Osmosis and Cells

a) Introduction In todays experiment you will investigate the effects of osmosis on cells. Recall that osmosis is the movement of water across a membrane (such as a cells membrane) and that the water always moves towards the side of the membrane with the higher solute concentration. It may be helpful to review the concepts of osmosis, diffusion, hypertonic, isotonic, and hypotonic from the last laboratory and your textbook/lecture outlines. b) Effect of Osmosis on Plant and Animal cells In this activity you will use your microscope to view the effects of osmosis on plant and animal cells that have been exposed to hypertonic, hypotonic, and isotonic saline solutions. Saline just means sodium chloride (NaCl), also known as table salt. Plant and animal cells look very different under the microscope. The animal cells we will use appear as empty circles or ovals surrounded by a thin membrane. There are organelles inside but the organelles are hard to see. Plant cells, on the other hand, are usually box-shaped and are filled with highly visible green chloroplast organelles. Furthermore, each plant cell is surrounded by a thick cell wall that animal cells do not have. When viewed under a microscope, osmosis has slightly different effects on plant and animal cells. Animal cells placed in a very hypotonic solution will gain water until they burst. Thus under the microscope you will not see any animal cells if the cells were placed in a hypotonic solution. Plant cells, on the other hand, are surrounded by a rigid cell wall. The cell wall stops them from bursting, so plant cells in a hypotonic solution will still be visible under the microscope. Animal cells will shrivel and shrink in hypertonic solutions because of water loss. Plant cells will also lose water in hypertonic solutions, but the cell wall is not affected by osmosis. Therefore the cell wall will remain in its normal box shape but the rest of the plant cell (the membrane, the chloroplasts, the nucleus, etc.) will shrivel and shrink inside the confines of the cell wall. 1) Obtain three microscope slides, three cover slips, and a wax marker. Label the slides A, B, and C. Also obtain 3 test tubes. Label these A, B, and C also.

2) On one of the countertops, find three solutions labeled A, B, and C. One of these solutions is a hypertonic saline solution, one is a hypotonic saline solution, and one is an isotonic saline solution. Your job is to figure out which solution is which by viewing the effects of the solutions on cells. Transfer 5 ml of each of the solutions into your labeled test tubes, then return to your desk.

3) Tear off three small leaves from the water plant elodea. Place one leaf into each of your test tubes. Allow the leaves to remain in the test tubes for a full five minutes. 4) After 5 minutes, remove each leaf and transfer it (along with a few drops of the solution it was soaking in) to the properly labeled microscope slide. Add a cover slip to each slide. 5) View each of the leaves under the microscope. In the first row of data table 1, sketch a typical plant cell from each slide. Label the cell wall, the membrane, and the chloroplasts in your sketches. 6) Wash the slides off and dry them. 7) Add two drops of the sheep blood to each of your test tubes. Allow the blood to remain in the solution for a full five minutes. 8) After 5 minutes, transfer a drop from each test tube to the proper slide. Add a cover slip to each slide. 9) View each of the slides under the microscope. (Remember that one solution will cause the blood cells to burst, and therefore it will have no visible cells under the microscope). In the second row of data table 1 sketch a typical blood cell from each slide. 10) Based on your results, fill in third row of data table one. Show your instructor your results before continuing.

c) Penetrating and non-penetrating solutes

Recall that diffusion is the tendency for solute particles to spread from areas of higher concentration to areas of lower concentrations until they are evenly distributed. There are many factors that can affect the rate at which a solute diffuses. For example, smaller molecules diffuse more rapidly than larger ones. The presence of a membrane can also affect a solutes diffusion rate because the solute may not be able to pass through the membrane. If the solute is able to pass through the membrane, we say the membrane is permeable to the solute and that the solute is a penetrating solute. If the solute is not able to pass through the membrane, we say that the membrane is impermeable to the solute and that the solute in a non-penetrating solute. Why is it important whether a solute can or cannot pass through a cells membrane? Because this can affect the solutes ability to cause osmosis.

