Exercise 4 CELL TRANSPORT: DIFFUSION AND OSMOSIS Group 4 GO, Loisirc MALUBAY, Justin Damian PRIMERO, Karl Emerson

QUITORIANO, Raia Biology 10 TFIJ2, Mr. Mark Jun Alcantara 8 July 2013

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ABSTRACT

Cell transport is the mechanism at which cells absorb and excrete foreign matter through a semipermeable membrane. Diffusion and osmosis is an example of passive transport wherein matter moves down the concentration gradient where there is no energy consumption. This paper is a follow-up of the experiment done which aims to describe a semipermeable membrane and explain its role in osmosis, to define hypoosmotic, hyperosmotic and isosmotic in terms of relative concentrations of osmotically active substances, and to explain the importance of diffusion and osmosis to cells. The significance of the study is to give a more concrete explanation of the processes in cells. Part A introduces the semipermeable membrane and the diffusion of molecules wherein a dialysis bag is subjected to certain solutions. Part B details the osmotic activity in animal cells wherein slides with blood is subjected to solutions with different molarities. Part C details the osmotic activity in plant cells where the Rhoeo discolor leaf is subjected to a sugar solution and water. Cells therefore have cell membranes which are semipermeable allowing the exchange of matter through cell transport. Students should not deviate from the procedure as it is written. 2. KEYWORDS Diffusion, osmosis, cell membrane, plasmolysis, hemolysis

INTRODUCTION Communication within the cell and between cytoplasm and the external environment is facilitated by the regulated movement of materials into or out of the cell. The cell membrane separates the cytoplasm from the extracellular environment both of which are aqueous solutions. These solutions contain dissolved substances, or solutes, in the dissolving medium, or solvent. The cell membrane and membranes of the cell organelles are

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semipermeable, which regulates the movement of solute. There are two types of cell transport: active and the passive. The active transport expends energy in the form of ATP to accomplish movement against the concentration gradient. On the other hand, passive transport is accomplished down the concentration gradient without the expense of ATP and consumes only the kinetic

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energy of the constant random motion of molecules inside the cell. Le Chateliers Principle in chemistry suggests that a system tends to shift towards equilibrium, where the net movement is equal to zero. Diffusion is the net passive movement of particles (atoms, ions or molecules) from a region in which they are in higher concentration to regions of lower concentration. Osmosis is a special example of diffusion. It is the diffusion of water through a partially permeable membrane from a more dilute solution to a more concentrated solution down the water potential gradient. The solutes that exhibit osmosis are called osmotically active substances (OAS). Osmolarity and tonicity are related, but different concepts. Thus, the terms ending in osmotic (isosmotic, hyperosmotic, hyposmotic) are not synonymous with the terms ending in tonic (isotonic, hypertonic, hypotonic). The terms are related in that they both compare the solute concentrations of two solutions separated by a membrane. The terms are different because osmolarity takes into account the total concentration of penetrating solutes and nonpenetrating solutes, whereas tonicity takes into account the total concentration of only nonpenetrating solutes. Penetrating solutes can diffuse through the cell membrane, causing momentary changes in cell volume as the solutes "pull" water molecules with them. Non-penetrating solutes cannot cross the cell membrane, and therefore osmosis of water must occur for the solutions to reach equilibrium. A solution can be both hyperosmotic and isotonic. For example, the intracellular fluid and extracellular can be hyperosmotic, but isotonic if the total concentration of solutes in one compartment is different from that of the other, but ions cannot cross the membrane, it cannot draw water with it, thus causing no net change in solution volume. Part A utilizes the osmotic activity of glucose, a six-carbon sugar, and tested through the Benedicts test. As the name of the test suggests the reagent is called the Benedicts

