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LAB TOPIC 5A Structure and Function of Living Cells  Adapted from Perry, Morton and Perry, Laboratory Manual for General Biology (8th Edition)    Laboratory Objectives:   After completing this exercise, you will be able to:  1

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LAB TOPIC 5A
Structure and Function of Living Cells 
Adapted from Perry, Morton and Perry, Laboratory Manual for General Biology (8th Edition) 
 
Laboratory Objectives:  
After completing this exercise, you will be able to: 
1.     Define cell, cell theory, prokaryotic, eukaryotic, nucleus, cytomembrane system,      organelle, multinucleate, cytoplasmic streaming, sol, gel, envelope; 
2.    List the structural features shared by all cells; 
3.    Describe the similarities and differences between prokaryotic and eukaryotic 
cells; 
4.    Identify the cell parts described in this exercise; 
5.    State the function for these cell parts; 
6.    Distinguish between plant and animal cells; 
7.    Recognize the structures presented in boldface in the procedure sections. 
 
Introduction 
Structurally and functionally, all life has one common feature: All living organisms are composed of cells. The development of this concept began with Robert Hooke's seventeenth-century observation that slices of cork were made up of small units. He called these units "cells" because their structure reminded him of the small cubicles that monks lived in. Over the next 100 years, the cell theory emerged. This theory has three principles: (1) All organisms are composed of one or more cells; (2) the cell is the basic living unit of organization; and (3) all cells arise from preexisting cells. 
 
Although cells vary in organization, size, and function, all share three structural features: (1) All possess a plasma membrane defining the boundary of the living material; (2) all contain a region of DNA (deoxyribonucleic acid), which stores genetic information; and (3) all contain cytoplasm, everything inside the plasma membrane that is not part of the DNA region. 
 
With respect to internal organization, there are two basic types of cells, prokaryotic and eukaryotic. Study Table 5-1, comparing the more important differences between prokaryotic and eukaryotic cells. The Greek word karyon means "kernel," referring to the nucleus. Thus, prokaryotic means "before a nucleus," while eukaryotic indicates the presence of a "true nucleus." Prokaryotic cells, those typical of bacteria, cyanobacteria, and archaea, are believed to be similar to the first cells, which arose on Earth 3.5 billion years ago. Eukaryotic cells, such as those that comprise the bodies of protists, fungi, plants, and animals, probably evolved from combinations of prokaryotes. This exercise will familiarize you with the basics of cell structure and the function of prokaryotes (prokaryotic cells) and eukaryotes (eukaryotic cells). 
 
Exercise 5.1   Prokaryotic Cells  
MATERIALS 
Per student:     Per student group (table): 
• dissecting needle     • culture of a cyanobacterium (either  
• compound microscope     Anabaena  or Oscillatoria) 
• microscope slide     Per lab room: (demonstration) 
• coverslip     • 3 bacterium-containing nutrient agar 
plates 
Per student pair: 
• 3 labeled demonstration slides of stained bacteria 
(coccus, bacillus, spirillum) 
? distilled water (dH20) in dropping bottle 
        
TABLE 5.1 Comparison of Prokaryotic and Eukaryotic Cells                      
 
Characteristic           
           Cell Type 
 
    Prokaryotic     Eukaryotic 
Genetic material     Located within cytoplasm, not bounded by a special 
membrane 
 
Consists of a single molecule of 
DNA          Located in nucleus, a double 
membrane-bounded compartment 
within the cytoplasm 
 
Numerous molecules of DNA combined with protein 
 
Organized into chromosomes 
Cytoplasmic structures     Small ribosomes 
  
Photosynthetic membranes arising from the plasma membrane (in some representatives only)          Large ribosomes 
 
Cytomembrane system, a system of connected membrane structures 
 
Organelles, membrane-bouned compartments specialized to perform specific functions      
Kingdoms represented     Eubacteria 
Archaea          Protista 
Fungi  
Plantae  
        Animalia 
Activities 
 
1.    Observe the culture plate with bacteria growing on the surface of a nutrient medium. Can you see the individual cells with your naked eye?_____________________ 
 
2.    Observe the microscopic preparations of bacteria on the demonstration microscope next to the culture plate. The three slides represent the three different common shapes of bacteria. Which objective lenses are being used to view the bacteria?_________________________ 
 
Can you discern any detail within the cytoplasm?_______________________________ 
 
In the space provided in 
 
through a microscope.) 
 
3.    Study Figure 5.2, two three-dimensional representations of bacterial cells. Now examine the electron micrograph of the bacterium Escherichia coli (Figure 5.3). Locate the cell wall, a structure chemically distinct from the wall of plant cells but serving the same primary function: to contain and protect the cell's contents. 
 
4.    Find the plasma membrane, which is lying flat against the internal surface of the cell wall and is difficult to distinguish. 
 
 
 
microscope. 
 
5.    Look for two components of the cytoplasm: ribosomes, electron-dense particles (they appear black) that give the cytoplasm its granular appearance, and a relatively electron-transparent region (appears light) containing fine threads of DNA called the nucleoid.  
 
Another type of prokaryotic 
cell is exemplified by cyanobacteria, such as Oscillatoria and Anabaena. 
Cyanobacteria (sometimes called blue-green algae) are commonly found in water and damp soils. They obtain their nutrition by converting the sun's energy through photosynthesis. 
 
