37 LABORATORY 3 ‘THE BEHAVIOR OF LIVING CELLS Geils are the building blocks of the human body. The activity of the human organism depends on the individual and collective actions of its many cells. Changes that disrupt cell structure can lead to the injury or death of living cells ‘The cell membrane forms the boundary thal separates the living cell from its external environment ‘All molecules that enter or leave the cell must either pass directly through the cell membrane of pass through channels that exist in the cell membrane. Since the cell membrane only allows certain molecules to pass through it, while excluding others, it is known as a selectively permeable or semipermeable membrane. Movement of substances into and out of cells through the cell membrane occurs by several transport mechanisms. The processes of diffusion and osmosis will be examined in the laboratory. 4. Diffuston Exercise Diffusion is generally defined as the movement of molecules from a region of high concentration toa region of lower concentration until a state of equilibrium is reached, As an example, if table salt (NaC) is dissolved in water, the salt dissociates and its ions disperse throughout the water molecules until a Uniform solution results. Diffusion is a very important mechanism within the human body. For example, the respiratory gases (oxygen and carbon dioxide) move across cell membranes by diffusion. 4, At the beginning of the lab period, fill @ test tube approximately two-thirds full of tap water. Using a pair of forceps, carefully drop an orcein crystal into the test tube, 2. Place the test tube into the test tube rack and leave it at room temperature. Do not disturb the tube throughout the lab period. Observe every half hour. \What happens tothe orcein crystal as the lab period progresses? Purple Lolose diFESes Tocovatnors tesy Whe Briefly explain how this experiment demonstrates cifusion Eystars Move from CTT Chattom of tube) to GV} Cop of whe VOD) C4oriver s reached (usiforrs purple Colour) ‘As a demonstration, an orcein crystal was dropped into each of two test tubes, One test tube was placed in a hot water bath and the other was placed in a bucket containing ice. At the end of the lab period, compate your test tube (which was at room temperature) to the demonstration test tubes. Record your observations in the space below. HOT- entnre the is purple - diffuses Fasrest COLD - \ess purge —Aikeuses Slows ROOM-_Ly between The: oTher WO when T tap , fT motor activing 1 collision w/ H2 0 Je Raster AERUSton rare38 Dialysis Tubing Exercise Recall that each cell is bounded by 2 selectively permeable cell membrane; that is, a membrane which allows some, but not all, substances to pass through, The permeability of the cell membrane to a specific molecule is partly related to the size of the submicroscopic pores which are present, Large molecules may be very slow in passing through the cell membrane or thelr passage may be completely blocked, Water is one of the few substances that can pass freely back and forth through a cell membrane. Diffusion refers to the passage of molecules from a region of high concentration to a region of lower concentration. As the water regulation of a cell is very important, the diffusion of water molecules through a cell membrane is given a special name, osmosis. Osmosis, then, by definition, is the diffusion of water molecules through a selectively permeable membrane. Water moves across the membrane from region of high water concentration to a region of lower water concentration. To demonstrate the processes of diffusion and osmosis, you will perform either Part A or Part B of the following experiment. Be sure to record the results obtained in both Parts A and B. You will be using starch and iodine solutions in this experiment. Starch is large organic molecule (polysaccharide), composed of many glucose units (monosaccharides) joined by covalent bonds. One property of starches is that they react with iodine molecules to produce @ dark blue color. Iodine is a small inorganic molecule. The dialysis tubing in this experiment represents the selectively permeable membrane of a cell The tubing permits the passage of small molecules such as iodine but restricts the passage of large molecules such as starch, The iodine solution has a high concentration of water molecules (a dilute solution) while the starch solution has a low concentration of water molecules (a more concentrated solution). Part A (Figure 1) 4. Fill a 500 ml beaker with approximately 300 mi of a starch solution. 2. Cuta strip of dialysis tubing 4 to 5 inches long. Handle the dialysis tubing only at the ends and as litle as possible, Moisten the tubing completely with tap water and tie one end closed with a string. Hold the tubing under running water and fil with water. If water runs out of the bottom, retie the string. Empty the tubing. |— lodine solution Dialysis bag is full Starch solution FIGURE 4. Initial Set-up of Part A39, 3. Fill the tubing full with an iodine solution and tie the other end with string. 