Diffusion of Betacyanin Pigment from Beetroot Vacuoles

Introduction

In this experiment, we will explore the factors that control the process of diffusion in a living cell. Under a variety of conditions, we will study the release of a red pigment from cells of beetroots. Using spectrophotometry, we can detect the movement of pigment from the cells into the surrounding solution.

The characteristic red color of beets (Beta vulgaris) is due to the accumulation of water-soluble pigments called betacyanins in vacuoles of the root cells. These pigments are separated from the cell’s surroundings by two membranes, the tonoplast of the vacuole and the plasma membrane at the cell’s surface. We will rely on treatments that affect the properties of these membranes to allow diffusion of betacyanins from the vacuole to a solution outside the cells.

In this procedure, you will treat beet samples with two salt solutions prior to using either detergent or ethanol to release the red betacyanin pigments from the beetroot vacuoles. The salt treatments last for 20 minutes. The following detergent or ethanol treatments last 24 minutes or until no more changes are observed. To use the lab period efficiently, a timing scheme for completing the absorbance readings at staggered intervals is provided in the laboratory instructions. This experiment is designed for groups of 4-5 students each.

The goals of this exercise are to:
1. Develop a consistent process for measuring the diffusion of a cellular component into the surrounding solution.
2. Process data in multiple formats to produce graphs that demonstrate the effects of experimental treatments on the course of diffusion.
3. Study the effects of three variables, time, salt concentration and ionic composition, on the rate of diffusion.
4. Relate experimental results to known properties of cellular components.
5. Formulate a laboratory report that presents the data and your interpretation of the data.

Experimental Procedure

1. Turn on and calibrate the spectrophotometer nearest you. Make sure it is set at 475 nm. Keep a cuvette of tap water handy to recalibrate it from time to time throughout the experiment.
2. Set up a series of 12 cuvettes containing fresh beet pieces. Use a weighing boat to measure 0.3 grams of beetroot per tube. Once you have a piece of the appropriate weight, remove it from the scale, slice it into 4-5 thinner pieces, and place it at the bottom of a cuvette. [Make sure to do any cutting in a weigh boat or other protective structure, NOT on the bench top.] The pieces should cover no more than one-half inch of the lower portion of the cuvette. Place about 5 milliliters of tap water into each tube to cover the beets until you are ready to begin the experiment.
Tap water is used to rinse away any pigment released due to cell injury during the cutting process.
3. Your group will be assigned a set of two salt treatments and one elution buffer. One salt treatment will be used for six of your tubes (First Salt for tubes 1-6), the other will be used for the remaining six tubes (Second Salt for tubes 7-12).
4. Use a 10% stock solution of your First Salt to prepare 5 mL solutions containing 1.0%, 2.5%, 5.0%, 7.5% and 10.0% of First Salt, each in a separate, clean cuvette. Recall that the required volumes can be determined by the following equation:
Concentration1 x Volume1 = Concentration2 x Volume2
Use the table below to record the volumes required for each solution:

Starting Concentration of First Salt Volume of First Salt solution (mL) Amount of Tap Water Added (mL) Total Volume
(mL) Final Concentration of First Salt
10% 5 mL 1.0%
10% 5 mL 2.5%
10% 5 mL 5.0%
10% 5 mL 7.5%
10% 5 mL 0 mL 5 mL 10.0%

5. Drain the tap water from six of your tubes. Make sure all of the beet pieces are retained in each tube. While inverted, blot the top of each tube on a paper towel to remove the last few drops of water. You may also use a transfer pipette to remove the water.
6. Place 5 milliliters of tap water in tube 1. Transfer the appropriate First Salt solution to each of tubes 2-6 as shown in the table below.
Tube Solution
1 Tap water
2 1.0% First Salt
3 2.5% First Salt
4 5.0% First Salt
5 7.5% First Salt
6 10.0% First Salt

7. After 20 minutes, drain the solution from each tube. Make sure all of the beet pieces are retained in each tube. While inverted, blot the top of each tube on a paper towel to remove the last few drops of solution. You may use a transfer pipette to remove the solutions but be sure to empty them in numerical order, by increasing concentration.
8. Add the solvent (either 40% ethanol or 60% detergent, as assigned by your instructor) in thirty second intervals according to the following time schedule. Apply a stopper to the tube and mix the contents by inversion.
This process sets up the experiment, no absorbance readings are taken during this time period. Time
(minutes) Tube Action
0 1 Add 5 ml solvent
0:30 2 Add 5 ml solvent
0:60 3 Add 5 ml solvent
1:30 4 Add 5 ml solvent
2:00 5 Add 5 ml solvent
2:30 6 Add 5 ml solvent

9. Read the absorbance of each tube at 475 nm, according to the following schedule. These absorbance readings will be staggered by the same thirty second interval as the solvent addition. To accomplish this process efficiently, assign the following roles to group members: timer, recorder, tube inverter and cleaner, absorbance reader. The latter two roles can be combined if there are only three group members. Just before each reading, invert the sample once to mix the color thoroughly. Make sure that the beet pieces have settled to the bottom of the cuvette, tapping the tube on the benchtop if needed, so that the pieces cover no more than one-half inch of tube height.