6.    With a dissecting needle, remove a few filaments from the cyanobacterial culture, placing them in a drop of water on a clean microscope slide. 
 
7.    Place a coverslip over the material and examine it first with the low-power objective and then using the high-dry objective (or oil-immersion objective, if your instructor says to). 
 
8.    In the space provided in Figure 5.4, sketch the cells you see at high power. Estimate the size of a single     cyanobacterial     cell     and     record     the magnification you used to make your drawing.       
 
9.    Now examine the electron micrograph of Anabaena (Figure 5.5), which identifies the cell wall, cytoplasm, and ribosomes. The cyanobacteria also possess membranes that function in photosynthesis. Identify these photosynthetic membranes, which 
 
10.    Look at the captions for Figures 
5.3     and     5.5.     Judging     by     the magnification     of     each     electron micrograph, which cell is larger, the bacterium     E.     coli     or     the cyanobacterium Anabaena? ______________________________ ______________________________ ______________________________ ______________________________
______________________________ 
 
Because the electron micrograph of 
Anabaena is of relatively low 
magnification, the plasma membrane is not obvious, but if you could see it, it would be found just under the cell wall. 
 
Exercise 5.2   Eukaryotic Cells  
 
MATERIALS 
Per student:     Per student pair: 
•    textbook     • methylene blue in dropping bottle 
•    toothpick          • distilled water (dH20) in dropping 
bottle 
• microscope slide     Per student group (table): 
•    coverslip    • Elodea in water-containing culture dish 
•    compound microscope     • onion bulb 
•    forceps     • tissue paper      
•    dissecting needle      
     Per lab room: 
•    model of animal cell 
•    model of plant cell 
 
A. Animal Cells Observed with the Light Microscope 
 
PROCEDURE:  Human cheek cells.   
 
1.    Using the broad end of a clean toothpick, gently scrape the inside of your cheek. Stir the scrapings into a drop of distilled water on a clean microscope slide and add a coverslip. Dispose of used toothpicks in the red Biohazard box in the front of the room.   
 
2.    Because the cells are almost transparent, decrease the amount of light to increase the contrast. Find the cells using the low-power objective of your microscope; then switch to the high-dry (40X) objective for detailed study. 
 
3.    Find the nucleus, a centrally located body within the cytoplasm of each cell. 
 
4.    Now stain your cheek cells with a dilute solution of methylene blue, a dye that stains the nucleus darker than the surrounding cytoplasm. To stain your slide, follow the directions illustrated in Figure 5.7. 
 
Figure 5.7.  Method for staining cells under cover slip.  Without removing the coverslip, add a drop of the stain to one edge of the coverslip. Then draw the stain under the coverslip by touching a piece of tissue paper to the opposite side of the coverslip. 
 
 
 
 
 
 
 
 
5.    In Figure 5.8, sketch the cheek cells, labeling the cytoplasm, nucleus, and the location of the plasma membrane.  (A light microscope cannot resolve the plasma membrane, but the boundary between the cytoplasm and the external medium indicates its location.) Many of the cells will be folded or wrinkled due to their thin, flexible nature. Estimate and record in your sketch the size of the cells. (The method for estimating size was presented in Week 4.) 
 
 
 
 
 
 
Figure 5.8 Drawing of human cheek cells 
Approximate size =                um 
Labels: cytoplasm, nucleus, plasma membrane 
 
 
B. Animal Cells as Observed with the Electron Microscope 
 
Studies with the electron microscope have yielded a wealth of information on the structure of eukaryotic cells. Structures too small to be seen with the light microscope have been identified. These include many organelles, structures in the cytoplasm that have been separated ("compartmentalized") by enclosure in membranes. Examples of organelles are the nucleus, mitochondria, endoplasmic reticulum, and Golgi bodies. Although the cells in each of the six kingdoms have some peculiarities unique to that kingdom, electron microscopy has revealed that all cells are fundamentally similar. 
 
Activities 
 
1.    Study Figure 5.9, a three-dimensional representation of an animal cell. 
2.    Obtain the plastic model of an animal cell.  With the aid of Figure 5.9, identify on the model the parts of the cell shown on the figure.   
 
 
 
Figure 5.9  Three-dimensional representation of an animal cell as seen with the electron microscope. 
 
3.    Figure 5.10 is an electron micrograph (EM) of an animal cell (kingdom Animalia). Study the electron micrograph and, with the aid of Figure 5.9 and any electron micrographs in your textbook, label each structure pointed at by a bar.   
4.    Observe carefully the membranes surrounding the nucleus and mitochondria. Note that these membranes seem double:  these two organelles are each bounded by two membranes, which are commonly referred to collectively as an envelope. 
 
  
 
Figure 5.10  Electron micrograph of an animal cell (1600x) 
Labels:  plasma membrane, cytoplasm, nuclear envelope, nuclear pore, chromatin, rough ER, smooth ER, Golgi body, mitochondrian 
 
5.    Using your textbook as a reference, list the function for the following cellular components: 
(a) plasma membrane ___________________________________________________ 
 
 
 
 
(b) 
cytoplasm__________________________________________________________  
 
 
 
 
(c)    nucleus (the plural is nuclei)  
___________________________________________  
 
 
 
 
(d)    nuclear 
envelope_____________________________________________________ 
 
 
 
(e)    nuclear 
pores________________________________________________________  
 
 
 
 
(f)    chromatin__________________________________________________________  
 
 
 
 
(g)    nucleolus (the plural is nucleoli)  
________________________________________  
 
 
 
 
(h)    rough endoplasmic reticulum 
(RER)_____________________________________  
 
 
 
 
(i)    smooth endoplasmic reticulum (SER)  
____________________________________  
 
 
 
 
(j)    Golgi 
body__________________________________________________________ 
 
 
 
 
(k)    mitochondrion (the plural is mitochondria) 
________________________________ 
 
 
 
 
C. Plant Cells Seen with the Light Microscope 
C.1. Elodea leaf cells 
Young leaves at the growing tip of Elodea are particularly well suited for studying cell structure because these leaves are only a few cell layers thick. 
 