4. Float the "bag’ filed with iodine solution in the beaker containing starch solution, 5, Observe after ninety minutes. Particularly note the color of the solution within the bag and beaker, and the amount of solution in the bag relative to the amount at the start of the experiment. In the space below, use a diagram to explain what you have observed in Part A of this experiment after 90 minutes. Your observations should include: any movement of water and the process by which these molecules moved; any movement of starch or iodine and the process by which these molecules moved; any volume changes in the bag and beaker and any color changes in the bag and beaker, Boke sfhoses ‘ = lo& Menenod Ov OF Ave. HAD, TP | O* Sractin ‘Pre Contents OP The. peaxer Wue/ Lace Vo moves vt of We Bag, by COSMAS SO U bag, valuene- Part B (Figure 2) 4, Filla 500 ml beaker with approximately 200 m! of an fodine solution. 2. Prepare the dialysis tubing as in Part A. Dialysis bag is 3/4 full tor + fst station + - | socine solution i ep + + FIGURE 2. Initial Set-up of Part B40 3. Fill the tubing 3/4 full with a starch solution and tie the other end with string 4. Float the "bag" filled with starch solution in the beaker containing iodine solution 6. Observe after ninety minutes. Particularly note the color of the solution within the bag and beaker, and the amount of solution in the bag relative to the amount at the start of the experiment. In the space below, use a diagram to explain what you have observed in Part B of this experiment after 90 minutes. Your observations should include: any movement of water and the process by which these molecules moved; any movement of starch or iodine and the process by which these molecules moved; any volume changes in the bag and beaker and any color changes in the bag and beaker. \ Xe lodroe O= storey Todme errecs The bas Yl agave process 08 Aikedon ; Joe maa comrtats oF bag he Wide / lace colour M Boo enacs hag by THe, M20 eee Se of oben Mi ‘bao, Volurne © Define: osmosis_prov'¥ oF B20 From CV) to LV] Mrrovah a semi- permeable Membrane Watt) equilbciumis Ceached diffusion PAYA OF Worecules Foor TTA to LY] Gon) equaiooy es is ceacked 3. Osmosis Across an Egg Membrane ‘The movernent of water across the cell membrane is critically important for cells. The intracellular molecules that are necessary for metabolic activities are often prevented from crossing the cell membrane. Consequently, if changes occur in the usual concentrations of these molecules, changes in water concentrations on both sides of the cell membrane can result. This situation can alter a cel's intemal volume of water, producing either a net gain or a net loss of water by the cell. As gaining water may cause a cell to swell and burst and losing water may cause a cell to shrivel and die, itis crucial that the balance of water between the cell and the fluid surrounding the cell be maintained. A solution's tonicity is a measure of the solution's ability to change the shape of cells by affecting the cells’ internal water volume. Two solutions that have the same concentration of dissolved particles and water molecules are isotonic solutions (iso means “the same"), A cell placed in an isotonic solution will maintain its normal shape and volume. A solution that contains a lesser concentration of dissolved particles and a greater concentration of water molecules than the call is a hypotonic solution (hypo means “below'). Placed in“4 this solution, water molecules enter the cell faster than they leave and the cell swells. The water molecules wil low by osmosis from the area of greater water concentraticn (outside the cell) to the region of lesser water concentration (inside the cell). A solution that contains a greater concentration of dissolved particles and a lesser concentration of water molecules than the call is a hypertonic solution (hyper means ‘above"). In such a solution, water molecules move out of the cell faster than they enter, causing the cell o shrink, Water will fow by osmosis from the area of greater water concentration (Inside the cel) fo a region of lesser water concentration (outside the cell), Quail eggs will be used to quantify water movement across a living semi-permeable membrane. Quail eggs have been immersed in a 5% acetic acid for 24 hours to remove the calcium carbonate shell. ‘Two membranes are just inside the shell ~ an outer and inner shell membrane surrounding the albumin (egg white). The two membranes provide an efficient defense against bacterial invasion and are made partly of keratin protein. Prior to the start of the experiment, the membrane bound egg is stored in a 28% sucrose "holding solution” which Is isotonic to the egg contents. 