1.4: Diffusion and Osmosis

The cell theory states that all living things are composed of cells and that cells only arise from other cells. Some cells are fairly simple in structure, while others are extremely complex. For example, some organisms are unicellular—they exist as a single cell, while multicellular organisms are composed of many cells that form tissues and organs. In either case, all cells share some common properties: the presence of DNA, intracellular proteins that enable the cell to perform its functions, and a plasma membrane. Some cells, known as eukaryotic cells, also contain membrane-bound organelles that allow a more complex level of functioning.

Homeostasis is defined as the maintenance of a stable internal environment. In order to maintain homeostasis, cells continually transport substances in and out across their cell (plasma) membrane.

The cell membrane serves as a “gatekeeper” and is the cellular structure that regulates the transport of materials into and out of the cell. The phospholipid bilayer architecture of the cell membrane allows certain molecules to pass through while keeping others out, therefore the cell membrane is selectively permeable (or semipermeable). Things that need to enter a cell for it to function properly include ions, nucleotides, sugars, oxygen, amino acids, water, vitamins, and some hormones. Cells also allow certain molecules like water, ions, and secreted proteins to leave. Additionally, cells must eliminate waste products like urea and carbon dioxide

In the following exercises, you will examine the semipermeable nature of the cell membrane. You will also explore the concept of tonicity, which refers to the solute concentration of a solution, and its inherent ability to influence the rate and direction of osmosis.

PART 1: DIFFUSION & OSMOSIS

Diffusion is the movement of molecules from an area in which they are high in concentration to an area in which they are low in concentration. Molecules move down a concentration gradient until they are equally distributed, or equilibrium is reached (Fig. 1). At equilibrium, there is no concentration gradient. Molecules still move once equilibrium is reached, but there is no net movement in any one direction.

Figure 1 - Diffusion of molecules from an area of high concentration to low concentration in coffee. Equilibrium is reached when the molecules are equally distributed. (Pixabay License; iirliinnaa from Pixabay)

Osmosis is a specific type of diffusion: the diffusion of water molecules across a semipermeable membrane. Like other molecules, water molecules diffuse down a concentration gradient, from an area of higher free water concentration to an area of lower free water concentration. This means that water will move across a semipermeable membrane, like the cell membrane, in the direction of the higher solute concentration. (In solution, high solute concentration = low free water concentration; conversely, low solute concentration = high free water concentration.) You will observe this concept in Part 2: Osmosis & Tonicity.

In living organisms, most substances are transported as solutes, dissolved in water, a solvent. For example, if we dissolve salt (\(\ce{NaCl}\)) in a beaker of water, salt (\(\ce{NaCl}\)) is the solute and water is the solvent. Examples of solutes in the human body include glucose, small proteins, and electrolytes like calcium and sodium ions. Waste products, such as \(\ce{CO2}\) and urea are also transported as solutes. Solutes are carried by body fluids, such as blood plasma, and pass into and out of cells through passive and active transport. In either case, the cell membrane will either inhibit or facilitate the process of diffusion: some molecules can easily diffuse across a plasma membrane and some cannot. For example, small, nonpolar molecules (such as \(\ce{CO2}\) and \(\ce{O2}\)) can cross a membrane by simple diffusion. Large molecules or polar molecules, however, cannot easily diffuse across a membrane. Cells must have specialized membrane-bound proteins that function to transport such substances across the membrane.

In Part 1: Diffusion & Osmosis, you will learn about diffusion and osmosis using dialysis membrane, a selectively permeable sheet of cellulose that permits the passage of water and small solutes, but does not allow larger molecules to diffuse across. This is because the membrane has microscopic pores that only allow small molecules through; anything larger than the size of the pores is prevented from crossing. Some of the solutes in this experiment, sucrose (\(\ce{C12H22O11}\)) and starch (\(\ce{(C6H10O5})n}\) are too large to pass through the pores of the dialysis tubing, but the solvent molecules (\(\ce{H2O}\)) and glucose (\(\ce{C12H22O11}\)), are small enough to pass easily.

Exercise 1: Molecular Weight and Diffusion Rate

Molecular weight is an indication of the mass and size of a molecule. The purpose of this experiment is to determine the relationship between molecular weight and the rate of diffusion through a semisolid gel. You will investigate two dyes, methylene blue and potassium permanganate.

