the 40 minutes, note the change in stiffness and record weight again and record observations in Table 3 of the Cell Membrane Structure and Permeability Lab Worksheet. 10. Rinse the dialysis tube and empty fluid into an empty beaker. 11. Obtain two sets of three small test tubes and prepare them as follows: a. Put 10 drops of the dialysis tube contents into each of the first set of three tubes.

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b. Put 10 drops of the solution from the culture dish into each of the second set of three tubes. 12. Use the following tests to search for starch, chloride ions, and sulfate ions in the two sets of test tubes: a. Starch Test: In the first test tube of each set, add three drops of iodine to test for starch. Starch will cause a color change. b. Chloride Test: In the second test tube of each set, add one drop of silver nitrate. Look for a white precipitate. c. Sulfate Test: In the third tube of each set, add three drops of 1% barium chloride. Barium chloride and sulfate ions yield insoluble barium fate that forms a white precipitate. Glucose Test: 1. Obtain three Clinistix test strips. 2. Using a transfer pipette, add two drops of water, two drops of culture dish fluid, and two drops of dialysis tube contents to separate sticks. a. Note: Green is a positive result compared to the negative control (water). 3. Record results in the Cell Membrane Structure and Permeability Lab Worksheet. Elodea Leaf Experiment: Materials:

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Young elodea leaf Compound light microscope 5% NaCl

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Dropper Glass microscope slide Cover slip

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Procedures: 1. Place a young elodea leaf on a slide, add a drop of deionized water, and place a cover slip over it gently. 2. Observe the cell on the outer edge of the leaf under a light microscope on low and high power. 3. Draw and label the figure in the Cell Membrane Structure and Permeability Lab Worksheet. 4. Removing the cover slip, add three drops of 5% NaCl, swap the old cover slip with a new one, and let the slide sit untouched for 5 minutes. 5. Observe the cells on the outer edge again under high power, and record observations by drawing the field of view in the Cell Membrane Structure and Permeability Lab Worksheet. 6. Draw a sketch of a single elodea cell that shows the position of the chloroplast and the central vacuoles in a plasmolyzed state. 7. Once observations are recorded, remove the leaf and rinse with tap water, and replace the leaf in the tray with tap water. 8. After 5 more minutes, observe the cells on the outer edge under high power. Record any change of the plasmolyzed state of a single cell. Beetroot Experiment: Materials:

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

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Hot plate 250 mL glass beaker Tongs

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Procedures: 1. Rinse nine diced beetroot cubes under water, being careful not to damage the cubes. 2. Put three cubes into three separate small test tubes, without bruising the cubes. 3. Prepare the test tubes as follows: a. Fill the first tube half full of water. b. Fill the second tube half full of water and set it in a boiling water bath until the water in the tube boils. c. In the third tube, add 1 mL of reagent alcohol and then add water so that the tube is half filled with liquid. 4. Shake all of the tubes thoroughly and allow them to sit undisturbed for about 30 minutes. 5. Compare the color of the liquid in the three tubes after 30 minutes and record the observations in table 4 of the Cell Membrane Structure and Permeability Lab Worksheet. Data Table 1 The Effect of Hypertonic/Hypotonic Solutions and Weight Solution

Weight Before Experiment

Weight After Experiment

Weight Change

Pure H20

80.40 g

80.04g

-.36g

30% Sugar

79.75g

80.11g

+.36g

10% Salt

72.68g

72.85g

+.17g

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

Inside Dialysis Tube Before Experiment

Outside Dialysis Tube

After Experiment

Before Experiment

After Experiment

Starc h

+

+

-

+

Chloride Ions

+

+

-

+

-

+

+

+

+

+

-

+

Sulfate Ions Glucose

Table 3 Documented Tautness (Stiffness) and Weight of Dialysis Bag Before and After Experiment Change in Characteristic of Dialysis Bag Stiffness (observation) Weight (mg)

Before Experiment

After Experiment

Difference

Fairly stiff

Less stiff

Lost a small amount of stiffness

24.62g

23.82

-.80g

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Figure 1: Elodea Leaf Experiment Drawings:

