Materials Included In Kit

Additional Materials Required

Prelab Preparation

1. This kit comes with 20 mL of 1% resazurin solution. This laboratory activity requires 0.5% solution.
2. Pour 10 mL of 1% resazurin into a clean 10-mL graduated cylinder and transfer it to a clean 50-mL beaker.
3. Using a 10-mL graduated cylinder measure 10 mL of distilled or deionized water.
4. Add the water to the 50-mL beaker containing resazurin and stir.
5. Place both beakers containing resazurin and bromcresol green where they are accessible to all lab groups on day 2 of this laboratory activity. Tip: Advise students to transfer their group’s dye from the main beaker to a weighing dish before transporting the dye to their lab station so they are not carrying the dye in pipets where it might easily spill.

Safety Precautions

Bromcresol green and resazurin will stain skin and clothing. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Exercise caution when handling hot agar. Remind students to wash their hands thoroughly with soap and water before leaving the laboratory. Please review current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. The agar, bromcresol green and resazurin waste may all be disposed of in the regular trash according to Flinn Suggested Disposal Method #26a. The test tubes may be washed and saved for future use.

Lab Hints

• Enough materials are provided in this kit for 30 students working in pairs or for 15 groups of students.
• To avoid congestion, set out several containers of dye solution.
• This laboratory activity requires a portion of laboratory work over the course of three days. Day 1 will take 45–50 minutes while days 2 and 3 should take around 10 minutes. The prelaboratory assignment may be completed before coming to lab, and the data compilation and questions may be completed the day after the lab. To save time the teacher may decide to prepare the agar-filled test tubes ahead of time.
• A magnetic stirrer/hot plate with a stir bar is ideal for mixing the agar solution if available.
• Stress to students the importance of heating the agar solution to a boil. If the water is not hot enough the agar will not go into solution and the gel will not solidify.
• Different concentrations of dye solutions are used because of different color intensities. Students are not given this information because many will find it confusing. If desired, an additional experiment may be conducted. Study two different concentrations of bromcresol green in the same sized test tube and the same agar concentration.

Teacher Tips

• The Diffusion and Osmosis Kit, Flinn Scientific Catalog No. AP8638, investigates selectively permeable membranes using dialysis tubing to study which molecules can or cannot pass through the membrane.
• Remind students of this activity later in the year when electrophoresis is studied. In particular, discuss how the size of molecules, ions and DNA fragments affects the distance traveled.

Answers to Prelab Questions

1. Two solutions are placed in a density box (a plastic box with a divider in the middle which can be removed). Solution A on the left contains 5% sodium chloride and solution B on the right contains 1% sodium chloride. If the divider is removed, will the sodium and chloride ions initially diffuse from Solution A to Solution B or from Solution B to Solution A? Explain your answer.

   The sodium and chloride ions will migrate from Solution A to Solution B because the solute in solution naturally moves down the concentration gradient.

2. Imagine that two dyes, vibrant violet and beautiful blue, are each added to separate 13 mm x 100 mm test tubes containing 0.2% agar. Vibrant violet has a molecular formula of C₁₄H₇NaO₆S and beautiful blue has a molecular formula of C₁₀H₅NaO₄S. Which dye molecule is larger? After 24 hours, which dye will have diffused further in the test tube? Explain.

   Beautiful blue will have diffused further because it is easier for the smaller molecules to migrate through the “physical barriers” of the medium.

Sample Data

Answers to Questions

1. Compare the diffusion distance between test tubes 1 and 2. Did the resazurin diffuse further in test tube 1 or 2? Explain why this occurred.

   The resazurin diffused further in test tube 1. Given that both test tubes contain the same concentration of agar and the same volume of resazurin, the only variable is the size or diameter of the test tube. The resazurin diffused further in the smaller (narrower) test tube because it did not have the ability to spread out as far horizontally as it did in the wider diameter.

2. Compare the diffusion distance between test tubes 1 and 3. In which test tube did the resazurin diffuse further? Explain why this occurred.