Molecule	Molecular Weight	Color
Methylene blue	300 grams/mole	blue
Potassium permanganate	150 grams/mole	purple

Employing Steps in the Scientific Method:

1. Record the Question that is being investigated in this experiment. ________________________________________________________________

2. Record a Hypothesis for the question stated above. ________________________________________________________________

3. Predict the results of the experiment based on your hypothesis (if/then). ________________________________________________________________

4. Perform the experiment below and collect your data.

Materials:

• Petri dish of agar semi-solid gel (Mueller Hinton agar plates, 150 x 15 mm) - make sure the agar has been allowed to come to room temperature
• Methylene blue solution (0.2% in 25% EtOH)
• Potassium permanganate solution (0.1% KMnO4)
• Small straws
• Small plastic metric ruler

Procedure:

1. Obtain a petri dish of agar

2. Take the plastic straw and gently stick it down into one side of the agar. Lift up the straw, withdrawing a small plug of agar. Repeat on the other side of the dish.

3. Using a 1mL transfer pipet, place a single drop of each dye into the appropriate agar well. (Fig. 2).

Figure 2 - Diffusion Experimental setup (by BB)

4. After 20 minutes, place a small, clear metric ruler underneath the Petri dish to measure the distance (diameter) that the dye has moved. Enter the data in Table 1.

5. Repeat step 4 at 40, 60, and 80 minutes.

Table 1 - Diffusion results

	Molecular Weight (g/mole)	Diameter after 20 min. (mm)	Diameter after 40 min. (mm)	Diameter after 60 min. (mm)	Diameter after 80 min. (mm)
Methylene blue
Potassium permanganate

Questions:

• What is the relationship between molecular weight and the rate of diffusion? Explain. ________________________________________________________________
• Go back and look at your initial hypothesis. Does your data support this hypothesis? Explain. _______________________________________________________________

Extension Activity: (Optional)

The results of this experiment can be presented graphically. The presentation of your data in a graph will assist you in interpreting your results. Based on your results, you can complete the final step of scientific investigation, in which you must be able to propose a logical argument that either allows you to support or reject your initial hypothesis.

1. Graph your results using the data from Table 1.
2. What is the dependent variable? Which axis is used to graph this data? ______________________________________________________________________

3. What is your independent variable? Which axis is used to graph this data? ______________________________________________________________________

Exercise 2: Diffusion Across a Membrane

Employing Steps in the Scientific Method:

1. Record the Question that is being investigated in this experiment. ________________________________________________________________

2. Record a Hypothesis for the question stated above. ________________________________________________________________

3. Predict the results of the experiment based on your hypothesis (if/then). ________________________________________________________________

4. Perform the experiment below and collect your data.

Materials:

• Dialysis tubing
• Plastic clips or string
• 5 x 400 mL beakers (or plastic cups)
• Electronic balance
• Weigh boats
• 15% Sucrose solution (MW sucrose = 342 g/mol)
• 30% Sucrose solution (MW sucrose = 342 g/mol)
• 30% Glucose solution (MW glucose = 180 g/mol)
• Graduated cylinders (10 mL and 100 mL)
• Wax pencil or sharpie
• 15% starch solution (MW = variable)
• Iodine solution (MW = 166 g/mol)
• Benedict’s reagent
• Hot plate or heat block

Procedure:

1. Cut 5 pieces of dialysis membrane approximately 10 cm long. Soak the pieces in tap water until they are soft and pliable (3-5 minutes). *This step may be done for you; check with your instructor.

2. Obtain 5 beakers (plastic cups) and label them #1 - 5. Fill each beaker with 150 mL of a solution as follows:

• Beaker #1 – H2O
• Beaker #2 – H2O
• Beaker #3 – H2O
• Beaker #4 – 30% sucrose solution
• Beaker #5 - H2O and 1mL Iodine solution

3. Set beakers aside.

4. Remove one piece of dialysis membrane from the soaking water and open it, forming a tube. Close one end of the tube with a plastic clip, a piece of string, or simply tie it with a knot (Fig. 3)

Figure 3 - Dialysis tube “bag” (By BB)

5. Fill the bag with 10 mL of H2O. Remove excess air, and close the other end of the bag with a plastic clip, a piece of string, or tie with a knot. Set aside on a paper towel.

6. Repeat steps 4 and 5 for the 4 remaining dialysis tubes, filling them with 10 mL of a solution as follows:

• Bag #2 – 15% sucrose

• Bag #3 – 30% sucrose

• Bag #4 – H2O

• Bag #5 - 5 mL 30% glucose solution and 5 mL 15% starch solution

7. Rinse off the outside of the bags with water and carefully blot dry.

8. Weigh bags #1 - 4 to the nearest 0.5g. Record the weights in Table 1 below, in the column labeled “0 min.”
9. Place each bag in the corresponding beaker (Bag #1 in Beaker #1, etc.). Make sure each bag is fully submerged in the solution.
10. Set a timer for 5 minutes.