reagent. Benedict's reagent is used as a test for the presence of reducing sugars. This includes all monosaccharides and many disaccharides, including lactose and maltose. Even more generally, Benedict's test will detect the presence of aldehydes, and alpha-hydroxy-ketones, including those that occur in certain ketoses. Thus, although the ketose fructose is not strictly a reducing sugar, it is an alpha-hydroxy-ketone, and gives a positive test because it is converted to the aldoses glucose and mannose by the base in the reagent. The copper sulphate in Benedict's solution reacts with reducing sugars. Benedict's solution can be used to tell if there is a sugar in a substance such as glucose in starch lamo. One litre of Benedict's reagent can be prepared from 100 g of anhydrous sodium carbonate, 173 g of sodium citrate and 17.3 g of copper(II) sulfate pentahydrate. It is often used in place of Fehling's solution. Benedict's reagent contains blue copper(II) ions (Cu2+) which are reduced to copper(I) ions (Cu+). These are precipitated as red copper(I) oxide which is insoluble in water. Benedict's Reagent provides a quantitative test for reducing sugars along with qualitative test. The color of the obtained precipitate gives an idea about the quantity of sugar present in the solution. A greenish precipitate indicates about 0.5% concentration; yellow precipitate indicates 1% concentration; orange indicates 1.5% and red indicates 2% or higher concentration. Part B explores the human blood in relation to solutions subjected to it and the processes that it undergoes, one is hemolysis. Hemolysis is the rupturing of erythrocytes (red blood cells) and the release of their contents (cytoplasm) into surrounding fluid (e.g., blood plasma). Hemolysis may occur in vivo or in vitro (inside or outside the body).. The ability of bacterial colonies to induce hemolysis when grown on blood agar is used to classify certain microorganisms. This is particularly useful in classifying streptococcal species. A substance that causes hemolysis is a hemolysin. The four solutions may either be isosmotic, hyperosmotic or hypoosmotic to the

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red blood cells. The cells may either lyse or shrivel or remain intact. A solution isosmotic and isotonic to the blood has a 0.15M. Part C distinguishes the plant cells from the animal cells in part B. Plant cells have cell walls made of strong material. Plasmolysis is the process in plant cells where the cytoplasm pulls away from the cell wall due to the loss of water through osmosis. This occurs in a hypertonic solution. The reverse process, cytolysis, can occur if the cell is in a hypotonic solution resulting in a lower external osmotic pressure and a net flow of water into the cell. Through observation of plasmolysis and deplasmolysis it is possible to determine the tonicity of the cell's environment as well as the rate solute molecules cross the cellular membrane. A plant cell in hypotonic solution will absorb water by endosmosis, so that the increased volume of water in the cell will increase pressure, making the protoplasm push against the cell wall, a condition known as turgor. Turgor makes plant cells push against each other in the same way and is the main line method of support in non-woody plant tissue. Plant cell walls resist further water entry after a certain point, known as full turgor, which stops plant cells from bursting as animal cells do in the same conditions. This is also the reason that plants stand upright. Without the stiffness of the plant cells the plant would fall under its own weight. Turgor pressure allows plants to hold their posture/form, and plants without turgor pressure (known as flaccid) wilt. The next is experimentation or the methods used then the discussion of results and lastly the conclusion. 4. EXPERIMENTATION Part A Diffusion of molecules through a selectively permeable membrane will be observed. The original procedure says that we need to make a dialysis bag (serves as the selectively permeable membrane) out of collodion but for practical purposes, longganisa skin was used.

The longganisa skin was filled by a mixture of four Pasteur pipettes of 15% glucose and starch solutions. In a 400-ml beaker, 300-ml water was poured in added with seven droppers of I2KI solution until the color turns golden-yellow. The skin was placed in the beaker making sure that its contents would not spill. In case there are spills, dispose the beaker solution and filled it again with the mixture. After 30 minutes, the bag was removed from the beaker solution. Physical changes were recorded. The Benedicts test was then performed to determine the presence of sugar in a solution. Two clean test tubes were labeled. Two Pasteur pipettes of the beaker solution were put in the first test tube while the same amount of the dialysis bag solution was put in the second test tube for testing. One drop of Benedicts reagent was added to each test tube. The test tubes were heated in a boiling water bath for 3 minutes. Results were then recorded. Part B Four clean microscope slides were labeled A, B C, and D. A drop of blood was then placed on slide D and then was observed under a microscope. In slide A, a drop of solution A was put and then added a drop of blood then observed under the microscope. The process was repeated for slides B and C. Observations were then recorded and tabulated. Part C A thin layer of the lower epidermis of Rhoeo discolor was stripped off and cut into two pieces using a scalpel and forceps. One of the specimen was mounted in tap water on a slide labelled E and then covered with coverslip. The cells of the leaf were observed under low power objective (LPO). The other specimen was mounted in 1.0 M sucrose solution on a slide labelled F, covered with coverslip, and then viewed under LPO. After two minutes, the two slides were compared. The specimen in slide F was then irrigated by placing tissue on one side of the coverslip then introducing water on the other