4, Atthe beginning of the lab period, using the plastic spoon, carefully remove en egg from the sucrose holding solution. Gently blot it dry with paper towel, and in the table below, record the weight to two decimal places Using the automated balance. Place the egg in the 42% sucrose solution and note the time. 2. Gently remove the second egg trom the holding solution and blot it dry and record the weight. Place that second egg inthe disiled water container and note the time 3, Leave the eggs in the solutions for two hours. 4, Alter two hours, remove the first egg from the 42% sucrose solution, gently blot it dry and record the weight. Place the egg back into the holding solution container. Carefully remove the second egg from the distlled water, gently blot it dry and record the weight. Return the egg to the holding solution with the first egg Initial weight (g) _| Weight after 2 hours (g)_| Weight change (g)_| De-calcified egg soaked BiaWy Pesaro Veg Do-calcitied egg soaked Tuseiony in distilled water Be oo Tee teem ei geee Which solution is hypotonic tothe egg contents?__ Dis Ved W¥2.0 Explain your observations, describing the direction of water flow. Ya} sucrose unrl\ couse ecyg te lose warte (urigl less) 20 moavis WV CT gr aden’ 00% of 49 & tbo Solu tien DiseWe d Yo causes gq ts gan B20 Cwegh moce). W20. vrD © 94 mons UTS gradseny From Salve Ken MW42 4, Volume Changes in Red Blood Cells When red biood cells are in an isotonic solution, such as blood plasma (which Is the fluid surrounding red blood cells), no net flow of water molecules ocours. As there is no net gain of loss of water, the red blood cell retains its normal shape and function, If red blood cells are placed in a hypotonic solution, such as distilled water, the water molecules. will enter the cell from the area of greater water concentration to the region of lesser water concentration, ‘As the water enters the cell, the red blood cell begins to increase in size. This swelling does not occur indefinitely, however. At a certain volume, the cell membrane ruptures and the contents of the red blood cell are released, leaving only fragments of the cell membrane behind. This phenomenon is known as hemolysis. Ifred blood cells are placed in a hypertonic solution, such as a concentrated salt solution, water will eave, The cell fends to shrink and lose its characteristic shape, a phenomenon known as crenation. Examine slide #49 of a human blood smear. To prepare this slide, @ drop of blood was spread thinly over the slide and then Wright's stain was applied. Red biood cells are numerous and appear as pale staining biconcave discs, which do not contain 2 nucleus, White blood cells are less numerous and have a purple stained nucleus. Proportionately, there are about 700 red blood cells for every white blood cell To illustrate the effects of isotonic, hypotonic and hypertonic solutions on living cells, animal blood willbe used. Its important to follow safe (aseptic) procedures when working with any type of body fluids. Bofore you begin this exercise, wipe down the bench top with the disinfectant spray provided. Each group ‘member should wear protective disposable gloves. 1. Using a grease pencil, label two clean glass slides A and B. 2, Add a drop of 0.9% sait solution to slide A Add a small drop of animal blood to the saline using a disposable glass pipette and stir the sample with a toothpick. Discard the toothpick in the autoclave bag provided. Carefully lower a coverslip onto the slide. Immediately examine the slide under the 40X ‘objective lens. Notice that the red blood cells should have a size and shape similar to slide #19. 3. Add a drop of concentrated (10%) salt solution to slide B. Add the blood and stir, Place on the coverslip. Carefully cbserve the red blood cells under high power. What is happening to the normally smooth disc shape of the red blood calls? Explain your observations describing the direction of water flow, Cells beard to Soewel, become “vaicsen- Wee” = cae & crenarien b/c We moves VE geadienk from ely te Souter 4, You will next need to fold a piece of fter paper in half. Add a drop of distilled water to the edge of the coverslip of slide B. Place the folded edge of the filter paper at the opposite side of the coversiip. It will absorb the saline solution and draw the distiled water across the cells. Watch the red blood cells, as they float across the surface. Explain what is happening to the size and shape of the cells. Celis Susi then burst? catled he molyars w/e W26 moved VED qraduent Kroon Soloricn to Cell The red blood cells intially should "plump up” but then they suddenly start to disappear. The red blood cells burst as the water floods into them, leaving "ghosts", membrane fragments, in their wake.43 \ich souton is hypertonic the red blood cets?