A study of diffusion through cell membranes

Introduction: (Initial Observation)

Diffusion is one of the methods by which water and some other liquids and gases can pass through the membrane of animal cells and plant cells. Random spontaneous movement of molecules caused by thermal agitation is known to be the cause of diffusion. In process of diffusion, molecules move from a region of higher to one of lower concentration. By diffusion water can move from one cell to the other cell and gradually travel across the plant or animal organ.

You may have noticed that after a rain, green plants look more fresh. The same is with human skin after being exposed to water for a few minutes. In all these examples water is entering the cells by diffusion.

If two solutions of different concentration are separated by a semi-permeable membrane which is permeable to to the smaller solvent molecules but not to the larger solute molecules, then the solvent will tend to diffuse across the membrane from the less concentrated to the more concentrated solution. Diffusion through a membrane is also called osmosis.

In this project you will study the factors that may affect diffusion through cell membrane in plant cells.

Information Gathering:

Gather information about diffusion and factors affecting the rate of diffusion. Read books, magazines or ask professionals who might know in order to learn about the diffusion through cell membrane. Keep track of where you got your information from.

Following are samples of information that you may find:

Osmosis

A semi-permeable membrane is easily penetrated by the water molecules but passing through is difficult for sugar molecules. If two liquids of different concentration, e. g. distilled water and a sucrose solution, are separated by a semi-permeable membrane, water molecules preferentially diffuse into the sugar solution to equalize the concentrations. We call a mainly one sided diffusion through membranes osmosis. If the concentrated solution is contained in an enclosed space, an excess pressure, known as the osmotic pressure, is produced due to the infiltration of the water molecules into this space. It eventually becomes so great that the suction power of the concentrated solution is neutralized and water molecules can no longer penetrate the membrane. However, if the concentration is increased, osmosis will start again. Osmotic processes play a large part in the regulation of water supply and in the absorption of nutrients by plants.

Osmosis with sausage skin

Use copper wire or string to ensure that the sausage skin is tight around the two rubber stoppers otherwise the solution will leak. You can use a parchment thimble fitted with a single stopper instead of the sausage skin. Mark the initial level of the solution in the tube and the level after successive twenty four hour periods. Note that the rate at which the solution rises in the tube gradually decreases with time.

Osmosis with a pig’s bladder

Dissolve two teaspoonfuls of sodium carbonate, washing soda, in a 600 mL water. Cut a section, 12 X 12 cm, from a pig’s bladder and steep it in the soda solution for 2 hours to degrease it. Then wash it well in running water for 15 minutes. Grease the lower edge of an osmosis bell with petroleum jelly both inside and outside. Stretch the degreased piece of pig’s bladder, still wet, over the greased opening and secure it by several tightly drawn rubber bands. Each band should go round twice. Close the neck of the osmosis bell with a rubber bung through the hole of which we have inserted a capillary tube. Use glycerine to ease its passage. Three quarters fill a culture vessel with distilled water. Hold the osmosis bell jar with its lower end in the water and blowing into the capillary tube check the seal. Remove the rubber stopper and capillary tube from the osmosis bell. Move the stopper further up the tube and inserted into the opening of a culture jar cover. Fill the osmosis bell almost to the top with a molar solution of sucrose. Push a second rubber stopper with a hole over the bottom of the capillary tube and then inserted in the top of the osmosis jar. Suspend the whole apparatus from the lid in the culture vessel filled with water. On inserting the rubber stopper into the neck of the osmosis jar the sucrose solution rises into the capillary. Attach a scale to the capillary tube. Note the reading and as soon as the meniscus begins to rise in the capillary tube record the reading at 15 minute intervals. The sucrose solution rises at a uniform rate in the capillary tube. The degreased pig’s bladder is a semi-permeable membrane, i.e. penetrating it is easy for water molecules but difficult for molecules of sucrose. Consequently, water passes into the sucrose solution and this causes excess pressure in the osmosis bell

Osmosis with a carrot or potato

Select a carrot or potato which has a large top and which is free of breaks in its surface. With a sharp knife, an apple corer, or cork borer cut a hole in the top of the carrot 5 cm in depth. Fill the cavity with a concentrated solution of sugar. Insert a tightly fitting 1-hole rubber stopper which carries two soda straws pushed together or a length of glass tubing. Put in a jar of water for a few hours. If the cut in the top of the carrot has not been even, it may be necessary to seal the cork in with wax dripped from a burning candle. Pupils enjoy competing to see who can attain the greatest height of water in the tube.