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side. After some moment, the sugar solution was completely replaced with water. The cells were again observed under LPO. 5. Data Results Bag starch and glucose solution cloudy white Beaker water and I2KI solution goldenyellow pale yellow orange

Original Contents Original Color

Cells are visibly larger than the normal blood cell. The average cell seems to have expanded as the outlines (dots in the center that carry oxygen) are more visible Cells set as the controlled set-up (Purely blood)

Final Color blue Color after yellow Benedicts test Table 1 Part A Results So lu ti o ns A Diagram of Cells

Description Table 2 Part B Results Cells are smaller than normal blood cell, uneven, seemingly deflated in appearance Cells seem to have more space between one another, although there are deviations, the average cell size seems to be as large as that of the normal blood cell (d) Mounting Medium Tap Water

Description After five minutes in tap water, the violet color inside the cell exhibited slight expansion, while the cell wall remained intact. After five minutes of exposure to sucrose solution, the violet color seen in the sample shrank thus became smaller leaving the cell wall in its original shape and position. The shrunk violet portion of the cell was entered by water and became bigger, pushing the cell wall outward.

Sucrose Solution

Distilled Water

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Diagram of Tap Water

Diagram of F before addition of sucrose

Diagram of F after addition of sucrose

the other hand, the water and I2KI mixture resulted to a golden-yellow solution. After 30 minutes of leaving the bag in the beaker, the color of both solutions changed. The color of the bag solution and the beaker solution turned bluish and pale yellow, respectively. The change in color from cloudy white to blue of the bag solution is due to the reaction of the diffused ion of I2KI in water, specifically iodide, and the starch. The same color was not observed in the beaker solution giving the idea that starch didnt diffuse out of the bag. Instead, the color of the beaker solution turned pale. This is because the I- ions were consumed as the reaction proceeds. It is already given that water will diffuse as it is the universal solvent. I2KI ions were able to pass through the bag. This is supported by the observed increase of the bag solutions volume. Unlike water and I2KI ions, starch molecules were not able to diffuse out of the bag since it is larger than the pores of the semipermeable membrane. The only substance left in question is glucose. In order to determine whether or not glucose diffused out of the bag, the Benedicts test was performed. It is a test primarily conducted to determine the presence and concentration of sugars in a solution. Table 1 shows the concentration of sugar in a solution via the resulting color changes. Solutions color Glucose concentration green ~0.5% yellow ~1.0% orange ~1.5% red ~2.0% or more Table 4 Part A Benedicts Test The resulting color of the bag solution is yellow while that of the beakers is orange. From the results, it is evident that both solutions contain glucose. Therefore, glucose can also pass through the pores of the bag. The large starch molecules may be accounted to why not all of the glucose molecules pass through the bag. Most probably the starch molecules blocked the pores of the bag disallowing all glucose molecules to pass through.

