ANATOMY AND PHYSIOLOGY 1 (2125)

ANATOMY & PHYSIOLOGY

Cell Structure and Function Investigation Manual

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CELL STRUCTURE AND FUNCTION

Table of Contents

2 Overview 2 Outcomes 2 Time Requirements 3 Background 7 Materials 8 Safety 9 Preparation 9 Activity 1 10 Activity 2 14 Disposal and Cleanup 14 Observations

Overview In this investigation, the student will explore the structure and function of the animal cell, particularly the selectively perme- able plasma membrane. The student will model the processes of simple diffusion and osmosis and assess the tonicities of aqueous solutions.

Outcomes • Identify the parts of an animal cell and describe their functions. • Describe the structure of the plasma membrane of the cell and

explain why it is selectively permeable. • Model the processes of simple diffusion and osmosis. • Calculate the rate of diffusion and determine how it is affected

by molecular weight. • Assess the relative tonicities of aqueous solutions.

Time Requirements Preparation .................................................................... 30 minutes Activity 1: Simple Diffusion ........................................... 60 minutes Activity 2: Osmosis ........................................................ 75 minutes

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Background The cell is the fundamental structural and functional unit of all living things. Although the human body is composed of an amazing variety of different, specialized cell types, all cells have certain characteristics in common. Most importantly, all animal cells possess three main components: a nucleus, a cytoplasm, and a plasma membrane.

The Nucleus The nucleus houses most of the genetic mate rial of the cell. Most of the time, the genetic material exists in the form of a threadlike complex of DNA and proteins known as

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chromatin. When a cell goes through the process of division, the chromatin coils up tightly to form compact structures called

chromosomes. Within the nucleus lies at least one nucleolus. This is where ribosomes, the machinery of protein synthesis, are assembled. The contents of the nucleus are separated from the rest of the cell by the nuclear envelope. This double membrane is penetrated by nuclear pores that permit materials to pass in and out of the nucleus.

The Cytoplasm The cytoplasm occupies the area between the nucleus and the plasma membrane. It consists of the cytosol (which is mostly water with dissolved ions and proteins), the protein filaments of the cytoskeleton, and a variety of organelles, which are specialized structures devoted to specific cellular tasks (Table 1).

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Table 1.

Structure Function Ribosomes Protein synthesis

Rough endoplasmic reticulum (rough ER) Processing and transport of proteins Smooth endoplasmic reticulum (smooth ER) Lipid and carbohydrate metabolism; detoxification Golgi apparatus Processing and transport of proteins, especially

secreted proteins Lysosomes Intracellular digestion Peroxisomes Catabolism of fatty acids Mitochondria ATP production

Centrioles Organization and movement of chromosomes during cell division

Cilia Movement Flagella Movement Microfilaments Cytokinesis; changes in cell shape; cell motility Intermediate filaments Strength and support for cells and tissues Microtubules Motility (internal components of cilia and flagella);

intracellular transport; chromosome movements during cell division

CELL STRUCTURE AND FUNCTION

Background continued The Plasma Membrane The plasma membrane of the cell (also called the cell membrane or the cytoplasmic membrane) surrounds and defines each cell and separates its internal environment from the external environment. The plasma membrane is composed primarily of phospholipids. A phospholipid molecule consists of a glycerol skeleton with two fatty acids and a phosphate group attached. The fatty acids are nonpolar hydrocarbon chains, and thus, they are hydro- phobic (i.e., repelled by water). The negatively charged phosphate group forms the polar head of the molecule and is hydrophilic (i.e., attracted to water). Recall that the cytosol within the cell is mostly water, and most cells of the human body are bathed in extracellular fluid, which is also mostly water. These conditions cause the phospholipid molecules to cluster together so that their hydrophilic heads are oriented toward the water and their hydrophobic tails exclude water. The resulting structure is a phospholipid bilayer: two layers of molecules, with the hydro- philic heads directed to the inside and outside of the cell and the hydrophobic tails sandwiched in between.

By themselves, the phospholipid molecules would form a relatively loose, fluid association, with a consistency similar to that of vegetable oil. However, phospholipid molecules are not the only type of molecule in the plasma membrane. In the membranes of animal cells, the phospho- lipids are stabilized by sterol molecules, such as cholesterol. Glycolipids, which have a carbohy- drate group instead of a phosphate group, are also present in the outer portion of the bilayer.