To understand why, consider a cell in an isotonic solution of non-penetrating solute. In the drawing below, the black dots represent the non-penetrating solute outside the cell and the white dots represent the solutes that occur naturally inside the cell. Since the solute concentration outside the cell is equal to the solute concentration inside the cell, no osmosis occurs:

Now consider a cell exposed to an isotonic solution of penetrating solute. Since the solute can pass through the membrane, some of it will diffuse into the cell. The

concentration of the solute inside the cell will increase. This means the cell will end up with more solute inside than outside, which will cause water to enter the cell by osmosis. The cell will eventually burst from the water it gains.

The cell membrane and the associated transport proteins found in the membrane are responsible for regulating the movement of thousands of different types of molecules into and out of the cell. The membranes of cells are usually selectively permeable. This means the membrane will allow some molecules to pass through but not others. d) Overview of penetrating vs. non-penetrating solute experiment In this activity you will be given several solutions, each containing a different solute molecule. For each solution, you will first hypothesize whether you think its solute molecule is penetrating (meaning it will be able to pass through a cell membrane) or nonpenetrating (meaning it will not be able to pass through a cell membrane). You will then carry out an experiment using real blood cells to test your hypothesis. How will you detect if a solute molecule can or cannot pass through the blood cell membrane? All the solutions you will use are isotonic, meaning the solute concentration of the solution is exactly equal to the solute concentration inside the cell. If the solute is a non-penetrating solute (in other words, if the blood cell membrane is impermeable to the solute molecule), the solute molecule will stay outside the cell. The cell will not gain or lose water because it is in isotonic conditions. (See first illustration on page 3). The blood cell solution will be a cloudy red because cells make solutions cloudy. If the solute is a penetrating solute (in other words, if the blood cell membrane is permeable to the solute molecule), the solute will diffuse through the membrane. Since this will add to the cells solute concentration, the solute concentration of the cell will become larger than the

solute concentration of the solution. Under these conditions, osmosis will cause water to enter to the blood cell. The cell will expand and eventually lyse (tear open). (See second illustration on page 3). When lysis occurs, the solution becomes transparent red (because the solution loses its cloudiness when the cells are destroyed). Therefore, if the blood cell solution becomes transparent, it means the solute was penetrating. If the blood cell solution stays cloudy, the solute was non-penetrating.

e) Making predictions about solute permeability To make predictions about which solute molecules are permeable and which are impermeable to the membrane, the three factors most important things to consider are the hydrophobic or hydrophilic nature of the solute molecule, the presence of membrane pores for the molecule, and the size of the solute molecule. Hydrophobic vs. hydrophilic solutes. Ions are extremely hydrophilic. Organic molecules that are polar (have many partial charges) tend to be hydrophilic molecules, but not as hydrophilic as ions. Hydrophilic means that the molecule is attracted to water and dissolve well in aqueous solutions. This is because the water is attracted to partial or ionic charges. In contrast, non-polar molecules (such as lipids) are hydrophobic. They have few if any partial charges and therefore do not dissolve well in water. An important concept is that hydrophilic molecules do not mix with hydrophobic molecules, and hydrophobic molecules do not mix with hydrophilic molecules. The phospholipids of the cell membranes serve as a barrier to the passage of hydrophilic solute molecules because hydrophilic molecules dont mix with the hydrophobic phospholipid molecules. Hydrophobic solute molecules, on the other hand, can easily pass through the cell membrane because they are not repelled by the hydrophobic phospholipids.

Alcohols are molecules with OH functional groups. Molecules with OH groups tend to be polar because of the partial charges on the hydrogen and oxygen atoms. However, the number of OH groups on a molecule affects the degree of polarity a molecule will exhibit. The more carbons with OH groups, the more polar the molecule will be. The fewer carbons with OH groups, the less polar the molecule will be. Ions, on the other hand, are so hydrophilic that they never pass directly through cell membranes at a significant rate.

Size of molecules can also affect their permeability. Surprisingly, some very small hydrophilic molecules can pass directly through the membrane, as long as they are not ions. Lastly, note that smaller molecules diffuse faster than larger ones. This can affect the speed of
cell lysis.