_\O. sant hypotonic to the red blood cols? ANSAINCA Ye __ssolonie to the red blood ces? O-S'L Sort b/c Wo aes MOV DE WANE AEBE appears Harmar Snape § Vowme ‘Once you have completed your observations, discard the three slides with their coverslips in the labeled disinfectant dish. Wash down the lab bench once again with the disinfectant spray and discard your gloves in the autoclave bag, Disruption of the Cell Membrane The living cell membrane Is called a phospholipid bilayer because it is composed of a double layer of phospholipid molecules with protein molecules immersed or fioating throughout the lipid portion. The cell membrane is disrupted if either the lipid or protein component is altered significanty. If the cell ‘membrane is disrupted the cell is no longer able to retain all the cellular contents. To ilusttate the composition and function of the cell membrane, you will be using a biological ‘model and disrupting the cell membrane with acetone and heat, Acetone is a lipid solvent and proteins characteristicaly denature (break down) at high temperatures, Cubed beet roots have been prepared by chopping a small portion of a raw beet and washing the cubes thoroughly with cool tap water to remove the pigments that are liberated by the cutting. The color of this material is the result of the anthocyanin pigments which are present in the beet cell cytoplasm. ‘The intact, living cell membrane is not permeable to these pigments. 41, Label three test tubes 1, 2 and 3, 2, Cover two cubes of beet root with 2 mi of distiled water in test tube 1 and leave as a contol 3, Cover two cubes of beet root with 2 ml of distilled water in test tube 2 and place on a heating plate for 15 minutes. 4, Cover two cubes of beet root with 2 mi of acetone in test tube 3 and let stand for 30 minutes with ‘occasional shaking, Be certain the test tube is covered with parafilm. Dispose of the contents of this tube in the discard jar, not down the drain. 5, Observe each test lube for the relative amount of pigment that is present in the solution. For each test tubs, explain what has happened to the cell membranes of the beet cells. Has the anthocyanin pigment been released from the cell? Briefly explain why these results were obtained, Test tube 4 - Control__Ce\\ membrane 18 wotach PAGQerenr Grows WSde celle H2O0 is clear Testtube 2-Heat__Cei\ Memiorane is Aiscuptta — Erolern COmMpENEoy CR mennorane 18 denatured by eax Damen’ escapes aod N2o Tens ced Test tube 3 - Acetone Cer sreanoecane is Siscupred - gia tomponen® OF membrane is dissolved by acetone Lament escapes and Acetone tocas ce A Make: Mgny ISS See ropes oF Plgment va acerone '6/C Qreeny 15 NSE Sole BEMComplete the following questions to review what you have learned about cell membrane permeability. © A dialysis tubing bag was constructed and filed with biuret solution. The bag was then placed ina beaker containing an albumin solution. Assume that the dialysis tubing is permeable to biuret molecules and impermeable to the albumin molecules (albumin is egg while protein). Also assume that the biuret solution contains a high concentration of water molecules and the albumin solution contains a low concentration of water molecules. Remember that the interaction of biuret and protein produces a violet color. Describe what you would observe at the ond of this experiment and include any movement of water and the process by which water molecules moved; any movement of biuret or albumin molecules and the process by which these molecules moved, any volume changes and any color changes in the bag and beaker. Breed (nes 1 C203) - 7 Prmaend (nos ¥ CA253) Brose leawts San via dvffusion & Colour Change is Seen va beaker, Woo learts bag vig moss S50 agw lose Volume Assume the dialysis tubing is permeable to both biuret molecules and albumin molecules. Describe what you would observe at the end of the experiment and explain what has occurred. Bol Bivrer f Albom move via diPFusws So Colour Change Sten bot bag Ah beaters W2o word leave baq Maly Vahl egal TRO) tA Gag Ook © You have condiicted an experiment in which you put a cube of fresh liver into each of four test tubes and treated them as indicated below. For each treatment describe what will happen to the liver cells, (Assume the liver cells behave in a similar manner to red blood calls and beet cube cells.) Test tube 4 - liver cube plus distilled water at room temperature. liver cells woud swell as Heo enters vir OSmosis dished W209 is Wypobom’ ts ler cells Test tube 2 - liver cube plus @ concentrated (10%) salt solution at room temperature. liver ces ‘would lose Heo via Osmosis and Sheive | lok saris bypetome te ler Celle Test tube 3 - liver cube plus acetone at room temperature. Liver cells Would lose their conkents b/ acetone dissolves lipid component Of cell membrane which dissupys Test tube 4 - liver cube plus water boiled for 5 minutes. liver cells Would lose thew Corkents be heat denatures the. Prot componeny of Celt membrane which discuphs ob