Proteins constitute a major component of the plasma membrane and play important roles

in cell signaling, adhesion, metabolism, and transport. Peripheral membrane proteins are weakly associated with the membrane, whereas integral membrane proteins are more firmly embedded. In fact, most integral membrane proteins are transmembrane proteins, meaning that they completely span the phospholipid bilayer and have exposed regions on both sides of the membrane. Many proteins are able to drift laterally within the phospholipid bilayer, which is why the plasma membrane is often described in terms of a fluid-mosaic model.

Cell Transport and Cell Size All living things take in nutrients and eliminate waste. These vital functions are facilitated at the cellular level by the selectively permeable (i.e., semipermeable) plasma membrane. The cell membrane permits the passage of mole- cules and ions of a certain size while restricting the passage of larger or differently charged molecules or ions. Some molecules, such as water, oxygen, and carbon dioxide, can move freely across the cell membrane’s lipid bilayer. These molecules move into and out of the cell by diffusion, which can be defined as the net movement of molecules or ions down a concen- tration gradient. Concentration is defined as the amount of a substance per unit volume, such as the mass of sucrose (table suguar) in a milliliter (mL) of water. So when a substance moves down a concentration gradient, it moves from a region of higher concentration to a region of lower concentration. Larger molecules, on the other hand, are excluded by the membrane and may enter or leave the cell only through processes mediated by dedicated transporter proteins located in the membrane.

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In Activity 1, you will observe how diffusion occurs in the absence of a membrane. However, diffusion across a selectively permeable membrane, such as the plasma membrane, is subject to certain conditions. Four main factors determine the rate of diffusion of molecules or ions across a membrane:

1. The steepness of the concentration gradient: The greater the difference between the concentrations on opposite sides of the membrane, the higher the rate of diffusion.

2. Temperature: Molecules and ions have more kinetic energy at higher temperatures. When molecules and ions move more rapidly, diffu- sion proceeds more rapidly.

3. The surface area of the membrane: The greater the surface area, the higher the rate of diffusion. A greater surface area allows more molecules or ions to cross the membrane at any point in time.

4. The type of molecule or ion diffusing: Large molecules (those of higher molecular weight) tend to diffuse more slowly than smaller mole- cules (of lower molecular weight). If the large molecules are contained within a selectively permeable membrane, they may not be able to diffuse at all. Ions may move more readily along a charge gradient; for example, a cation (positively charged ion) may diffuse more quickly toward a region rich in anions (nega- tively charged ions) than toward a region with an overall positive charge.

Osmosis is the diffusion of water molecules across a selectively permeable membrane. The

four factors listed above also apply to osmosis. The net movement of water molecules in osmosis is to the side of the selectively perme- able membrane having the higher concentration of solute, and, therefore, the lower concentration of water. The cytoplasm is an aqueous solution, consisting of water with dissolved molecules and ions. If the cell is surrounded by solute- free, pure water, the concentration of water is actually lower inside the cell compared with the outside, and the net movement of water will be into the cell. If the cell is in a solution with a high solute concentration, the concentration of water may be higher inside the cell compared with the outside, causing the net flow of water to be out of the cell.

The terms hypertonic, hypotonic, and isotonic are used to compare aqueous solutions of varying solute concentration in which the solute cannot cross the membrane. If the solutions have the same concentration of solute, they are called isotonic (iso-, “same”). When two solu- tions have different concentrations of a solute, the one with the higher solute concentration is called hypertonic (hyper-, “above”), and the one with the lower solute concentration is called hypotonic (hypo-, “below”). The hypertonic solution, which contains a higher solute concen- tration than the comparison solution, can also be thought of as having a lower concentration of water. In contrast, the hypotonic solution has a lower concentration of solute, but a higher concentration of water. Because the solute cannot cross the membrane, osmosis occurs between solutions of different tonicities. The water will move from the solution in which it is

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CELL STRUCTURE AND FUNCTION

Background continued more concentrated to the solution in which it is less concentrated. In other words, water will move from the hypotonic solution to the hyper- tonic solution. In Figures 1 and 2, the larger black circles represent the solute molecules, and the smaller open circles represent the water molecules. The vertical center line represents a selectively permeable membrane. In Figure 1, the concentration of solute molecules is higher on the left side of the membrane, so the left side is hypertonic relative to the right side. The right side is hypotonic relative to the left side. Figure 2 shows that the left and right sides are at equilibrium and are isotonic relative to each other. The concentration of molecules on both sides of the membrane is equal.