Osmosis with a thistle funnel and cellophane

(i) Tie a piece of cellophane over the mouth of a thistle funnel, invert the funnel and partly fill it with a concentrated sugar solution. Make sure the string around the funnel and cellophane is tied tightly so that no leaks occur. Now place the funnel with its mouth in a beaker of water and clamp it to a stand. Set the apparatus aside and notice the level of the sugar solution in the stem of a thistle funnel from time to time during the day and also next day. (ii) Repeat with sugar solution in the beaker and water in the thistle funnel. You should notice that the water level now falls in the funnel. Water particles are obviously diffusing between the membrane into the solution of sugar. In osmosis, the diffusion of water molecules is always from pure water to the solution; the result is to dilute the solution. Experiments in which a dilute solution of sugar is separated from a concentrated solution in the apparatus would show that water molecules diffuse from the dilute solution to the concentrated solution making the latter more dilute.

Osmosis with an egg

(i) Carefully remove the shell of an egg by dissolving it in dilute hydrochloric acid – the shell is largely made of calcium carbonate – leaving the egg enclosed in the thin outer skin – a membrane. Now place it in pure water. It will swell because water passes into it by osmosis – the liquid in contact with the inner surface of the membrane is an aqueous solution. (ii) Place a similar egg in a concentrated salt solution. It will shrink. Water passes out of the egg solution into the salt solution because the latter is more concentrated. (ii) Similar effects, due to diffusion of molecules by osmosis through membranes, can be observed if you place each of the following in pure water and leave them for some time. Then place them into a concentrated solution of sugar or salt (a) prunes, (b) wheat seeds, (c) dried apricots. In each case the object gains water and swells when placed in pure water: and loses water, and consequently shrinks, when in the concentrated solution.

Osmosis with dialysis tubing

Tie a length of dialysis tubing filled with a sugar solution to a capillary tube with strong thread and watch the rise in liquid level. However in this osmometer it is difficult to achieve a water tight junction between the dialysis tubing and the capillary tube because the thread is not elastic and the wall of the glass capillary tube is slippery. So you may observe a small initial rise in the level of sugar solution in the capillary tube followed by a steady drop, usually caused by leakage at the junction when sufficient hydrostatic pressure has been built up in the liquid column.

Osmosis with dialysis tubing and a polypropylene connector

Make a water tight junction between the dialysis tubing and the glass tubing using a polypropylene connector with a wide end of bore diameter 10 mm and a narrow tapered end of bore diameter 5 mm, (or fix the dialysis tubing to the T-shape connector with a rubber band, taking care not to trap air bubbles in the dialysis tubing when filling with the sugar solution). (i) Tie a knot tightly at one end of a length of soaked dialysis tubing about 16 em long. (ii) Fix the other end of the dialysis tubing to the wide end of a polypropylene connector by winding a rubber band tightly around it to form a water tight junction. (iii) Fill the dialysis tubing with a sugar solution. (iv) Join the tapered end of the polypropylene connector to a T-shape connector with rubber tubing. Join the other two ends of the T-shape connector to a 10 mL syringe filled with the sugar solution and to a calibrated 1 cm3 pipette. (v) Rinse the outer wall of the dialysis tubing with water to remove any trace of sugar solution. Immerse the dialysis tubing into a beaker of water. (vi) Move the plunger of the syringe to adjust the position of meniscus of the sugar solution in the pipette to a suitable position. Start taking measurements. When the meniscus reaches the top of the pipette, it can be moved to the starting position by adjusting the plunger so that further measurements can be made. Add a tiny amount of Congo red to the sugar solution to see the liquid column in the pipette. Investigations can include the following: (a) The effect of temperature or solute concentration on the initial rate of osmosis of a sugar solution. (b) Compare the initial rates of osmosis of different solutions, e.g. starch, sucrose and glucose solutions. (c) Changes in the rate of osmosis with time. Use a 1 meter long capillary tube instead of the 1 cm3 pipette. Use a Hoffman clip to close the rubber tubing that connects the syringe to the T-shape connector to prevent the syringe plunger being pushed outwards by hydrostatic pressure. This investigation shows the rate of osmosis is affected by the rising hydrostatic pressure as the height of the liquid column in the capillary tube increases. As the rate of osmosis approaches zero, the hydrostatic pressure exerted by the liquid column indicates the osmotic potential of the solution.