   The resazurin diffused further in test tube 1. The test tube size and resazurin volume were the same. The concentration of agar was 0.2% in test tube 1 and 2.0% in test tube 2. The lesser concentration of agar in test tube 1 provided less interference, allowing the molecules to diffuse further.

3. Compare the diffusion distance between test tubes 1 and 4. Which dye diffused further? Explain why this occurred.

   The resazurin (in test tube 1) diffused further than bromcresol green (in test tube 4). The molecular weight of bromcresol green is approximately three times greater than that of resazurin. The smaller resazurin molecules were able to diffuse further.

4. What other factors, not tested in this lab, might affect the diffusion distance.

   Factors which also affect diffusion distance are polarity, ionic charge or the amount of dye applied to the medium.

5. Gel electrophoresis is the process which separates DNA based on the distance different size DNA fragments migrate in an agarose gel medium in an electric field. Negatively charged DNA migrates through the gel towards the positively charged electrode. The following four fragments—760 base pairs (bp), 3000 bp, 178 bp and 1800 bp. Arrange the DNA fragments in the order they would appear in the electrophoresis gel beginning at the negative terminal (where the DNA is loaded) and moving to the positive terminal.

   Since the DNA is negatively charged all fragments of DNA migrate towards the positive terminal. The smallest fragments will migrate the furthest through the gel and be closest to the positive terminal. Therefore the fragments would appear in the following order: 3000 bp, 1800 bp, 760 bp and 178 bp.

References

Villani, P. Investigating the Process of Diffusion Using an Analytical Puzzle. The American Biology Teacher: 2007, Vol 69, No. 7, pp 411–415.

Introduction

Have you ever placed a drop of food coloring into a glass of water and watched the color swirl around and dissipate throughout the water? This process is known as diffusion. What additional factors will affect the rate or extent of diffusion?

Concepts

• Diffusion
• Concentration gradient

Background

Molecules have an intrinsic kinetic energy, which means that they are in a state of constant motion. When a group of molecules is placed into a new environment, they will naturally move from an area of higher concentration to an area of lower concentration. This process is known as diffusion. Diffusion is explained using the thermodynamic property called entropy. Entropy is commonly defined as the tendency of all things to move toward a more disorganized state. In the absence of other forces, substances such as ions or molecules will move from an area of greater concentration (more organized) to an area that is less concentrated (less organized) (see Figure 1). This is also known as moving along a concentration gradient. Diffusion or dispersion of a solute in water increases entropy by producing a more random mixture than exists when there are localized concentrations of solute. It is important to understand that each substance diffuses down its own concentration gradient, unaffected by the concentration differences of other substances.

Diffusion was investigated by physiologist Adolf Fick (1829–1901) during the mid-nineteenth century. From his investigations Fick proposed a mathematical equation to express how various physical parameters affect the rate of diffusion of a compound. Fick’s Law of Diffusion is expressed below as Equation 1. The variable J represents the diffusion flux or the number of molecules moving from one point to another per unit of time. It is traditionally expressed in units of moles/m²s. D is the diffusion coefficient or diffusivity which describes how fast or slow an object diffuses. It is expressed in units of area/time. A is the cross-sectional area. ΔC represents the change in concentration. The concentration is the amount of mass in a given volume. Δx is the distance the object diffuses.

The diffusion coefficient, D, takes into account three major factors among several smaller factors. First, it accounts for the major effect of temperature, which reflects the amount of kinetic energy in the system. Temperature has a significant effect on the rate of diffusion. The second factor that contributes to the diffusion coefficient is the size of diffusing particles. Smaller particles diffuse faster than larger particles because large particles are more likely to collide with other particles, which slows them down. The third factor is the viscosity of the medium in which the particles move. A highly viscous medium will decrease the rate of diffusion. As an analogy, think of how easy it would be to paddle a boat through fresh water versus a thick muddy swamp.

Experiment Overview

The purpose of this activity is to compare the rate of diffusion of dye molecules in agar. The effects of three variables will be investigated—the concentration of the agar gel, the diameter of the test tube and the size of the dye molecule.