Diagram of F after addition of distilled water

Table 3 Part C Results

6. DISCUSSION Part A The mixture of starch and glucose in the dialysis bag produced a cloudy white solution. On

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In summary, water, I2KI and glucose passed through the bag while starch being a large molecule was not able to pass through it. Part B Blood cells on A can be observed to have shriveled, which can be attributed to A being a hypertonic. Due to the greater amount of solute outside the blood cell, the water would theoretically flow out to balance the concentration outside the cell, reaching equilibrium. Blood cells on B seem to have appeared to be similar to that of D's indicating the solution's isotonic nature. With an isotonic solution, the solute inside the cell and that of its surrounding environment is alike, causing the net movement of water to be 0. Blood cells on C seem to appear larger than that of D's. This is due to the absorption of water by the cell due to the solution's hypotonic nature. Since the red blood cell has more solutes than its environment, water is flown inside to balance the concentration of the cell, reaching equilibrium. Since the cell has absorbed water, it will swell and most likely, burst. Part C The slight expansion of the violet portion, which is the vacuole of the cell, is an example of cytolysis. Cytolysis is the net flow of water from a less concentrated environment to the more concentrated fluid in the cell. This phenomenon can be explained by osmosis, the flow of water from a hypoosmotic solution to a hyperosmotic solution through a semipermeable membrane. It can be said that the hypoosmotic solution is tap water as it may contain very small amount of dissolved substances such as minerals and dissolved gases. The hyperosmotic solution is the cells vacuole, solutes of which are waste products and small molecules which are in higher concentration than that of tap water. In contrast, when exposed to sucrose solution, the cell exhibited plasmolysis, the opposite of cytolysis. This happens when water flows from the less concentrated fluid in the cell to the more concentrated liquid in the

environment. It can be concluded that the hypoosmotic solution is the cells vacuole. The hyperosmotic solution is the sucrose solution, which has a relatively high concentration of 0.1M. When the sucrose solution was replaced by distilled water, vacuole swelled, pushing the cell wall outward. Unlike the animal cell, the plant cell did not burst because the cell wall is rigid and strong. Distilled water has no solute. Therefore, water comes rushing into the cell in order to attain equilibrium. However, this cannot happen due to the absence of solute in the water, but the vacuole eventually stopped swelling when the water potential, the tendency to give out water, of the cell is equal to that of distilled water. Plasmolysis is the shrinking of the protoplasm in a plant or bacterial cell away from the cell wall, caused by loss of water through osmosis. 7. Guide Questions Part A 1. Explain your results in terms of permeability of the dialysis tubing to the substances being tested. The dialysis bag allows molecules to pass through it only if these molecules are smaller in size compared to the pores of the bag. In this case, water, I2KI and glucose all have small molecules passed throgh the dialysis bag unlike the starch having a large molecule. 2. From your results, predict the size of I2KI molecules relative to the glucose and starch. The molecules of I2KI molecule is smaller relative to the size of the starch and glucose since I2KI molecules were able to pass through the dialysis bag. 3. What colors would you expect if the experiment started with glucose and I2KI inside the bag and starch in the beaker? We have already concluded that in terms of sizes, the smallest substance was water, then the I2KI molecules, glucose, the membrane pores, and the largest substance was the starch molecules. From there, we can say that placing glucose and I2KI inside the bag while the starch and water in the beaker would yield us to the same observation of the bags permeability.

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Therefore, glucose and I2KI molecules will move out of the bag while water will move in. The starch molecules are too big to pass through the bags pores leaving it outside the bag. The beaker solution will turn blue while the bag solution will be pale. Part B I. Explain your results. Blood cells on A can be observed to have shriveled, which can be attributed to A being a hypertonic . Due to the greater amount of solute outside the blood cell, the water would theoretically flow out to balance the concentration outside the cell, reaching equilibrium. Blood cells on B seem to have appeared to be similar to that of D's indicating the solution's isotonic nature. With an isotonic solution, the solute inside the cell and that of its surrounding environment is alike, causing the net movement of water to be 0. Blood cells on C seem to appear larger than that of D's. This is due to the absorption of water by the cell due to the solution's hypotonic nature. Since the red blood cell has more solutes than its environment, water is flown inside to balance the concentration of the cell, reaching equilibrium. Since the cell has absorbed water, it will swell and most likely, burst. II. Based on the results, which of the three solutions is Hyperosmotic - A (since A has more solute particles in the outside environment, the movement of water go outside of the cell to attain equilibrium) Hypoosmotic- C (since C has less solute particles in the outside environment, the net movement of water go into of the cell to attain equilibrium) Isosmotic- B (since B has the same amount of solute particles in the outside environment, the net movement of water goes outside of the cell to attain equilibrium) III. Define Hemolysis and Crenation.