Time
(minutes) Tube Read Abs475 Value
to Plot Time
(minutes) Tube Read Abs475 Value
to Plot
3:00 1

3 minute
time point 15:00 1

15 minute
time point
3:30 2 15:30 2
4:00 3 16:00 3
4:30 4 16:30 4
5:00 5 17:00 5
5:30 6 17:30 6
6:00 1

6 minute
time point 18:00 1

18 minute
time point
6:30 2 18:30 2
7:00 3 19:00 3
7:30 4 19:30 4
8:00 5 20:00 5
8:30 6 20:30 6
9:00 1

9 minute
time point 21:00 1

21 minute
time point
9:30 2 21:30 2
10:00 3 22:00 3
10:30 4 22:30 4
11:00 5 23:00 5
11:30 6 23:30 6
12:00 1

12 minute
time point 24:00 1

24 minute
time point
12:30 2 24:30 2
13:00 3 25:00 3
13:30 4 25:30 4
14:00 5 26:00 5
14:30 6 26:30 6

10. Continue for 27 and 30 minute time points if the absorbance values are still increasing for your samples.

Use STEPS 11-17 for treatment and measurements using your SECOND SALT solution.

11. (Step 4 Repeated for Second Salt) Use a 10% stock solution of your Second Salt to prepare 5 mL solutions containing 1.0%, 2.5%, 5.0%, 7.5% and 10.0% of Second Salt, each in a separate, clean cuvette. Recall that in a separate, clean cuvette. Recall that the required volumes can be determined by the following equation:
Concentration1 x Volume1 = Concentration2 x Volume2
Use the table below to record the volumes required for each solution:
Starting Concentration of Second Salt Volume of First Second solution (mL) Amount of Tap Water Added (mL) Total Volume
(mL) Final Concentration of Second Salt
10% 5 mL 1.0%
10% 5 mL 2.5%
10% 5 mL 5.0%
10% 5 mL 7.5%
10% 5 mL 0 mL 5 mL 10.0%

12. (Step 5 Repeated for Second Salt) Drain the tap water from six of your tubes. Make sure all of the beet pieces are retained in each tube. While inverted, blot the top of each tube on a paper towel to remove the last few drops of water. You may also use a transfer pipette to remove the water.
13. (Step 6 Repeated for Second Salt) Place 5 milliliters of tap water in tube 1. Transfer the appropriate Second Salt solution to each of tubes 2-6 as shown in the table below.
Tube Solution
7 Tap water
8 1.0% Second Salt
9 2.5% Second Salt
10 5.0% Second Salt
11 7.5% Second Salt
12 10.0% Second Salt

14. (Step 7 Repeated for Second Salt) After 20 minutes, drain the solution from each tube. Make sure all of the beet pieces are retained in each tube. While inverted, blot the top of each tube on a paper towel to remove the last few drops of solution. If using a transfer pipette, be sure to empty the tubes in numerical order, by increasing concentration.
15. (Step 8 Repeated for Second Salt) Add the solvent (either 40% ethanol or 60% detergent, as assigned by your instructor) in thirty second intervals according to the following time schedule. Apply a stopper to the tube and mix the contents by inversion.
This process sets up the experiment, no absorbance readings are taken during this time period. Time
(minutes) Tube Action
0 7 Add 5 ml solvent
0:30 8 Add 5 ml solvent
0:60 9 Add 5 ml solvent
1:30 10 Add 5 ml solvent
2:00 11 Add 5 ml solvent
2:30 12 Add 5 ml solvent

16. (Step 9 Repeated for Second Salt) Read the absorbance of each tube at 475 nm, according to the following schedule. These absorbance readings will be staggered by the same thirty second interval as the solvent addition. To accomplish this process efficiently, assign the following roles to group members: timer, recorder, tube inverter and cleaner, absorbance reader. The latter two roles can be combined if there are only three group members. Just before each reading, invert the sample once to mix the color thoroughly. Make sure that the beet pieces have settled to the bottom of the cuvette, tapping the tube on the benchtop if needed, so that the pieces cover no more than one-half inch of tube height.