This shows a picture of the 4 potato halves. Number 1 has salt added to the cavity scooped out, 2 has sugar, whereas 3 is left empty and 4 (which has been boiled) also has salt.

After about 1 hour, it can be seen that liquid has formed in numbers 1 & 2, but not in 3. Number 4 remains practically the same.

DEMONSTRATION OF OSMOSIS USING VISKING TUBING

“Visking” tubing is a form of processed cellulose or cellophane which has pores in it through which water (and other small molecules) can pass, so it can be considered as a PARTIALLY PERMEABLE MEMBRANE. If it is sealed at one end, attached to a glass tube, and filled with a liquid such as sugar solution, and immersed in another liquid such as water, then water should pass through the visking tubing and cause the level of liquid to rise inside the glass tube.

What has caused the change in levels within the visking osmometers?

Movement of water NOT SOLUTION

What is the difference between the results from the levels inside the two setups?
Container 1: liquid level rose
Container 2: liquid level fell

What do you think has happened to the volume of liquids in the surrounding containers?
Container 1: liquid level fell
Container 2: liquid level rose

Apart from liquid movement, what else does the change in liquid levels indicate?
Buildup of pressure

DEMONSTRATION OF OSMOSIS IN POTATO TISSUE

Potato osmometer.

Each potato half is subjected to a different experimental treatment. Salt and sugar both dissolve to make a strong solution. Boiling damages the cell structure, especially the cell membrane.

Observe and record the level of any liquid forming in the cavities.

Return to see and record the results of this experiment next day.

Note only the presence or absence of liquid.

Do not worry about apparent decay or odd colors, and do not bother referring to starch.

Question/ Purpose:

What do you want to find out? Write a statement that describes what you want to do. Use your observations and questions to write the statement.

Possible questions that you may choose to study are:

• How does temperature affect diffusion through cell membrane?
• How does concentration of a solution affect the rate of diffusion through cell membrane.

Identify Variables:

When you think you know what variables may be involved, think about ways to change one at a time. If you change more than one at a time, you will not know what variable is causing your observation. Sometimes variables are linked and work together to cause something. At first, try to choose variables that you think act independently of each other.

For the question “How does temperature affect diffusion through cell membrane?”, this is how you may define variables:

Independent variable (also known as manipulated variable) is the temperature.

Dependent variable (also known as responding variable) is the relative rate of diffusion of water through cell membrane.

Controlled variables are light and wind. (We control light and wind as two environmental factor that may affect our experiment results. We make sure that all of our experiments are performed at identical light and wind conditions.)

Hypothesis:

Based on your gathered information, make an educated guess about what types of things affect the system you are working with. Identifying variables is necessary before you can make a hypothesis.

This is a sample hypothesis:

Since heat energy increases the agitation and random movements of molecules, I expect a higher rate of diffusion in higher temperatures.

Experiment Design:

Design an experiment to test each hypothesis. Make a step-by-step list of what you will do to answer each question. This list is called an experimental procedure. For an experiment to give answers you can trust, it must have a “control.” A control is an additional experimental trial or run. It is a separate experiment, done exactly like the others. The only difference is that no experimental variables are changed. A control is a neutral “reference point” for comparison that allows you to see what changing a variable does by comparing it to not changing anything. Dependable controls are sometimes very hard to develop. They can be the hardest part of a project. Without a control you cannot be sure that changing the variable causes your observations. A series of experiments that includes a control is called a “controlled experiment.”

Experiment 1: Does heat change the diffusion ability of the cell membrane?

Introduction: In this experiment you will test to see if the cells of a steamed/ cooked carrot allows diffusion of water. You will also compare salt and sugar for their effects in diffusion of water.