Pores are open channels through the membrane. They are a tunnel though the membrane that is formed by a type of protein called a membrane transport protein. The cell uses pores to import and export hydrophilic molecules that cannot pass directly through the phospholipid bilayer. Note that pores are usually very specific for just the one molecule they transport. In other words, a pore for a certain solute molecule will only transport that solute molecule. It would not transport any other solute molecule. Cells do not have pores for every solute molecule. Cells usually have pores only for very important molecules that must be transported in and out on a regular basis. Below is a table of the solutions that you will use in this experiment. After each one, hypothesize (a) Whether or not lysis will occur (Remember, in this experiment lysis occurs when the cell membrane is permeable to a solute molecule), and (b) If you predict that lysis will occur, how quickly will cell lysis occur? Show your instructor your predictions before continuing. Be ready to defend your predictions to your instructor. Solute Molecule: NaCl Number of carbon atoms None Number of Will cause OH groups lysis (Y/N) None ______ (but has ions) 1 1 3 6 6 ______ ______ ______ ______ ______ Speed of lysis (slow/medium/fast)

Methanol Isopropanol Glycerol Mannitol Glucose

1 3 3 6 6

f) Testing predictions about solute permeability 1) Obtain three screw cap test tubes. Pipette 5 ml of the first solution into the tube.

2) Mix the sheep blood gently before taking samples. Then add two drops of blood to the tube, Immediately cap and invert to mix, and record the time the blood was added in the data table 2 (minutes and seconds). 3) The tubes will be turbid (cloudy) at first. If lysis occurs, the solution in the tube will become transparent. Record the time at which the tube becomes transparent in the appropriate column in the table. If the tube remains turbid for 5 minutes, you can assume that no lysis has occurred. 4) Calculate the hemolysis time (the time for the blood cells to lyse) by subtracting the time the blood was added from the time the tube became transparent and record the number of minutes and seconds. 5) Wash out the tube with water, then repeat steps 1 4 using the other solutions. 6) Show your instructor your results when you have finished testing all solutions. 6) When done, clean all test tubes with water using the test tube brush.

f) Results tables Data table 1 (Osmosis effects on plant and animal cells)

Solution A

Solution B

Solution C

(Plant cell)

(Plant cell)

(Plant cell)

(Animal cell)

(Animal cell)

(Animal cell)

Based on its osmosis effects, solution A is _______tonic

Based on its osmosis effects, solution B is _______tonic

Based on its osmosis effects, solution C is _______tonic

Data table 2 (Tests of penetrating and non-penetrating solutes)

Tube number 1 2 3 4 5 6 Solute molecule NaCl Methanol Isopropanol Glycerol Mannitol Glucose Time blood was added Time solution became transparent Hemolysis time (minutes: seconds)

g) Review questions 1) Define the following terms: Diffusion Osmosis Selectively permeable membrane Penetrating solute Non-penetrating solute Saline solution Lysis Hemolysis Polar molecule Alcohol

Pore Membrane transport protein

2) A hypertonic/hypotonic (circle one) solution will cause a cell to gain water until it bursts. 3) One of the solutions you tested caused animal cells to burst but not plant cells. What feature of plant cells prevented them from busting?

4) Is NaCl a penetrating or a non-penetrating solute? Justify your answer based on your results from data table 1.

5) The solutions of red blood cells were all initially turbid (cloudy). Some of the solutes you tested caused them to become clear. This happened because the cell membrane was permeable/impermeable (circle one) to the solute.

6) Explain, in terms of osmosis and diffusion, the mechanism (the steps) by which some solutes in your experiment caused lysis of the blood cells.

7) If you placed a cell in a hypertonic solution of a non-penetrating solute, the cell would shrink/expand/stay the same (circle one). Justify your answer using concepts from todays laboratory.

8) If you placed a cell in a hypertonic solution of a penetrating solute, the cell would shrink/expand/stay the same (circle one). Justify your answer using concepts from todays laboratory.

9) Explain, in terms of hydrophobic and hydrophilic, why the membrane is a barrier to some solutes but not to others.

10) Are all alcohols hydrophilic? Why or why not?

11) Suppose that, using an experimental procedure similar to todays, you found that molecules A and B both caused hemolysis, but that molecule A caused hemolysis much faster than molecule B. What might you conclude is the major difference between molecules A and B?

12) Mannitol and glucose are about the same size molecules, yet one was permeable to the membrane and one was not. Hypothesize why. Hint: In nature, glucose is a very common sugar but mannitol is a very rare sugar.

13) Glycerol and isopropyl alcohol are about the same size molecules, yet one was permeable to the membrane and one was not. Hypothesize why.