Time
(minutes) Tube Read Abs475 Value
to Plot Time
(minutes) Tube Read Abs475 Value
to Plot
3:00 7

3 minute
time point 15:00 7

15 minute
time point
3:30 8 15:30 8
4:00 9 16:00 9
4:30 10 16:30 10
5:00 11 17:00 11
5:30 12 17:30 12
6:00 7

6 minute
time point 18:00 7

18 minute
time point
6:30 8 18:30 8
7:00 9 19:00 9
7:30 10 19:30 10
8:00 11 20:00 11
8:30 12 20:30 12
9:00 7

9 minute
time point 21:00 7

21 minute
time point
9:30 8 21:30 8
10:00 9 22:00 9
10:30 10 22:30 10
11:00 11 23:00 11
11:30 12 23:30 12
12:00 7

12 minute
time point 24:00 7

24 minute
time point
12:30 8 24:30 8
13:00 9 25:00 9
13:30 10 25:30 10
14:00 11 26:00 11
14:30 12 26:30 12

17. (Step 10 Repeated for Second Salt) Continue for 27 and 30 minute time points if the absorbance values are still increasing for your samples.

Writing Laboratory Report 2:
Diffusion of Betacyanin Pigment from Beetroot Vacuoles

The second lab report is intended to build on skills you practiced in the first report and to add some new ones. You will write more statements that identify the precise relationships between variables when you formulate conclusions for the graphs that you prepare. You will continue to analyze error, but by estimating its magnitude rather than calculating the standard deviation. (The proper procedure is to repeat data until you have a reproducible, statistically significant result, but we don’t have time to do this for every experiment.) You will learn to perform calculations for graphing data in alternative ways in order to obtain additional information. You will again analyze all graph shapes to seek the underlying natural mechanisms that can be deduced from them. And you will be asked to connect the results of the diffusion experiment to your knowledge of the structural components of cells. The overall focus of the report is to explore the factors that control the rate of diffusion, using movement of betacyanin from beetroot cells as the model system.

Laboratory Report 2 will emphasize presentation and interpretation of data collected from the betacyanin diffusion analysis. In addition to composing a scientific title for your report, there will therefore be three major sections, results, discussion and conclusion. Refer to the lab manual section on “How to Write a Scientific Report” for general information on what to include in these sections. Specific details relating to this report are provided below.

For this report, provide a Title that will identify the purpose of the experiments, including the environmental factors that were manipulated and the types of measurements that were made, along with the organism or system studied. Recall that a title should be self-explanatory, giving the reader precise information about what is contained in the report.

For this report, the Results section will contain a text and four figures as described below:
1. Text: Present a paragraph that introduces the reader to the way in which the data will be presented and describes what the reader should notice about each figure. You can comment on the mathematical relationships between the variables that are shown in the figures. Provide objective information, without any interpretation in this section. Note that the results text is separate from all of the figure captions.
2. Figure 1: Prepare a graph of Absorbance versus Time for the data collected after your treatment with the First Salt. Plot the absorbance values for each tube as a set and use the same axes to display all six sets of data. It is helpful to use a different symbol for each data set and to provide a key for these symbols in the figure caption. Use a graphing program like Excel to insert a trend line for each set of data, and keep a record of the trend line equations for calculations pertaining to Figures 3 and 4. Be sure that the independent variable (Time) is on the X-axis and the dependent variable (Abs475nm) is on the Y-axis. Write a full caption, including a title and brief description of the data presented, and place it below the figure. It should describe the central concept of the graph and show the source and purpose of the data. Providing the equations for the trend lines in the captions is optional. In summary, there should be six sets of points, absorbance values from tubes 1-6, with their associated trend lines on Figure 1.