11. At the end of 5 minutes, remove bags 1 - 4 from their beakers, blot excess fluid, and record the mass (in grams) in Table 1.

12. Return the bags to the appropriate beaker, and wait another 5 minutes.

13. Repeat steps 11 - 13 every 5 minutes and record the weights in Table 1.

Table 1. Osmosis - mass (g) over time for dialysis bags

Time (min.)		0	5	10	15	20
Beaker #1 (water)	Bag #1 (water)
Beaker #2 (water)	Bag #2 (15% sucrose)
Beaker #3 (water)	Bag #3 (30% sucrose)
Beaker #4 (30% sucrose)	Bag #4 (water)

14. Calculate the total weight change (weight change = final weight – initial weight) for each bag. Record the values in Table 2. Calculate the rate (g/min) of osmosis for each bag by dividing the weight change by the time change. Since all 4 bags were recorded for a total of 20 minutes, the time change for all 4 bags is 20 minutes. Record the rate of osmosis for all 4 bags in Table 2.

Table 2. Rate of osmosis

	Weight Change (g)	Time (min)	Rate (g/min)
Bag #1 (water)
Bag #2 (15% sucrose)
Bag #3 (30% sucrose)
Bag #4 (water)

15. Make observations about bag #5 and beaker #5 in Table 3.
16. Remove several mL of liquid from bag #5 and beaker #5 and add each to separate test tubes.

17. Add several drops of Benedict's solution to each of the two test tubes and heat to 100 degrees Celsius in a boiling water bath or heat block for 2 - 5 minutes. Record the test results in Table 3.

Table 3. Selective Permeability

	Appearance of liquid after 20 minutes	Appearance of liquid after heating
Bag #5 (5 mL 30% glucose solution and 5 mL 15% starch solution)
Beaker #5 (H2O and 1mL Iodine solution)

Questions

1. Did the weight of each bag (#1 - #4) change significantly over 20 minutes? Explain.

2. In which bag(s) was there a net movement of water?

3. Explain what is meant by “net movement”.

4. Which carbohydrate molecules (glucose, sucrose, starch) were not able to move across the membrane? Explain.

5. In terms of solvent (water) concentration, water moved from the area of _______________ concentration to the area of __________________ concentration across a selectively permeable membrane, which is defined as ________________________.

6. What can you conclude about the movement of Iodine, glucose, and starch across the dialysis membrane based on your results in Table 3? Support your answers for each with the observation from bag #5 and beaker #5.

• Iodine -
• Glucose -
• Starch -

7. We used the dialysis tubing to simulate a cell membrane. How is the dialysis tubing functionally the same as a cell membrane?

8. We used the dialysis tubing to simulate a cell membrane. How is the dialysis tubing functionally different from a cell membrane?

Extension Activity: (Optional)

The results of this experiment can be presented graphically. The presentation of your data in a graph will assist you in interpreting your results. Based on your results, you can complete the final step of scientific investigation, in which you must be able to propose a logical argument that either allows you to support or reject your initial hypothesis.

1. Prepare a line graph using the data from Table 2.

2. What is the dependent variable? Which axis is used to graph this data? ______________________________________________________________________

3. What is your independent variable? Which axis is used to graph this data? ______________________________________________________________________

PART 2: OSMOSIS & TONICITY

Tonicity is the relative concentration of solute (particles), and therefore also a solvent (water), outside the cell compared with inside the cell.

• An isotonic solution has the same concentration of solute (and therefore of free water) as the cell. When cells are placed in an isotonic solution, there is no net movement of water.

• A hypertonic solution has a higher solute (therefore, lower free water) concentration than the cell. When cells are placed in a hypertonic solution, water moves out of the cell into the lower free water solution.

• A hypotonic solution has a lower solute (therefore, higher free water) concentration than the cell. When cells are placed in a hypotonic solution, water moves into the cell from the higher free water solution.

IMPORTANT NOTE: Notice that all of the above definitions have ‘solution’ as the noun. Sometimes the noun will refer to the cell instead of the solution. For example, a hypotonic cell will experience a net movement of water out of the cell. What this means is that if the ‘solution’ is hypotonic, the cell is hypertonic and vice versa.