Osmotic pressure is the measure of a solu- tion’s tendency to gain water when separated from pure water by a selectively permeable membrane. A solution’s osmotic pressure is proportional to its solute concentration; the greater the solute concentration, the greater the osmotic pressure and, therefore, the greater the tendency for the solution to gain water. In isotonic solutions, water diffuses across the membrane from one solution to another at an

equal rate in both directions. There is no net osmotic movement of water and no net osmotic pressure.

Water enters our cells passively through osmosis. For instance, most water absorption in the digestive tract occurs in the large intes- tine, and there are no channels in the plasma membranes of intestinal cells that actively transport water. While water transport relies on osmosis, there are membrane channels that actively transport sodium and other ions into the cytoplasm, using ATP for energy. In order to manipulate the characteristics of osmosis, the concentration of solutes can be increased in the cells of the intestinal lining such that the cyto- plasm becomes hypertonic relative to the lumen of the large intestine. Then, water flows into the cells by osmosis.

The kidneys regulate the water balance in our bodies. Like the large intestine, the movement of water by osmosis is regulated by the active transport of salts. In addition, some cells of the kidneys have selective channels called aqua- porins, which allow water to move across the membrane very quickly in response to osmotic pressure.

In Activity 2, you will use dialysis tubing to simulate the plasma membrane of a cell. The flat, transparent dialysis tubing has microscopic pores that permit the passage of water, but not larger solutes such as sugars.

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Figure 1. Figure 2.

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Materials Included in the materials kit:

Ruler

4 Plastic cups, 10 oz

Sucrose, 100 g packet

5 Pipets

Petri dish Potassium permanganate, 1 g

Methylene blue, 1 g

Teaspoon

2 Micro spoons

3 Weigh boats

3 Pieces of dialysis tubing, 8"

Agarose, 30 mL

3 Beakers, 250 mL

Grease pencil

Graduated cylinder, 100 mL

Graduated cylinder, 10 mL

Reorder Information: Replacement supplies for the Cell Structure and Function investigation (item number 580506) can be ordered from Carolina Biological Supply Company.

Call: 800.334.5551 to order.

Needed but not supplied: • Tap water • Timing device • Paper towels • Digital camera or mobile device

capable of taking digital photos • Pot holder or mitt

(recommended but not required)

CELL STRUCTURE AND FUNCTION

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Safety Do not eat, drink, or chew gum while performing these activities. Wash your hands with soap and water before and after performing the activities. Clean up the work space with soap and water after completing the investigation. Keep pets and children away from lab materials and equipment.

Read all of the instructions for these laboratory activities before beginning. Follow the instructions closely and observe established laboratory safety practices, including the use of appropriate personal protective equipment (PPE).

Wear safety goggles, gloves, and a lab apron while performing this laboratory investigation. Work in close proximity to a sink or other source of running water. A kitchen sink sprayer or a shower may serve as an emergency eyewash station if needed.

Potassium permanganate is an oxidizing agent.

Methylene blue is an irritant of the skin, eyes, and respiratory passages; exposure may result in drowsiness

or dizziness and may impair fertility or, if preg- nant, cause harm to an unborn child. If either substance is inhaled, seek fresh air immediately and seek medical attention. In case of contact with the eyes, rinse immediately with plenty of water and seek medical attention. In case of contact with skin, wash immediately with soap and rinse with plenty of water. If skin irritation results, seek medical advice or attention. If swal- lowed, call a poison center and/or seek medical attention immediately.

ACTIVITY 1

Simple Diffusion Preparation 1. Read the procedure description thoroughly

and become familiar with the kit materials and procedure steps prior to beginning.

2. Put on PPE (safety goggles, gloves, and lab apron) and wear throughout the rest of the preparation and procedure.

3. To prepare the Petri dish containing agarose: a. Loosen, but do not remove, the cap of the

agarose bottle. b. Using a microwave, heat the agarose at 30-second intervals until the agarose is completely melted. The agarose

must have no lumps and should pour easily. The container will be hot, so you may wish to use a potholder or oven mitt.

c. Place the Petri dish on a level surface. Remove the lid and set it aside.

d. Pour all of the melted agarose slowly into the Petri dish, making sure the bottom of the dish is evenly covered and there are no bubbles in the agarose.

e. Allow the Petri dish to sit undisturbed and uncovered for 25–30 minutes,

or until the agarose is completely solidified. 4. Turn the Petri dish upside down and use the

grease pencil to draw a line down the center of the bottom of the dish, as shown in Figure 3. Then turn the Petri dish right side up (agar facing up).