Activities 
1.    With a forceps, remove a single young leaf, mount it on a slide in a drop of distilled water, and cover with a coverslip. 
2.    Examine the leaf first with the low-power objective. Then concentrate your study on several cells using the high-dry objective. Refer to Figure 5.11. 
 
3.    Observe the abundance of green bodies in the cytoplasm. These are the 
chloroplasts, organelles that function in photosynthesis and that are typical of green plants. 
4.    Locate the numerous dark lines running parallel to the long axis of the leaf. These are the air-containing intercellular spaces. 
5.    Find the cell wall, a structure distinguishing plant from animal cells, visible as a clear area surrounding the cytoplasm. 
 
 
 
Figure 5.11  Elodea cells (400X) 
 
6.    After the cells have warmed a bit, notice the cytoplasmic streaming taking place. Movement of the chloroplasts along the cell wall is the most obvious visual evidence of cytoplasmic streaming. Microfilaments are responsible for this intracellular 
 
 
 
 
7.    Remember that you are looking at a three-dimensional object. In the middle portion of the cell is the large, clear central vacuole, which can take up from 50% to 90% of the cell interior. Because the vacuole in Elodea is transparent, it cannot be seen with the light microscope. 
8.    The chloroplasts occur in the cytoplasm surrounding the vacuole, so they will appear to be in different locations, depending on where you focus in the cell. Focus on the upper or lower surface and observe that the chloroplasts appear to be scattered throughout the cell. 
9.    Now focus in the center of the cell (by raising or lowering the objective with the fine focus knob), and note that the chloroplasts lie in a thin layer of cytoplasm along the wall. 
10.    Locate the nucleus within the cytoplasm. It will appear as a clear or slightly amber body that is slightly larger than the chloroplasts. (You may need to examine several cells to find a clearly defined nucleus.) 
11.    Describe the three-dimensional shape of the Elodea leaf 
cell_________________________________________________________________ 
 
____________________________________________________________________ 
 
12.    What are the shapes of the chloroplasts and 
nucleus?________________________  
 
____________________________________________________________________
_ 
 
13.    Now add a drop of methylene blue stain to make the cell wall more obvious. Add the stain as shown in Figure 5.7. 
14.    Look for the very, very tiny mitochondria. (If you have an oil-immersion lens on your microscope, ask your professor for instructions on how to use that lens.) 
15.    Compare the size of the mitochondria to 
chloroplasts:__________________________________________________________
_ ____________________________________________________________________
_ 
 
C.2. Onion scale cells    
1. Make a wet mount of a colorless scale of an onion bulb, using the technique described in Figure 5.12. The inner face of the scale is often easiest to remove. 
 
                                                                 
 
 
 
 Figure 5.12 Method for obtaining onion scale cells. 
 
a    Cut an onion bulb into quarters.  
 
b    Remove one of the fleshy "scale" leaves. 
 
c    Snapping the "leaf" backward usually  produces a ragged piece of epidermis. 
 
 
d    Remove a small piece of epidermis and spread it evenly in a drop of water on a slide. 
 
e    Gently lower a coverslip to prevent trapping air bubbles Examine with your microscope. Add more water to the edge gof the coverslip with an eye dropper if the slide begins to dry. 
 
2.    Observe your preparation with your microscope, focusing first with the low-power objective. Continue your study, switching to the medium power and finally the high-dry objective. Refer to the photograph (Figure 5.13, next page). 
3.    Identify the cell wall and cytoplasm. 
4.    Find the nucleus. 
5.    Examine the nucleus more carefully at high magnification. Within it, find one or more nucleoli (the singular is nucleolus). Nucleoli are rich in a nucleic acid known as RNA (ribonucleic acid), while the nucleus as a whole is largely DNA 
(deoxyribonucleic acid), the genetic material. 
6.    You may see numerous oil droplets within the cytoplasm, visible in the form of granulelike bodies. These oil droplets are a form of stored food material. You may be surprised to learn that onion scales are actually leaves! Which cellular components present     in     Elodea     leaf     cells     are     absent     in     onion     leaf cells?_________________________ 
 
7.    If you are using the pigmented tissue from a red onion, you should see a purple pigment located in the vacuole. In this case, the cell wall appears as a bright line. 
8.    In Figure 5.14, sketch and label several cells from onion scale leaves. 
 