Hemolysis- is the deterioration of the red blood cells membrane commonly detected by a red or pink tinge in the plasma. This deterioration is due to the entrance of the solution (mostly pure water) and other substances. Since this solution is hypotonic, water or the said hypotonic substance would enter into the blood cell to achieve equilibrium, causing it to expand and burst. This bursting is hemolysis (hemo-blood lysis-breakage) Crenation- is the formation of different etchings on the cell wall due to the exposure to a hypertonic solution. Since the environment has more solute than the red blood cell, the movement of water is outward to attain equilibrium, causing the cell to decrease in size and shrink. IV. What impact would be the principles investigated in the experiment on the blood cells have on medical procedures, such as intravenous feeding? Intravenous feeding is done by suspending the drug or the material, such as water in a saline solution. The most common type of intravenous feeding is crystalloid injections, which can either be hypertonic, hypotonic or isotonic. Hypertonic crystalloids are used to increase blood volume, much like colloids injected intravenously. This is due to the crystalloid's higher tonicity compared to the blood plasma, the water moving outside the red blood cell to dilute the high concentrations of electrolytes within the intravenous fluid. For hypotonic crystalloids, their lower tonicity compared to the blood plasma causes the water from the outside of the cells to enter the cells to dilute the electrolytes inside the blood cell. Hypotonic solutions are used mainly to counter cell dehydration. V. Explain the basis of meat preservation with salt or sugar. Salt and sugar are both materials used to regulate the amount of water inside the meat via osmosis. With sat/sugar added, the meat gradually loses water, causing it to shrivel. The lack of water inside the meat product would cause it to become sturdier, causing it less likely

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to collapse; since water is also a breeding ground for bacteria (since bacteria thrives with moisture), salt/sugar can be used as an effective ingredient in removing water, hence preserving the duration of food. Part C 1. Explain your results The slight expansion of the violet portion, which is the vacuole of the cell, is an example of cytolysis. Cytolysis is the net flow of water from a less concentrated environment to the more concentrated fluid in the cell. This phenomenon can be explained by osmosis, the flow of water from a hypoosmotic solution to a hyperosmotic solution through a semipermeable membrane. It can be said that the hypoosmotic solution is tap water as it may contain very small amount of dissolved substances such as minerals and dissolved gases. The hyperosmotic solution is the cells vacuole, solutes of which are waste products and small molecules which are in higher concentration than that of tap water. In contrast, when exposed to sucrose solution, the cell exhibited plasmolysis, the opposite of cytolysis. This happens when water flows from the less concentrated fluid in the cell to the more concentrated liquid in the environment. It can be concluded that the hypoosmotic solution is the cells vacuole. The hyperosmotic solution is the sucrose solution, which has a relatively high concentration of 0.1M. When the sucrose solution was replaced by distilled water, vacuole swelled, pushing the cell wall outward. Unlike the animal cell, the plant cell did not burst because the cell wall is rigid and strong. Distilled water has no solute. Therefore, water comes rushing into the cell in order to attain equilibrium. However, this cannot happen due to the absence of solute in the water, but the vacuole eventually stopped swelling when the water potential, the tendency to give out water, of the cell is equal to that of distilled water. 2. Define plasmolysis. Plasmolysis is the shrinking of the protoplasm in a plant or bacterial cell away from the cell wall, caused by loss of water through osmosis.

VI. CONCLUSIONS AND RECOMMENDATIONS A cell is a system in which transport of materials occur actively or passively and generally shifts toward equilibrium. The system may be affected with stresses from the outside environment, through the concentration gradient of the system to the surroundings. Diffusion and osmosis are necessary to maintain a biological balance with the organisms immediate environment. Active transport is important for the distribution of nutrients throughout the plants. The group recommends not deviating from the manual as it may have different results. Also, observe the slides carefully as it may have changed over a small amount of time. VII. REFERENCES Kuncaite, D. (2009). Diffusion and Osmosis. Retrieved from: http://www.eportfolio.lagcc.cuny.edu /scholars/doc_fa09/eP_fa09/Daiva.ku ncaite/documents/scb201_week3.do c. Laboratory manual of biology 10: General biology I hereby certify that I have given substantial contribution to this report. _____________________ GO, Loisirc _____________________ MALUBAY, Justin Damian _____________________ PRIMERO, Karl Emerson _____________________ QUITORIANO, Raia

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