LAB 5—DIFFUSION & OSMOSIS

OBJECTIVES

• Practice applying hypothesis testing, and further your understanding of the basic principles underlying the scientific method and experimental design.
• Identify independent and dependent variables in an experiment.
• Describe diffusion and osmosis.
• Practice graphing data obtained in the lab and designing useful data tables.
• Understand and be able to define the following terms: diffusion, osmosis, concentration gradient, tonicity, hypotonic, hypertonic, isotonic, turgor, selectively permeable, semipermeable, osmotic pressure, turgor pressure, plasmolysis, hypothesis, prediction, theory, control, independent variable, dependent variable, controlled variable, quantitative variable, qualitative variable.

PRELAB

1. Draw a “flow chart” (in your lab notebook) to diagram what you will do (and in what order) in this Lab.

Exercise A – Plasmolysis in green leaf cells

• Predict what you think will happen and why, when the fresh water around the leaf is replaced by salt water. Predict what will happen with respect to osmosis and also with respect to what will happen to the cell itself.

Exercise B – Osmosis and dialysis tubing

• Predict what will happen inside the tubing comparing the three concentration treatments.

Exercise C – ‘Osmosis Egg as Model Cell’

• What hypothesis is being tested by this “experiment”/demonstration?
• What is the dependent variable?
• What is the independent variable?
• Are they quantitative or qualitative?

EXERCISE A – INTRODUCTION

In this lab you will explore the processes of diffusion and osmosis.  Diffusion can occur across a semipermeable membrane, however diffusion also occurs where no barrier (or membrane) is present.  A number of factors can affect the rate of diffusion, including temperature, molecular weight, concentration gradient, electrical charge, and distance.  Water can also move by the same mechanism.  This movement of water is called osmosis.

PROCEDURES

1. Please work in pairs for Exercises A and B and work in groups of 4 for Exercise C.
2. BEFORE YOU BEGIN make a group plan about what you will do and who will be doing what.  Your Flow Chart that you made for the Prelab Assignment will be useful for this.  Note that one of the exercises requires using a microscope and each student should make their own microscopic observations and drawing.
3. All data/observations must be written in your lab notebook.

Exercise A: Plasmolysis – Osmosis in a Living System (plant leaf)

Plant cells rely on pressure exerted outwardly by the fluid in the their vacuole to help them maintain their shape, much as a tire must be pumped up with air to provide the outward pressure that maintains the tire’s shape.  The pressure exerted by the vacuole’s fluid is called turgor pressure.  That pressure is maintained by the cell by adjusting the concentration of solutes in the vacuole’s fluid.  In this exercise you will not be testing a hypothesis.  Instead, you will observe what happens when you replace the fresh water surrounding a specimen of plant cells with a salt solution.

1) Obtain a small section of a leaf from an aquatic plant and put it on a clean slide.  Place a drop of water on the specimen on the slide, cover it with a cover slip, and examine the leaf first at scanning (40X), the low power (100X) and then at high power (400X).  Locate a region of healthy cells where there are only one or two layers of cells.  Be sure that you can clearly see the individual cells.  Sketch several adjacent cells at 400x in your lab notebook.  Remember to include total magnification and a title that identifies the organism.  Label the structures that you can see such as cell walls, nuclei, vacuoles, and chloroplasts.  DO NOT move the slide while doing the next step; you will want to observe the same cells.

2) While touching one edge of the cover slip with a piece of Kimwipe to draw off the water, add a drop of 15% salt solution to the slide next to the opposite edge of the cover slip. Be sure that the salt solution moves under the cover slip.  Observe how the cell responds. After a few minutes, sketch the same cells you sketched in before (in step a).  Label the cell structures again, including the cell or plasma membrane. Be sure to label both sketches “before adding saltwater” and “after adding saltwater”.

Include in your lab notebook answers to the following questions:

• Describe your observations (i.e. what happened?) when the water in which the cells were mounted was replaced by the salt solution. Refer to the cell structures that you labeled.
• Assuming that the cells have not been killed, what should happen if the salt solution were to be replaced by water? Describe what you would likely observe (i.e., make a prediction), and explain.
• Can osmosis likely cause plant cells to burst? Explain, comparing plant and animal cells.

For photomicrographs of images similar to what you observed in lab, you can do a “Google” image search for ‘Elodea plasmolysis’.  These images will not be the same as your observations and drawings that you must do from your slides.

EXERCISE B – INTRODUCTION

Exercise B: Demonstrating osmosis using semi-permeable tubing

The water movement through a membrane is called Osmosis.

All living cells are surrounded by a selectively permeable membrane, which contains transmembrane proteins with hydrophilic interiors (channels) to allow smaller polar molecules to pass such as water, ions, sugars and amino acids. Especially favored is the movement of water molecules through many aquaporins or water channels.

In this exercise, you will use synthetic or human-made membranes, which were first developed in Seattle, and used in artificial kidneys or dialysis machines. We will use them to demonstrate the osmotic movement of water molecules into concentrated sugar solutions.

Materials:  (per group of 2)

• One large tray
• One large beaker (500mL)
• Three 16cm dialysis tubes
• Ring stand with clamps x3
• Three 10mL Falcon Tubes
• Scissors
• Three stretches of thread or cord
• Green tubing clamp (flat)

PROCEDURES

In this portion of the lab, please work in groups of 2 at your lab bench.