 
                                                                  nucleolus      
                                  cell wall               nucleus cytoplasm  
 
Figure 5.13 Onion bulb leaf cells (67x).                      Figure 5.14 Drawing of onion scale            Labels: cell wall, cytoplasm, nucleus, nucleolus     cells     (____________ X). 
                Labels: cell wall, cytoplasm, nucleus 
 
D. Plant Cells as Seen with the Electron Microscope 
The electron microscope has made obvious some of the unique features of plant cells. Activities 
 
1.    Study Figure 5.15, a three-dimensional representation of a typical plant cell. 
2.    Obtain the plastic model of a plant cell.  With the aid of Figure 5-15, identify on the model the parts of the cell shown on the figure.   
3.    Now examine Figure 5.16, a transmission electron micrograph from a corn leaf. Label all of the structures listed. Caution: Many plant cells do not have a large central vacuole. This is one of them. Notice that the chloroplast has an envelope, just as do the nucleus and mitochondria. 
 
 
 
4.    With the help of Figure 5.15 and any transmission electron micrographs and text in your textbook, list the function of the following structures. 
 
(a) cell wall 
___________________________________________________________ 
 
 
 
 
(b) 
chloroplast_________________________________________________________  
 
 
 
 
(c)    vacuole 
____________________________________________________________  
 
 
 
 
(d)    vacuolar membrane 
__________________________________________________  
 
 
 
 
(e)    plasma membrane ___________________________________________________  
 
 
 
 
(f)    cytoplasm__________________________________________________________  
 
 
 
 
(g)    nucleus 
____________________________________________________________ 
 
 
 
 
(h)    nuclear 
envelope_____________________________________________________  
 
 
 
 
(i)    nuclear 
pores________________________________________________________  
 
 
 
 
(j)  
chromatin___________________________________________________________  
 
 
 
 
(k)    nucleolus (the plural is nucleoli)  
________________________________________  
 
 
 
 
(l)    Rough endoplasmic reticulum (RER) 
_____________________________________  
 
 
 
 
 
  
Figure 5.15 Three-dimensional representation of a plant cell as seen with the electron microscope. 
 
(m)    smooth endoplasmic reticulum (SER) 
___________________________________ 
 
 
 
 
(n)    Golgi body  
_________________________________________________________  
 
 
 
 
(o) 
mitochondrion_______________________________________________________  
 
 
 
      
 
Figure 5.16 Electron micrograph of a corn leaf cell (2700x). 
Labels: cell wall, chloroplast, vacuole, vacuolar membrane, plasma membrane, nuclear envelope, chromatin, nucleolus, endoplasmic reticulum (ER), Golgi body, mitochondrion 
Name:_____________________________ Section Number __________________ 
 
EXERCISE 5 :  Structure and Function of Living Cells  
POST-LAB QUESTIONS 
 
5.1 Prokaryotic Cells 
1.    Did all living cells that you saw in lab contain mitochondria? 
 
2.    Below is a high-magnification photomicrograph of an organism you observed in this exercise. Each rectangular box is a single cell. What organelle is absent from each cell that makes it "prokaryotic?"  
      
 
                                                                      (75OX). 
5.2 Eukaryotic Cells 
3.    Is it possible for a cell to contain more than one nucleus? Explain. 
 
 
4.    When students are asked to distinguish between an animal cell and a plant cell, they typically answer that plant cells contain chloroplasts and animal cells do not. If you were the professor reading that answer, what sort of credit would you give and why? 
 
5.    Describe a major distinction between most plant cells and animal cells. 
 
 
 
 
 
6.    Observe the electron micrograph to the right. 
Is the cell prokaryotic or eukaryotic? 
Identify the labeled structures. 
A._________________ 
B._________________ 
C._________________                                                                 
                              (480OX). 
7.    Look at the photomicrograph to the right, which was taken with a technique that gives a three dimensional impression. Identify the structures labeled A, B, and C. 
 
A.__________________________________ 
 
B.___________________________________ 
 
C.___________________________________ 
 
8.    Is the electron micrograph below a plant or an animal cell? 
 
Identify structures labeled A and B.                                
                                                                                  A.___________________ 
 
B ___________________. 
9.    What are the numerous "wavy lines" within the cell (labeled C) 
________________ 
 
____________________________________________________________________
_ 
      
 
                                                            A          B                    C          (15,OOOX) 
10.    What structure(s) found in plant cells is (are) primarily responsible for cellular support? 
 
Food for Thought 
 
11.    What structural differences did you observe between prokaryotic and eukaryotic cells? 
 
12.    Are the cells in the electron micrograph below prokaryotic or eukaryotic? How do you know? 
 
 
 
 
 
 
  
 
                     (Photo by J. J. Cardamone, Jr., University of Pittsburgh/BPS.)  
 
LAB TOPIC 5B 
 
 
Diffusion, Osmosis, and the Functional 
Significance of Biological Membranes 
Adapted from Perry, Morton and Perry, Laboratory Manual for General Biology (8th Edition) and Peter Lanzetta, Membranes, Osmosis and Diffusion (Bio 13 Laboratory Manual 2004) 
 
Laboratory Objectives:  
After completing this exercise, you will be able to 
1. Define solvent, solute, solution, selectively permeable, diffusion, osmosis, concentration gradient, equilibrium, turgid, plasmolyzed, plasmolysis, turgor pressure, tonicity, hypertonic, isotonic, hypotonic;, 2. Describe the structure of cellular membranes; 
3.    Distinguish between diffusion and osmosis; 
4.    Determine the effects of concentration and temperature on diffusion; 
5.    Describe the effects of hypertonic, isotonic, and hypotonic solutions on red blood cells and Elodea leaf cells. 
 
 
 
* Before coming to lab, read this exercise and the appropriate section of your text,  and complete the Pre Lab Questions. 
 