Materials

Prelab Questions

1. Two solutions are placed in a density box (a plastic box with a divider in the middle which can be removed). Solution A on the left contains 5% sodium chloride and solution B on the right contains 1% sodium chloride. If the divider is removed, will the sodium and chloride ions initially diffuse from Solution A to Solution B or from Solution B to Solution A? Explain your answer.
   {12732_PreLab_Figure_7}
2. Imagine that two dyes, vibrant violet and beautiful blue, are each added to separate 13 x 100 mm test tubes containing 0.2% agar. Vibrant violet has a molecular formula of C₁₄H₇NaO₆S and beautiful blue has a molecular formula of C₁₀H₅NaO₄S. Which dye molecule is larger? After 24 hours, which dye will have diffused further in the test tube? Explain.

Safety Precautions

Bromcresol green and resazurin will stain skin and clothing. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Exercise caution when handling hot agar. Wash hands thoroughly with soap and water before leaving the laboratory. Please follow all laboratory safety guidelines.

Procedure

Day 1. Preparing the Agar Medium

1. Place a clean 200-mL beaker on the balance and press the tare button on the balance so the measurement reads 0.00 g.
2. Measure 0.2 g of agar in the beaker.
3. Using a 100-mL graduated cylinder measure 100 mL of distilled or deionized water.
4. Add the 100 mL of distilled water to the beaker containing 0.2 g of agar. This will be the 0.2% agar solution.
5. Stir the mixture with a stirring rod and set aside.
6. Place a clean 100-mL beaker on the balance and press the tare button so the measurement reads 0.00 g.
7. Measure 0.6 g of agar in the beaker.
8. Using a 100-mL graduated cylinder measure 30 mL of distilled water.
9. Add the 30-mL of distilled to the beaker containing 0.6 grams of agar. This will be the 2.0% agar solution.
10. Stir the mixture with a stirring rod and set both the 0.2% agar and 2.0% agar beakers on a hot plate.
11. Turn the hot plate on high. Continue to heat and intermittently stir until the solution boils and agar dissolves completely into each solution. Tip: If available, a magnetic stirrer/hot plate combination with a stir bar may be used.
12. While the agar dissolves use a permanent marker to label four test tubes according to Table 1.
    {12732_Procedure_Table_1_Test tube labels}
13. Once the 0.2% agar has dissolved use a hot vessel gripping device to carefully pour the agar into test tubes 1, 2 and 4. Fill test tubes to about 1 cm from the top.
14. Once the 2.0% agar has dissolved, carefully pour the agar into test tube 3, about 1 cm from the top.
15. Allow the agar-filled test tubes to sit 20–30 minutes and cover with Parafilm.
16. Allow test tubes to remain undisturbed for 12–24 hours in a test tube rack to allow the agar to set.

Day 2. Preparing Dye Solutions

1. Obtain test tube 1.
2. Using a graduated pipet, measure 0.2 mL of resazurin solution.
3. Gently add the 0.2 mL of resazurin dropwise to the center of test tube 1. Note: The resazurin must be added dropwise gently to the middle of the agar. Do not forcefully squirt solution into the test tube or the laboratory activity results may be skewed.
4. Obtain test tube 2.
5. Repeat steps 2 and 3 using test tube 2.
6. Obtain test tube 3.
7. Repeat steps 2 and 3 using test tube 3.
8. Obtain test tube 4.
9. Using a clean graduated pipet, measure 0.2 mL of bromcresol green solution.
10. Gently add the 0.2 mL of bromcresol green to test tube 4.
11. Once all dye solutions have been added to the appropriate test tube, cover each tube with Parafilm and let sit for 24 hours.
    {12732_Procedure_Table_2_Summary of the test tube sizes and contents}

Day 3. Measuring Diffusion Distance

1. Obtain a metric ruler and a white sheet of paper.
2. Obtain test tube 1 and lay it flat on a white piece of paper.
3. Measure the distance from the top of the agar where the dye was initially added to the location where the dye color is no longer visible.
4. Repeat steps 1–3 with test tubes 2–4.
5. Consult your instructor for appropriate disposal procedures.

Student Worksheet PDF

12732_Student1.pdf