Exercise 1: Observing Osmosis in Potato Strips

Employing Steps in the Scientific Method:

1. Record the Question that is being investigated in this experiment. ________________________________________________________________

2. Record a Hypothesis for the question stated above. ________________________________________________________________

3. Predict the results of the experiment based on your hypothesis (if/then). ________________________________________________________________

4. Perform the experiment below and collect your data.

Materials:

• Potatoes
• Cork borer
• Petri dish
• Wax pencil or Sharpie
• Metric ruler
• 10% NaCl solution
• 0.9% NaCl solution
• Forceps and scalpel

Procedure:

1. Obtain a potato and use a cork borer to prepare 3 cylinders of potato. Push the borer through the length of the potato. When the borer is filled, use the flat end of a wooden skewer to gently push out the potato cylinder into the petri dish. Use the scalpel to cut each potato cylinder into a length of 5 cm.

2. With a wax pencil or Sharpie, label 3 test tubes (#1, #2, #3).

3. Using the metric ruler, mark each tube at the 10 cm mark level from the bottom of the tube.

4. Set up three test tubes with 10 cm of solutions as follows:
   1. Tube #1 - distilled water
   2. Tube #2 - 10% sodium chloride (NaCl)
   3. Tube #3 - 0.9% NaCl

5. Place one potato cylinder into each test tube and allow them to soak for about 15 minutes in the solutions.

6. You can now move on to Exercises 2 and 3 while your potatoes soak.

7. After the elapsed time, observe each strip for limpness (water loss, flaccid) or stiffness (water gain,turgor).

Questions

1. Which tube contained the limp (flaccid) potato strip? Explain.

2. Which tube contained the stiff (turgid) potato strip? Explain.

3. Which solution is isotonic to the inside of the potato cell?

4. What happened to the potato strip in the isotonic solution?

Osmoregulation in Living Cells

Some organisms, known as osmoregulators, have special adaptations to keep tight control over their internal osmotic conditions while still others, known as osmoconformers, are able to live in a variety of osmotic conditions. Most living cells, however, are often at the mercy of their surrounding osmotic environment. Many freshwater plants live in an isotonic or hypotonic environment, so they have no adaptations to protect them from a hypertonic environment. Likewise, mammalian red blood cells live in the isotonic plasma inside your circulatory system so they have no protection from either a hypertonic nor a hypotonic environment.

A plant cell is surrounded by a rigid cell wall, so when the cell is placed in a hypotonic environment, the net flow of water is from the surrounding medium into the cell, and it simply expands to the cell wall and becomes turgid. When the same plant cell is placed in a hypertonic environment, water leaves the central vacuole and the cytoplasm shrinks. This causes the cell membrane to pull away from the cell wall. In this situation, the plant cell will undergo plasmolysis and die. Animal cells have no cell wall so when they are in a hypotonic environment they will expand and fill with water until they burst in a condition known as lysis. Figures 4 and 5 demonstrate these conditions.

Figure 4 - Red blood cells in varied osmotic environments.(Diffusion and Osmosis. (2021, February 28). Retrieved April 1, 2021, from https://chem.libretexts.org/@go/page/34826 CCBY)

Figure 5 - Plant cell tonicity. The turgor pressure within a plant cell depends on the solution's tonicity in which it is bathed. (credit: modification of work by Mariana Ruiz Villareal CCBY)

Exercise 2: Observing Osmosis in Elodea Cells

Employing Steps in the Scientific Method:

1. Record the Question that is being investigated in this experiment. ________________________________________________________________

2. Record a Hypothesis for the question stated above. ________________________________________________________________

3. Predict the results of the experiment based on your hypothesis (if/then). ________________________________________________________________

4. Perform the experiment below and collect your data.

Materials

• Microscope
• Blank slides and coverslips
• DI water
• 10% salt solution
• Forceps
• Elodea leaf

Procedure

1. Place a drop of water onto a clean microscope slide.
2. Using the forceps, gently tear off a small leaf from the Elodea plant.
3. Prepare a wet mount by placing the Elodea leaf into the drop of water on your slide.
4. Place a coverslip onto the slide.
5. Use the scanning (4x) objective to bring the Elodea cells into focus. You may not be able to observe individual cells at this power.
6. Switch to the low power (10x) objective. The Elodea cell walls should be visible. They will look like dark green grid lines. Use the fine focus adjustment to focus the specimen.
7. Once you think you have located an Elodea cell, switch to the high power (40x) objective and refocus using the fine focus adjustment.
8. Next, add several drops of 10% salt (NaCl) solution to the edge of the coverslip to allow the salt to diffuse under the coverslip. Observe what happens to the cells (this may require you to search around along the edges of the leaf). Look for cells that have been visibly altered.
9. Record your observations in the following table. The cells in distilled water should look similar to the figure below.