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

Color of Solution Light red tint

Boiled Tube

Dark red Red traces to almost clear solution

Tube with Reagent Alcohol

Analysis The occurrence of osmosis was observed by placing a de-shelled egg in a solution of pure H2O, 30% sugar, and 10% salt. When the egg was placed in a solution of 30% sugar the weight increased by .36g, indicating a hypotonic solution, not supporting the hypothesis. The egg tonicity was recorded to be hypotonic because the solutes in the solution presented a higher

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osmotic concentration; resulting in water to enter the cell through osmosis to balance the concentrations of solutes allowing glucose molecules to pass through the membrane. Both the H20 and the 30% Sugar solution saw a net increase in weight, thus indicating the samples were hypotonic. The osmotic concentration of solutes inside the cell was lowest in the 10% salt sample, resulting in the smallest weight gain of .17g. In the dialysis bag experiment, only sulfate ions were able to pass from the surrounding solution into the dialysis bag. Through diffusion, sulfate ions passed through the membrane along the concentration gradient. The diffusion of sulfate ions did not require any energy to enter the cell because sulfate ions were not present inside the bag, so the ions collided and moved into the cell to reach equilibrium. Chloride ions and glucose exited the bag because there was no concentration of chloride and glucose were not present in the sodium sulfate solution outside the bag, so they diffused outside the bag to the area of lower concentration. Starch appeared to not diffuse in or out the cell. Starch being the largest of the molecules tested is evidence that the size of molecules effect how readily transports of the molecules cross a membrane. Sodium ions were not tested because the ions were present in the solution both inside the bag and outside the bag prior. The hypothesis for this experiment was only partially supported as chloride ions did in fact diffuse outside the tube and sulfate ions diffused inside the dialysis tube. However, glucose also diffused from inside the dialysis bag to the outside. When the salt solution was added to the leaf, water was shown to leave the cell wall through osmosis, because the cells shrunk due to water leaving the cell to approach an equal osmotic concentration causing plasmolysis, supporting the hypothesis. If the cells of the leaf remained in the salt for several hours, water will continue to diffuse out of all the cells, until all water leaves the cell, thus killing them. Likewise, if a leaf is unable to receive any water the

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plant will wilt as all water will completely exit the cells. However, plasmolysis was shown to be reversible if the cells did not undergo extreme plasmolysis. When the leaf was then placed in a solution containing pure water the cells expanded larger than the initial cell size. The rigidity of a plant cell is due to the cell wall as it was shown to both expand and contract. Anthocyanin, a red pigment found in beetroots is not permeable in cell membranes. Compared to the control tube, the cubes placed in boiling water and alcohol changed to a darker red, supporting the hypothesis. They changed to a darker red because the anthocyanin left the cell due to the cell membrane being destroyed. No anthocyanin was released in the control tubes because it is not permeable to the cell membrane, thus no pigment is released. When the beetroots were boiled, the most amount anthocyanin was released indicating that a drastic increase of temperature severely damages the cell membrane. The heat increase denatures the transport proteins in the cell membrane allowing substances in the cell that are normally nonpermeable to be released to the environment. The heat increase also increases energy and speed of molecules which will result in more motion, thus increasing diffusion. When placed in isopropyl alcohol more pigment was released. Isopropyl alcohol is soluble in lipids, which makes most of the cell membrane. So, when isopropyl alcohol is dissolved in the phospholipid bilayer the membrane is weakened. Conclusion This experiments in this lab are designed to test the diffusion, function of the cell membrane, and the permeability of specific substances within it. The hypothesis that a de-shelled egg placed in a 30% sugar solution will increase the mass of the egg, the elodea leaf will undergo plasmolysis were supported by the data. The hypothesis that only chloride and sulfite ions will diffuse through the cell membrane was supported by

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the data but did not account for all solutes diffused. Lastly, the beetroot did in fact release pigment, however it did not release more pigment than boiling water. Effect of diffusion between hypertonic and hypotonic was learned in this lab. No noticeable errors were detected in the data collected.

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References Grand Canyon University. (2016). Structure and Functions of Macromolecules. Retrieved January 28, 2020 from https://lc.gcumedia.com/bio181l/laboratory-manual-for-general-biologyi/v2.1/#/chapter/1 Reece, J. B., Urry, L. A., Cain, M. L., Wasserman, S. A., & Minorsky, P. V. (2017). Campbell Biology (11th ed.). Boston, MA: Pearson....