 
 
Introduction 
 Water is a great environment. Earthly life is believed to have originated in the water. Without it, life as we know it would cease to exist. Living cells are made up of 75-85% water. Virtually all substances entering and leaving cells are dissolved in water, making it the solvent most important for life processes. The substances dissolved in water are called solutes and include such substances as salts and sugars. The combination of a solvent and dissolved solute is a solution. The cytoplasm of living cells contains numerous solutes in solution. 
 All cells possess membranes composed of a phospholipid bilayer that contains different kinds of embedded and surface proteins. Look at Figure 5-1 (next page)to get an idea of the complexity of a cellular membrane.  
 Membranes are boundaries that solutes must cross to reach the part of the cell where they will be used  They control what substances travel into and out of the cell. They are selectively permeable, allowing some substances to move easily (water, oxygen, carbon dioxide) while completely excluding many others (too large, or many with charges). The simplest way by which solutes enter the cell is diffusion, the movement of solute molecules from a region of high concentration to one of lower concentration. Diffusion occurs without the expenditure of cellular energy. Once inside the cell, solutes move through the cytoplasm by diffusion, sometimes assisted by cytoplasmic streaming. 
 
 
             (cytoskeletal                        ADHESION            channel        channel       channel        transport              RECEPTOR      RECOGNITION   LIPID              proteins beneath the                  PROTEIN               protein        (open)         (closed)         protein            PROTEIN         PROTEIN         BILAYER           plasma membrane)                                            TRANSPORT PROTEINS 
                                                                     CYTOPLASM 
 
                                     PLASMA MEMBRANE 
 
 
Figure 5.1 Artistic rendering of cutaway view of part of the plasma membrane (After Starr, 2000). 
 
 Water (the solvent) also moves across the membrane in a process called osmosis. Think of osmosis as a special form of diffusion, one occurring from a region of higher water concentration to one of lower water concentration. When the same molecules have different concentrations in two regions, they are said to create a concentration gradient. Diffusion and osmosis take place down concentration gradients.  
 Over time, the concentration of solvent and solute molecules becomes the same in both regions, and  the gradient ceases to exist. The system is said to be at equilibrium. Molecules are always in motion and randomly colliding, even at equilibrium. However, at equilibrium there is no net change in their concentrations. 
 These exercises introduce you to cellular membrane characteristics, and how membranes use the principles of diffusion and osmosis. 
 
EXERCISE 5.2 
Diffusion 
Solutes move within a cell's cytoplasm largely because of diffusion. However, the rate of diffusion (the distance diffused in a given amount of time) is affected by such factors as temperature and the size of the solute molecules. In this experiment, you will discover the effects of these two factors in agar, a substance much like cytoplasm and used to simulate it in this experiment. 
 
Procedure 
1.    Work in a group.  Open the plastic petri dish containing agar that is at your bench. Using a forceps (tweezers), place one crystal of potassium permanganate (KMnO4) on the agar to one side of the dish. The molecular weight of this molecule is 158.03. Opposite the potassium permanganate place a crystal of methylene blue. The molecular weight of methylene blue is 319.9.   Methylene blue's crystals are like tiny slivers so scoop several on a wooden splint and shake off the extra. Be very deliberate and try to place only one crystal of each. Mark the cover with your group initials.  
 
If requested by your instructor, determine the rate of motion of each substance, using the following formula:                  rate of diffusion = distance/elapsed time (min) 
 
Which of the solutes diffused the slowest (regardless of temperature)?           d) 
Which diffused the fastest?                                         e) 
What effect did temperature have on the rate of diffusion?                f) 
Make a conclusion about the diffusion of a solute in a gel, relating the rate of diffusion to the molecular weight of the solute and to temperature.                                g) 
 
 
2.    Go to the demonstration table and look at the graduated cylinders of water that contain a crystal of potassium permanganate. Written on each cylinder is the time the crystal was added.  
In which cylinder has the diffusion of dye reached equilibrium?           h) 
How do you know it reached equilibrium?                            i) EXERCISE 5.3 
Osmosis 
Cell membranes only allow the free passage (diffusion) of certain molecules like water, oxygen and carbon dioxide. All other atoms, ions and molecules must enter and leave by other means, such as membrane channels. Let us consider a container (as illustrated in Figure 5.4) which is divided in two by an artificial selectively permeable membrane. In the diagram the membrane's pores are indicated by spaces in the line between the two sides. In essence, artificial membranes like the one pictured are molecular filters. The pores in these artificial membranes are so small that they allow only the passage (diffusion) of molecules smaller in size than the diameter of the pore.  
On either side of the membrane pictured are two solutions, one (A) containing more dissolved molecules than the other (B). The dissolved molecules are indicated by dark dots. If we were to remove the membrane and directly expose the two solutions to one another (Figure 5.4 B) the solute molecules would diffuse from the side in which they are in greater number (the more concentrated side) to the side in which they are fewer in number. The dissolved molecules would diffuse until they were in equal concentration throughout the container -- that is until they were in equilibrium. 
 