1. Cut three 16-cm-long strips from the dialysis tube. It is dry and flat and needs to be soaked in warm water.
2. Get three 10 ml-sized Falcon Tubes and remove the lower tip with a razor blade or scissors while leaving a cone-shaped opening.
3. You now push one open end of the wet dialysis tubing over the cone-shaped end of the Falcon Tube. It will resist at first but finally fit snugly over the tube since both have similar diameters. Wrap some thread or cord around the tube fixing it firmly to the Falcon tube.
4. At the other end of the dialysis tube you fold it twice and clamp it closed with a green tubing clamp. This should be done so that you have an exposed length of dialysis tube of about 10 cm. Attach the Falcon tube to a ring stand and a clamping fixture.

Fill it slowly with tap water to test for stability and potential leaks.

5. Repeat this procedure to produce two more identical sets.
6. You are now ready to fill the three tubes with different solutions. One tube will contain a Pancake syrup (fully or 1.0 x concentrated), another tube with diluted Pancake syrup (0.5 x concentrated), and the third tube with water (as a control). Fill all three tubes to a similar height and mark the upper meniscus with black marker line.
7. You are set to start the experiment by lowering the filled dialysis tubes completely into a large (500 ml) beaker with water. Record time zero in the table below.

DATA ANALYSIS & INTERPRETATION

Include in your lab notebook answers to the following questions:

• Graph the results with 3 curves (placet the dependent variable on the y axis). Make sure to include a legend to tell the differences between treatments.
• Why does the water flow into the dialysis tubes rather than the sugar flowing out and into the water of the beaker?
• If the Falcon tubes would be closed, the liquid level could not rise. What would happen in this case? Then, compare what might happen if this was an animal cell.

EXERCISE C – OSMOSIS & TONICITY

Materials:  (per group of 4)

• 1 large tray                               •  3 – 400 ml beakers
• 3 weigh boats                          •  1 electronic balance
• 3 decalcified eggs                   •  paper towels

In this portion of the lab, please work in the same groups of four at your lab bench.

There will be three solutions available in lab. Your objective is to determine the tonicity of these solutions relative to the egg “model cells”.  These solutions may be isotonic, hypotonic or hypertonic relative to the egg.  Models or simulations are used extensively in science to study phenomenon that may otherwise be difficult to study.  We will model cells by using eggs.  The egg shells have been removed (decalcified) by soaking them in vinegar, and the remaining egg membrane is permeable to water but not sugar.

Work over the large tray when you are handling the eggs!

Your group should choose three solutions for your three beakers.  You will dry and weigh the three eggs before placing them in the beakers.  As the egg sits in the solution, it will either gain weight, lose weight, or remain the same weight.  By weighing the eggs, you will be able to determine the tonicity of each unknown solution relative to each egg.  Each egg should be in solution at total of 30 minutes.  Your group will need to determine how often to weigh your eggs. You will need at least 5 measurements for each egg in each solution, more may be better.  For your measurements, you will gently remove from its solution (work over the tray) and weigh every X minutes.

Include the following in your lab notebook:

1. Hypothesis

• • What hypothesis is being tested by this experiment?  Remember that this should mention both the independent and dependent variables and explain the phenomenon.

2. Prediction
   • Remember to use the “If … then …” format.  Remember that the “then…” part is stated with respect to the dependent variable (what you predict will happen to the dependent variable). Explain why you made your prediction based on your knowledge from lecture, readings, etc.
3. Variables

• • What is the dependent variable? Is it a quantitative or qualitative variable?
  • What is the independent variable? Is it a quantitative or qualitative variable?

4. Results/Data Collection
   • Record all of your experimental data in the table(s) that you created for your prelab.

PROCEDURE

1. Using the wax pencil, label beakers, to identify the solution(s), for example solution “A”, solution “B” or solution “C”. Fill each beaker half full (approximately 200 ml) with the appropriate solutions (A, B or C). Also label the threeweigh boats (A, B, or C) with a wax pencil.
2. Carefully dry off the eggs. Handle the eggs one at a time OVER the tray throughout the experiment, in case the egg breaks!
3. Carefully weigh each egg (A, B or C) in its weigh boat.
4. Record the initial weights of the egg in solution A, B and/or C in data tables in your lab notebook (remember to include units).
5. Carefully place each egg in its corresponding beaker. Note the position of each egg in its respective solution. Record the time at which you placed each egg in its solution.
6. After each egg has been in its solution for the required amount of time, carefully remove the egg from the beaker. Working over the tray (in case the egg breaks), dry off the egg with paper towels, and weigh the egg in its weigh boat.
7. Collect and record your data. Record the times and weights in the data table in your lab notebook.
8. Note the final weight of each egg compared to the initial weight. What is the difference for each egg.

DATA ANALYSIS

• Complete your data tables (in your lab notebook).
• Graph these data by hand in your lab notebook. Each student must produce their own graph. Include the weight of each egg (in grams), time or time elapsed (in hours:minutes or minutes), a key, and an informative caption.

INTERPRETATION

• • What are the tonicities of your three solutions relative to the solutions inside the cells? Explain your conclusion.
  • What can you conclude about the rates of osmosis, based on your data and your graph?
  • Is the rate constant (over 30 minutes or more) for each egg?
  • What do you think would happen if you allowed the egg to sit in each solution for hours?

ERROR ANALYSIS

• • What mistakes occurred? If none occurred, what mistakes could have occurred?
  • What other sources of error can you think of, besides any mistakes?