5. Fill a micro spoon about half full of potassium permanganate. Use a pipet to suction up the permanganate crystals and deposit the contents gently onto the surface of the agarose in the center of one side of the

dish. Take care not to scatter any crystals of potassium permanganate across the surface of the agarose.

6. Use a pipet to suction an equivalent amount of methylene blue and deposit the contents onto the surface of the agarose in the center of the opposite side of the dish.

If scattering does occur, you may wish to try again using a clear area of the agarose surface. Just make a note of the new dye location and make sure it is as far from the other dye as possible.

7. Put the ruler underneath the Petri dish, and record the initial diameter of each dye ring (in mm) in Data Table 1. Start the timer. Take a photo of the Petri dish.

Figure 3.

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ACTIVITY

A

ACTIVITY

ACTIVITY 1 continued 8. Record the diameter of each dye ring in Data Table 1 at 15-minute intervals for 1 hour. At the end of the hour,

take a final photo of the Petri dish. 9. Determine the rate of diffusion for each dye

using the following formula:

where time is the time in minutes when the dye front reaches the edge of the Petri dish or the hour had elapsed.

10. Based on the information recorded in Data Table 1, determine whether the two dyes diffused at the same rate or at different rates.

ACTIVITY 2 Osmosis Preparation 1. Read the procedure description thoroughly

and become familiar with the kit materials and procedure steps prior to beginning.

2. Put on PPE (safety goggles, gloves, and lab apron) and wear throughout the rest of the preparation and procedure.

3. Prepare one solution of 40% sucrose and two solutions of 20% sucrose:

a. Label two 250-mL beakers “20% sucrose” and one 250-mL beaker “40% sucrose.”

b. Add five level teaspoons (20 g) of sucrose to a “20% sucrose” beaker. Add warm tap water to approximately the 90-mL mark on the beaker and stir with the teaspoon until the sugar is completely dissolved. Pour into the 100-mL graduated cylinder, and carefully add more water to reach a total volume of 100 mL. Pour back into the beaker and rinse the graduated cylinder and teaspoon with tap water.

c. Repeat Step 3b to produce another beaker of 20% sucrose solution.

d. Add 10 level teaspoons (40 g) of sucrose to the “40% sucrose” beaker. Add warm tap water to approximately the 90-mL mark on the beaker and stir with the teaspoon until the sugar is completely dissolved. Pour into the 100-mL graduated cylinder, and carefully add more water to reach a total volume of 100 mL. Pour back into the beaker and rinse the graduated cylinder and teaspoon with tap water.

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Rate of diffusion (mm/min) = final diameter – initial diameter

time (min)

A

e. Allow all three sucrose solutions to cool to room temperature before starting the procedure.

4. Label three weigh boats “A,” “B,” and “C” for each treatment.

5. Label three plastic cups “A,” “B,” and “C” for each treatment.

Procedure 1. Use the 100-mL graduated cylinder to measure 90 mL of 20% sucrose solution. Pour into Cup “A.” 2. Use the 100-mL graduated cylinder to measure 90 mL of 20% sucrose solution. Pour into Cup “B.” 3. Use the 100-mL graduated cylinder to measure 90 mL of 40% sucrose solution. Pour into Cup “C.” 4. Fill another clean plastic cup with

tap water. Place the flat, transparent dialysis tubing in the water. Allow the dialysis tubing to remain in the water for at least 30 seconds.

5. Remove the dialysis tubing from the water, and gently roll the end of the tubing between your index finger and thumb. This should cause the dialysis tubing to open. Slightly wetting your fingers will make opening the dialysis tubing easier. Opening Dialysis Tubing https://players.brightcove.

net/17907428001/HJ2y9UNi_default/ index.html?videoId=4573412134001

6. Continue rolling the tubing down its length until the tubing is completely open. Tie

a simple knot on one end of the dialysis tubing, as shown in Figure 4. This knot should be as close to the end as possible. Repeat this process for all three pieces of dialysis tubing.

Figure 4.

7. Use the 10-mL graduated cylinder to measure out 6 mL of 20% sucrose solution. Use a pipet to transfer all 6 mL into a piece of dialysis tubing. You may need to pour the last milliliter or so into the open end of the tubing.

8. Carefully remove almost all of the air from the space above the sucrose solution, leaving only a small bubble, so the tube will float when placed in its designated cup. To expel the air, gently squeeze upward with the thumb and index finger of one hand while supporting the filled tubing with the other hand.