Solution	Appearance of Elodea Cells
Distilled water (0% NaCl)
10% NaCl

Figure 6 Wet Mount of an Elodea Leaf Cell (Burran, S., & DesRochers, D. (2021, March 19). Cells. Retrieved April 1, 2021, from https://chem.libretexts.org/@go/page/24084)

Questions

1. Which solution is hypertonic to an Elodea cell? Use your observations to support your answer.

2. Would you expect pond water to be isotonic, hypotonic, or hypertonic to Elodea cells? Explain your answer.

3. Explain what happens to a plant cell that undergoes plasmolysis.

Exercise 3: Observing Osmosis in Red Blood Cells (Erythrocytes)

Employing Steps in the Scientific Method:

1. Record the Question that is being investigated in this experiment. ________________________________________________________________

2. Record a Hypothesis for the question stated above. ________________________________________________________________

3. Predict the results of the experiment based on your hypothesis (if/then). ________________________________________________________________

4. Perform the experiment below and collect your data.

Materials

• Microscope
• Wax pencil or Sharpie
• Blank slides and coverslips
• DI water
• 10% NaCl solution
• 0.9% NaCl solution
• Sheep red blood cells

Procedure

1. Obtain 3 test tubes and label them (#1, #2, #3) with a wax pencil or Sharpie.
2. Set up the three test tubes with 5 mL of solutions as follows:
   1. Tube #1 - Distilled water
   2. Tube #2 - 10% NaCl
   3. Tube #3 - 0.9% NaCl
3. Using a new transfer pipette, add 2 drops of sheep blood to each tube and swirl gently to mix the contents.
4. Hold each test tube up to a sheet of paper with printed text. Attempt to read the print through each tube and record your results in the following table.

Test Tube / Solution	Appearance of Solution	Can you read print?
#1 - Distilled water
#2 - 10% NaCl
#3 - 0.9% NaCl

5. Label 3 microscope slides (#1, #2, #3) with a wax pencil or Sharpie.
6. Prepare wet mounts of each tube by placing a drop of the solution in each tube (#1 - 3) on the appropriate microscope slide (#1 -3). Add a coverslip to each slide.
7. View slide #1 through the microscope using the scanning (4x) objective first. Focus the image using the coarse adjustment. Then view the blood cells under low power and then high power. Only use the fine focus adjustment to focus the specimen.
8. Observe slide #2 and slide #3 in the same manner.
9. Record the appearance of the red blood cells in each solution in the following table.

Solution	Appearance of RBCs
#1 - Distilled water
#2 - 10% NaCl
#3 - 0.9% NaCl

Questions

1. Which solution allowed you to read print through the solution? Explain.

2. Which solution is hypertonic to the RBCs? Use your observations to support your answer.

3. Which solution is hypotonic to the RBCs? Use your observations to support your answer.

4. Which solution is isotonic to the RBCs? Use your observations to support your answer.

Questions for Review

1. Define diffusion. What is the energy source for diffusion? Is diffusion considered an active or passive process? Explain.

2. Name a molecule that diffused through the artificial membrane (dialysis tubing) that we used in the laboratory. Can diffusion occur without a membrane? Give an example to support your answer.

3. What is osmosis? Is it an active or a passive process? Explain.

4. Fill in the blank in the following statements.
   1. A solution that has a lower solute concentration than another solution is said to be ______________________ when compared with the second solution.
   2. A solution that has the same solute concentration as another solution is said to be ______________________ when compared with the second solution.
   3. A solution that has a higher solute concentration than another solution is said to be ______________________ when compared with the second solution.

5. What does it mean when a membrane is selectively permeable?

Practical Challenge

6. Briefly explain what happens to a red blood cell when placed into the following solutions.
   1. Isotonic solution –
   2. Hypertonic solution –
   3. Hypotonic solution -
7. The concentration of glucose inside Elodea cells is 5 mM. What is the solution in moles (M)? What will happen to an Elodea cell if it is placed in a 1 M glucose solution? Explain.

8. Apply what you learned in the lab to explain why it is said that marine organisms, which live in saline environments, literally live in a desert environment.