 
            A                                            B                                              C 
Figure 5.4 
 
 If one solution (Figure 5.4 A) was a 6% solution and the other was an equal amount of a 2% solution, when the solutions reached equilibrium (Figure 5.4 C) we would have a 4% solution. 4% represents the average between 2 and 6 (2 + 6 = 8/2 = 4).  Let us repeat the scenario we set up in Figure 5.4 but this time leave the membrane between the two solutions. The large dissolved molecules “try" to diffuse but the pores of the membrane are too small. Look at Figure 5.5. Two solutions of different concentrations separated by a membrane will still reach equilibrium, not by the process just described but by a related series of events. Even though the dissolved solute molecules cannot cross the membrane, the much smaller water (solvent) molecules can cross. The water molecules diffuse from the less concentrated solution to the more concentrated solution -- from B to A. Now this is not really a violation of the laws of diffusion (diffusion always proceeds from an area of high concentration to an area of lower concentration). Solution B has a lower concentration of solute but a higher concentration of water (a 2% solution is 2% solute and 98% water) while solution A has a higher solute concentration (6%) but a lower water concentration (94%) So water diffusing from B to A actually obeys the laws of diffusion since it is moving from where there is more water to where there is less. 
 
  
 
Figure 5.5 
 
 When water is removed from a solution the solution becomes more concentrated. A pan of sugar water is left out on a warm day, as some of the water evaporates the solution becomes more concentrated or sweeter. On the other hand if you add water to a solution it becomes more dilute. As water molecules leave side B and go to side A, side B becomes more concentrated while side A becomes more dilute. There is no change in the numbers of dissolved molecules. In side B the molecules are dissolved in less water while in side A they are dissolved in more water. 
 
 Look at Figure 5.5 b. The diffusion of water across the membrane continues until side B is concentrated up to 4% and side A is diluted down to 4%. When the two solutions are at equal concentrations the solutions are in equilibrium and there is no further net movement of water. This process of water diffusion across a semipermeable membrane between two solutions of different concentrations is called osmosis. 
 
 When solutions of different concentrations are compared to each other the solution with the higher solute concentration is said to be hypertonic while the solution of lower concentration is hypotonic. (Remember O and low; this may help you remember hypotonic and therefore hypertonic).  
 
 No solution is just hypotonic or hypertonic by itself; it must be compared to another solution. For example a 10% salt solution is neither hypo or hypertonic: in fact it is both according to the solution it is compared with; if it is compared to a 3% solution, it is hypertonic: while it is hypotonic to a 15% solution. When two solutions are of equal concentration they are said to be isotonic. Remember when a hypotonic and hypertonic solution are separated by a semipermeable membrane, osmosis occurs, with water diffusing from the hypotonic to the hypertonic side until the solutions are isotonic (in equilibrium). Go to Table 5.3 at the end of the Lab Topic and fill it in, according to the directions.   
 
EXERCISE 5.3A 
Osmosis On Line.  Your instructor may direct you to an exercise on Diffusion and Osmosis accessible from the KBCC web site.   
 
EXERCISE 5.3B 
Selective Permeability of Membranes 
Dialysis is the process of molecular filtration through a selectively permeable membrane. Generally, at the same time the solute molecules are diffusing and separating by their size (through the membrane pores) the process of osmosis (movement of water) takes place as well. An example of a selectively permeable membrane within a living cell is the plasma membrane.  In this experiment, you will learn about osmosis and the dialysis process using dialysis membrane tubing, a selectively permeable cellulose tube that has very tiny holes or pores that permit the passage of water, but obstruct the passage of larger molecules.    
 
 
MATERIALS 
 
Per student group (4):       
• one 25-cm length of dialysis tubing, soaking in dH2O     • balance 
• string or waxed dental floss     • Lugol’s iodine solution 
• scissors     •  
 
Procedure 
1.    Take a piece of dialysis tubing from the beaker of water in which it is soaking. The tubing feels like a flat piece of cellophane. Rub the tubing between your fingers to open the tube. Fold over one end of the tube and tie it tightly with string or dental floss.  Be sure to be thorough because any leaking through the seal you have made will ruin the exercise. 
2.    Slip the open end of the bag over the stem 
with 25 mL of the starch/NaCl solution. (Figure 5.6). Fold over and tie the open end carefully.   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
                                                                             
  Figure 5.6 Method for filling and tying dialysis bags. 
 
3.    Rinse the outside of the bag in the dishpan of dH2O, blot the outside of the bag and weigh it to the nearest tenth of a gram with a triple beam balance. Place the weighed bag in a large beaker and pour enough dilute Lugol's iodine solution to cover the bag. 
After one hour note the color of the solution in the bag and the solution in the beaker. 
Record your observations.                                                                                    j) 
      Explain.                                                                            k) 
4.    After noting the color, remove ~ 5 ml of fluid from the bathing solution and place it into a clean test tube. Add several drops of silver nitrate (AgNO3) to the test tube, mix and note the reaction. Silver nitrate is a test for sodium chloride (NaCl).                  l)  
      Based on the results above what can you say about Cl- ions and iodine molecules?   m) 
      What can you say about starch molecules?                                                                 n) 
5.    Remove the bag from the beaker, blot dry and weigh. What has happened to the 
weight of the bag after the hour?                                                                               o)  
      Explain.                                                                                                                     p)   
      (Hint: compare the concentrations of solute inside and outside the bag.) 
      
Let us now apply some of the principles we have just learned to the membranes of living cells. 
 
EXERCISE 5.3C 
Plasmolysis in Plant Cells 
Plant cells are surrounded by a rigid cell wall.  Remember from Lab Topic 5A that many 
 
plant cells have a large central vacuole, surrounded by a vacuolar membrane.  The vacuolar membrane is selectively permeable. Normally, the solute concentration within the cell's central vacuole is greater than that of the external environment. Consequently, water moves into the cell, creating turgor pressure, which presses the cytoplasm against the cell wall. Such cells are said to be turgid. In this experiment, you will discover the effect of external solute concentration on the structure of plant cells. 
 