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ACTIVITY

ACTIVITY 2 continued 9. Twist the top inch of the tubing and tie a

simple knot at the top end, as shown in Figure 5.

10. Rinse the outside of the dialysis tubing with water, and gently blot it dry with a paper towel.

11. Place the dialysis tubing in the weigh boat labeled “A.”

12. Repeat steps 7–10 with the next piece of dialysis tubing. Place the tubing in the weigh boat labeled “C.”

13. Rinse the 10-mL graduated cylinder with tap water. Use it to measure out 6 mL of 40% sucrose solution. Use a new pipet to transfer all 6 mL into the last piece of dialysis tubing. Pour the last bit into the open end of the dialysis tubing if necessary. Repeat Steps 8–10, and place the tubing in the weigh boat labeled “B.”

14. Take a photo of all three pieces of filled dialysis tubing sitting in their weigh boats.

15. Measure the volume of each solution in each tube by displacement:

a. Add 80 mL of tap water to the 100-mL graduated cylinder.

b. Drop tubing “A” into the graduated cylinder and make sure the solution inside the tubing is completely submerged.

c. Observe the new water level and record the value as “initial volume” in Data Table 2.

d. Remove the tubing from the graduated cylinder and discard the water. Dab each end of the tubing with a paper towel to remove any excess water, and place the tubing in its designated weigh boat. continued on next page

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Figure 5.

c. Observe the new water level and record the value as “final volume” in Data Table 2.

d. Remove the tubing from the graduated cylinder, squeeze any excess water out from the ends of the tubing, and place the tubing in its designated weigh boat. Discard the water.

e. Perform Steps 21a–d for tubing pieces “B” and “C.”

22. Determine the change in the volume of solution within each piece of dialysis tubing and record in Data Table 2.

23. Determine the percentage change in the volume of solution within each piece of dialysis tubing and record in Data Table 2.

24. Based on the results, determine whether the solution in each piece of tubing was isotonic, hypotonic, or hypertonic relative to the solution in the cup. Record your conclusions in Data Table 2.

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e. Perform Steps 15a–d for tubing pieces “B” and “C.”

16. Transfer the tubing pieces into the labeled cups containing the appropriate sucrose solutions as follows:

a. Cup “A”: 20% sucrose solution in the cup and 20% sucrose solution in the dialysis tubing.

b. Cup “B”: 20% sucrose solution in the cup and 40% sucrose solution in the dialysis tubing.

c. Cup “C”: 40% sucrose solution in the cup and 20% sucrose solution in the dialysis tubing.

17. Make sure the dialysis tubing is completely submerged, and start the timer.

18. Allow the tubing to sit in the cups for 1 hour. 19. Remove each piece of tubing from its cup,

dab each end of the tubing with a paper towel to remove any excess water, and place the tubing back into its corresponding weigh boat.

20. Take a photo of all three pieces of filled dialysis tubing sitting in their weigh boats. 21. Measure the new volume of the solution in

each piece of tubing. a. Add 80 mL of tap water to the 100-mL

graduated cylinder. b. Drop tubing piece “A” into the graduated

cylinder and make sure it is completely submerged.

ACTIVITY

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Disposal and Cleanup 1. Keep your PPE (safety goggles, gloves, and

lab apron) on throughout the disposal and cleanup process.

2. Carefully pour all liquids down the drain, flushing with excess tap water for at least 1 full minute.

3. The weigh boats, plastic cups, pipets, and dialysis tubing should be disposed of in the household trash.

4. Secure the lid on the Petri dish of agarose, wrap in a plastic bag, and dispose of in the household trash.

5. Wash and dry the teaspoon, micro spoons, beakers, and graduated cylinders.

6. Store remaining materials in the materials kit bag or equipment set.

7. Sanitize the work space and wash your hands.

Data Table 2.

Treatment A Treatment B Treatment C

Solution in dialysis tubing 20% sucrose 40% sucrose 20% sucrose

Solution in cup 20% sucrose 20% sucrose 40% sucrose

Initial volume (Vi) (mL)

Final volume (Vf) (mL)

Change in volume (Vf−Vi) (mL)

Percent change in volume (change in volume/Vi) x 100

Hypotonic, isotonic, or hypertonic

Observations

Time (min)

Diameter, Potassium

Permanganate (mm)

Diameter, Methylene Blue (mm)

0 15 30 45 60

Data Table 1.

NOTES

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ANATOMY & PHYSIOLOGY Cell Structure and Function

Investigation Manual

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