3. Figure 2: Prepare a graph of Absorbance versus Time for the data collected after your treatment with the Second Salt. Plot the absorbance values for each tube as a set and use the same axes to display all six sets of data. It is helpful to use a different symbol for each data set and to provide a key for these symbols in the figure caption. Use a graphing program like Excel to insert a trend line for each set of data, and keep a record of the trend line equations for calculations pertaining to Figures 3 and 4. Be sure that the independent variable (Time) is on the X-axis and the dependent variable (Abs475nm) is on the Y-axis. Write a full caption, including a title and brief description of the data presented, and place it below the figure. It should describe the central concept of the graph and show the source and purpose of the data. Providing the equations for the trend lines in the captions is optional. In summary, there should be six sets of points, absorbance values from tubes 7-12, with their associated trend lines on Figure 2.
4. Figure 3: Prepare a graph of Rate of Diffusion versus Time for treatments with one concentration of the First Salt and the same concentration of the Second Salt. Rate of Diffusion is calculated as the slope of the plots in Figures 1 and 2, that is, rate is represented by change in absorbance per unit time. The process for determining the slope is given below:
a. Choose data from treatment with any ONE salt concentration to study. For example, perhaps you are interested in the results with 5% of your First Salt treatment. For this graph, you should also separately analyze the results with 5% of your Second Salt treatment.
b. Calculate the slope of the trend line at multiple intervals along the absorbance versus time plot (from Figure 1 or 2) for the selected salt concentration. Use two-minute time intervals for the change in X values. Use the trend line equation to determine the Y values before calculating the difference. Calculate at least 10 slope values throughout the time span of the experiment.
For example, suppose the trend line for absorbance versus time with
1% CaCl2 treatment is Y = 0.3517Ln(X) – 0.2444.
Then at 3 minutes, Y=0.142 and at 5 minutes, Y=0.322.
The difference in Y values is 0.322-0.142 = 0.180.
The difference in X values is 5-3 = 2.
The slope is the change in Y over the change in X = 0.18/2 = 0.09.
c. Plot each slope as a Y value. For the corresponding X value, take the midpoint of the time interval studied. For example, the slope from the 3-5 minute time interval would be the Y value plotted against an X value of 4 minutes.
d. It is also possible represent the rate by taking the derivative of the absorbance versus time trend line. If you choose this method, you can use a graphing program to plot the equation for the derivative directly.
e. Once you have calculated the slope values for your First Salt treatment, you should repeat the process to analyze the trend line at the same concentration of your Second Salt.
Be sure that the independent variable (Time) is on the X-axis and the dependent variable (Rate of Diffusion) is on the Y-axis. Write a full caption, including a title and brief description of the data presented, and place it below the figure. In summary, Figure 3 should have two sets of points, one showing slope values for the selected concentration of First Salt and one for slope values from the same concentration of Second Salt, with their associated trend lines.

5. Figure 4: Prepare a graph of Rate of Diffusion versus Salt Concentration that compares your results for treatments with the First Salt and the Second Salt. Choose one time interval to study for all of the absorbance versus time graphs in Figures 1 and 2. Ideally the interval should be short and occur early in the experiment. Find the rate of diffusion during this same time interval for all trend lines in Figures 1 and 2, using the slope calculation described for Figure 3. Then plot each rate (slope) against the salt concentration used to treat the beetroot cells. Be sure that the independent variable (Salt Concentration) is on the X-axis and the dependent variable (Rate of Diffusion) is on the Y-axis. Write a full caption, including a title and brief description of the data presented, and place it below the figure. In summary, Figure 4 should have two sets of points, one showing slope values for the range of concentrations of the First Salt (six points, one each for tubes 1-6) and one for slope values for the range of concentrations of the Second Salt (six points, one each for tubes 7-12), with their associated trend lines.

For this report, the Discussion section should be an integrated interpretation of the data presented. Although the specifics are provided here in outline form, your discussion should be written as a series of paragraphs that form a cohesive unit for describing your data analysis.

1. Figures 1 and 2: Describe the relationship between the variables that is observed in these graphs. What is the basis for this relationship? Consider events at the cellular and molecular levels that provide the mechanism for this relationship. Be sure to compare what is happening early in the experiment to events at later time points. Assess whether the use of salt treatments demonstrated differences from samples treated with water only. Also be certain to contrast the patterns in Figures 1 and 2, highlighting any differences. How well does the data fit the expected pattern? By what criteria are you evaluating the fit of the data? What are some possible sources of error that could have contributed to deviations from the expected values?
2. Figure 3: When interpreting this graph, focus on comparing and contrasting the patterns for treatment with First Salt and Second Salt. Describe the relationship between the variables that is observed in this graph. What is the basis for this relationship? Consider events at the cellular and molecular levels that provide the mechanism for this relationship. Be sure to compare what is happening early in the experiment to events at later time points. How well does the data fit the expected pattern? By what criteria are you evaluating the fit of the data? What are some possible sources of error that could have contributed to deviations from the expected values?
3. Figure 4: In analyzing this graph, focus on the effects of treatment with increasing salt concentrations on the rate of diffusion. Also be certain to compare and contrast treatments with the First Salt and Second Salt. Consider the chemical properties of the salts you studied and how these salts would interact with components of beetroot cells. Describe the relationship that is observed and propose a mechanism to account for that relationship. How well does the data fit the expected pattern? By what criteria are you evaluating the fit of the data? What are some possible sources of error that could have contributed to deviations from the expected values?

4. Either within the body of your discussion or as a summary, be sure that you have addressed how the diffusion process worked in your study of beetroots. Why were you able to observe diffusion in this system? What was the action of the solvent? What was the purpose of the salt treatments? Did one of the two salts have a greater effect than the other? Be sure to connect the actions of the salts and solvent to their affects on cellular structures in beetroots.

The Conclusion section should be a concise summary of the relationship(s) observed between the variables studied. Be sure that each relationship is precisely described, using mathematical terms when possible. Your conclusion should attempt to integrate what has been learned about the factors that influence the rate of diffusion.