MATERIA
LS 
 
Figure 5.7 Turgid Elodea cells (400x).  
(Photo by J. W. Perry.)  
Labels: cell wall, chloroplasts in cytoplasm, nucleus, central vacuole 
  
 
 
Per student pair:     Per classroom: 
• forceps     • Elodea in tap water 
• 2 microscope slides      
• 2 coverslips      
• compound microscope      
• dropping bottle of dH2O      
• dropping bottle of 20% sodium chloride (NaCl)      
 
Procedure  
1.    Work in pairs.With a forceps, remove two young leaves from the tip of an Elodea plant. 
2.    Mount one leaf in a drop of distilled water on a microscope slide and the other in 20% NaCl solution on a second microscope slide. Place overslips over both leaves. 
3.    Observe the leaf in distilled water with the compound microscope. Focus first with the mediumpower objective and then switch to the high-dry objective.  
4.    Label the photomicrograph of turgid cells (Figure 5.7) 
5.    Now observe the leaf mounted in 20% NaCl solution. After several minutes, the cell will have lost water, causing it to become plasmolyzed. (This process is called plasmolysis.) Label the plasmolyzed cells shown in Figure 5.8. 
 
 
 
 
 
 
 
 
 
 
Figure 5.8 Plasmolyzed Elodea cells (400x). (Photo by J. W. 
Perry.)  
Labels: cell wall, chloroplasts in cytoplasm,  
plasma membrane, space  (between cell wall and plasma membrane) 
 
 
 
 
 
 
 
 
 
Were the contents of the vacuole in the Elodea leaf in distilled water hypotonic, isotonic, or hypertonic compared to the dH2O?                                                                          q)  
Was the 20% NaCl solution hypertonic, isotonic, or hypotonic relative to the cytoplasm?        r) If a hypotonic and a hypertonic solution are separated by a selectively permeable membrane, in which direction will the water move?                               s) 
Name two selectively permeably membranes that are present within the Elodea cells and that were involved in the plasmolysis process.                                                                 t) 
 
EXERCISE 5.3D 
Osmotic Changes in Red Blood Cells 
 
Animal cells lack the rigid cell wall of plant cells. The external boundary of an animal cell is the selectively permeable plasma membrane. Consequently, an animal cell increases in size as water enters the cell. However, since the plasma membrane is relatively fragile, it ruptures when too much water enters the cell. This is because of excessive pressure pushing out against the membrane. Conversely, if water moves out of the cell, the cell becomes plasmolyzed and looks spiny.  In this experiment, you will use red blood cells to discover the effects of osmosis in animal cells. 
 
MATERIALS 
 
Perstudent:      
• compound microscope      
Per student group (4):      
• 3 clean screw-cap test tubes     • 3 disposable plastic pipets  
• test tube rack          • 3 clean microscope slides 
• metric ruler     • 3 coverslips      
• china marker     • toothpicks 
• bottle of 0.9% sodium chloride (NaCl)     Per lab room:  
• bottle of 10% NaCl     • bottle of sheep blood (in ice bath)  
• bottle of dH2O      
 
 
Procedure 
Work in groups of four for this experiment, but do the microscopic observations individually. 
1.    Observe the scanning electron micrographs in Figure 5.9 
 
 
Figure 5.9  
Scanning electron micrographs of red blood cells.  
(Photos from M. Sheetz, R. Painter, and S. Singer. Reproduced from The Joumal of Cell Biology, 1976, 70:193, by 
copyright permission of the Rockefeller University Press and M. Sheetz.) 
a.    Red blood cells in      
b.    Red blood cells in    c. Red blood cells in a an isotonic solution        a hypertonic solution   a hypotonic solution  ("normal")                    
("crenate")          
                
Figure 5.9a illustrates the normal appearance of red blood cells. They are biconcave disks; that is, they are circular in outline with a depression in the center of both surfaces. Cells in an isotonic solution will appear like these blood cells. 
Figure 5.9b shows cells that have been plasmolyzed. (In the case of red blood cells, plasmolysis is given a special term, crenation; the blood cell is said to be crenate.) Figure 5.9c represents cells that have taken in water but have not yet burst. (Burst red blood cells are said to be hemolyzed, and of course they can't be seen.) Note their swollen, spherical appearance. 
2.    Obtain three clean screw-cap test tubes. 
3.    Lay test tubes 1 and 2 against a metric ruler and mark lines indicating 5 cm  from the bottom of each tube.   
4.    Fill each tube as follows: 
Tube 1:   5 cm of 0.9% sodium chloride (NaCl) 
     5 drops of sheep blood 
Tube 2:   5 cm of 10% NaCl 
     5 drops of sheep blood 
5.    Lay test tube 3 against a metric ruler and mark lines indicating 0.5 cm and 5 cm from the bottom of the tube.  
6.    Fill tube 3 to the 0.5 cm mark with 0.9% NaCl, and to the 5 cm mark with dH2O. Then add 5 drops of sheep blood. Enter the contents of each tube in the appropriate column of Table 5.5 at the end of the Lab Topic. 
 
7.    Replace the caps and mix the contents of each tube by inverting several times  
(Figure 5.10a). 
  
 
Figure 5.10  Method for studying effects  of different solute concentrations  on red blood cells. 
 
 
8.    Hold each tube flat against the printed page of your lab manual.  (Figure 5.10b). Only if the blood cells are hemolyzed should you be able to read the print. 
9.    In Table 5.5, record your observations in the column "Print Visible?" Alternative:   
10.    Number three clean microscope slides. 
11.    With three separate disposable pipets, to the first slide add one drop of  water, to the second, one drop of 0.9% sodium chloride (NaCl), and to the third, one drop of  10% NaCl. 
12.    With a toothpick, dip into the sheep blood and mix the toothpick into one of the drops on the slides.  Repeat for each slide with a different toothpick..   
13.    Cover each drop with a coverslip.  Or, make three slides, one each from the test tubes prepared above. 
14.    Observe the three slides with your compound microscope, focusing first with the medium-power objective and finally with the high-dry objective. (Hemolyzed cells are virtually unrecognizable; all that remains are membranous "ghosts," which are difficult to see with the microscope.) 
15.    In Figure 5.11, sketch the cells from each tube. Label the sketches, indicating whether the cells are normal, plasmolyzed (crenate), or hemolyzed.      
 
 
Figure 5.11 Microscopic appearance of red blood cells in different solute concentrations (_____X). Labels: normal, plasmolyzed (crenate), hemolyzed 16. Record the microscopic appearance in Table 5.5. 
17. Record the relative tonicity of the sodium chloride solutions you added to the test tubes in Table 5.5. 
Why do red blood cells burst when put in a hypotonic solution whereas Elodea leaf cells 
do not?                                                                                                                          u) 
 
After completing all experiments, take your dirty glassware to the sink and wash it as directed. Invert the test tubes in the test tube rack so they drain. Reorganize your work area, making certain all materials used in this exercise are present for the next class. 
 
 
 
 
Name: _____________________________     Section:  ___________________________   
PRE-LAB QUESTIONS (Circle correct answer)
 
 1. A solvent is 
(a)    the substance in which solutes are 
     dissolved 
(b)    a salt or sugar 
(c)    one component of a biological 
     membrane 
(d)    selectively permeable 
 
2.  If one were to identify the most important compound for sustenance of life, it would probably be  (a) salt 
(b)    BaC12 
(c)    water (d) I,KI 
 
3.  Diffusion 
(a) is a process requiring cellular energy (b) is the movement of molecules from a region of higher concentration to one of lower concentration (c) occurs only across selectively permeable membranes 
(d)  is none of the above 
 
4. Cellular membranes (a) consist of a phospholipid bilayer containing embedded proteins (b) control the movement of substances 
into and out of cells (c) are selectively permeable 
(d) are all of the above 
 
5. An example of a solute would be 
(a)    Janus green B 
(b)    water 
(c)    sucrose 
(d)    both a and c 
 
 
6. Dialysis membrane is 
(a) selectively permeable (b) used in these experiments to simulate cellular membranes  
(c)    permeable to water but not to sucrose 
(d)    all of the above 
      
7. Specifically, osmosis  (a) requires the expenditure of cellular energy  
(b)    is diffusion of water from one region to another  
(c)    is diffusion of water across a selectively permeable membrane  
(d)    is none of the above 
 
8. Which of the following reagents does not fit with the substance being tested for?  
(a)    Biuret reagent protein  
(b)    BaCl2, starch  
(c)    AgN03 chloride ion  
(d)    albustix protein 
 
9.    When the cytoplasm of a plant cell is pressed against the cell wall, the cell is said to be 
(a)    turgid 
(b)    plasmolyzed 
(c)    hemolyzed 
(d)    crenate 
 
10.    If one solution contains 10% NaCl and another contains 30% NaCl, the 30% solution is (a) isotonic (b) hypotonic (c) hypertonic (d) plasmolyzed, with respect to the 10% solution 
 
Answer Sheet Exercise 5.1 a) 
 
b) 
 
c)   
 
Exercise 5.2 
Table 5.2                      Rm  Temp  Cold Temp 
Solute (crystal)     Distance (mm)     Distance (mm)     Rate of diffusion 
Potassium permanganate KMnO4     15 min 30 min 45 min           
Methylene blue     15 min 30 min 45 min           
     Groups at room temperature 
Final  
Distance (mm)     Groups at ice  temperature 
Final  
Distance (mm)      
Potassium permanganate     1. 2. 3.     1. 2. 3.      
Average                
Methylene blue     1. 2. 3.     1. 2. 3.      
Average                
d) 
e) 
f) 
g) 
 
 
 
 
h) 
i)  Exercise 5.3   
                                                               TABLE 5.3 
Fill in columns 3, 4, 5 and 6 from the information provided in columns 1 and 2; a and b are filled in for you as an example of what you are to do. 
  
 
Exercise 5.3 B 
j) 
k) 
  
l) 
 
m) 
 
n) 
 
o)   
 
p) 
 
 
 
 
Exercise 5.3C q) 
 
r) 
 
s) 
 
t) 
 
Exercise 5.3D 
 
TABLE 5.5 Effect of Salt Solutions on Red Blood Cells 
Tube     Contents     Print Visible?     Microscopic 
Appearance of Cells     Tonicity of  
External Solutiona 
1 
                                   
2 
                     
3 
                     
aWith respect to that inside the red blood cell at the start of the experiment. 
 
u)