Procedure:

1. Get 4 clear glass or plastic tubes about 10 cm (4 inches) long each. The diameter of tubes must be less than 1 cm (3/8 inch).
2. Get 4 same size fresh carrots.
3. Use steam or boiling water to cook one of the carrots for 10 minutes.
4. Use a drill bit to make one 2 cm deep hole on the top of each carrot (where the stem used to be). The diameter of the hole must be the same as the diameter of the tube so the tubes will fit snugly.
5. Insert the tube in the hole of the first uncooked carrot, place the carrot in a cup half filled with water, and label it as “Fresh/Nothing”. This is also your control experiment.
6. Place some salt (about 1 gram) in the hole of the second fresh carrot, place the carrot in a cup half filled with water, and label it as “Fresh/Salt”.
7. Place some sugar (about 1 gram) in the hole of the third fresh carrot, place the carrot in a cup half filled with water, and label it as “Fresh/Sugar”.
8. Place some salt (about 1 gram) in the hole of the cooked carrot, place the carrot in a cup half filled with water, and label it as “Cooked/Salt”.

9.Make sure the level of water in the cups are high enough to cover at least 3/4th of each carrot.

10.Inspect the carrots every few hours and the next day. Look for accumulation of water in the tubes.

11.Does the cell membrane of cooked carrot allow diffusion?

12.Which of the two salt and sugar solutions cause a stronger force of diffusion?

13.Does diffusion happen in the carrot with an empty hole?

Experiment 2: How does temperature affect diffusion through cell membrane?

Introduction: In this experiment you will test the effect of temperature on the rate of diffusion through cell membrane.

Procedure:

Repeat the experiment number 1 with 4 fresh carrots and four clear tubes; however, all four carrots will remain uncooked and all four carrots will have sugar in their holes. Use aluminum foil to cover all cups with the carrot inside them. Aluminum foil will protect your experiment setup from light. Label your setups as cold, normal, warm and hot. You may also use thermometers to measure and record the actual temperatures.

Also cover the tops of the tubes with paper towel to make sure that no water is evaporated.

Place one cup in the refrigerator, second cup at room temperature, third cup in a warm room and the last cup in a hot room (or next to a heater).

Possible temperatures that you may try in Celsius degrees are 5, 20, 35 and 50; however, you may not be able to create such temperatures and satisfy with anything close to these temperatures.

After 24 hours revisit all setups and record the level of water in the tube. For best results you may repeat this experiment a few times and get average of your results. Your results table may look like this:

Make a graph:

You may also present your experiment results in the form of a bar graph. Make one vertical bar for each temperature. The height of bar represents the height of water in tubes (relative diffusion rate through the cell membrane). To make you graph more visible, you may increase the height of bars by a factor of 10 or 20. In other words 15 mm of water in tube may have a bar that is 150 millimeters tall. Make sure to name each bar with a word such as “Cold” or with a temperature such as “5ºC”.

Materials and Equipment:

List of material may be extracted from the experiment section.

Results of Experiment (Observation):

Experiments are often done in series. A series of experiments can be done by changing one variable a different amount each time. A series of experiments is made up of separate experimental “runs.” During each run you make a measurement of how much the variable affected the system under study. For each run, a different amount of change in the variable is used. This produces a different amount of response in the system. You measure this response, or record data, in a table for this purpose. This is considered “raw data” since it has not been processed or interpreted yet. When raw data gets processed mathematically, for example, it becomes results.

Calculations:

If you do any calculations, please write your calculations in this section of your report.

Summary of Results:

Summarize what happened. This can be in the form of a table of processed numerical data, or graphs. It could also be a written statement of what occurred during experiments.

It is from calculations using recorded data that tables and graphs are made. Studying tables and graphs, we can see trends that tell us how different variables cause our observations. Based on these trends, we can draw conclusions about the system under study. These conclusions help us confirm or deny our original hypothesis. Often, mathematical equations can be made from graphs. These equations allow us to predict how a change will affect the system without the need to do additional experiments. Advanced levels of experimental science rely heavily on graphical and mathematical analysis of data. At this level, science becomes even more interesting and powerful.

Conclusion:

Using the trends in your experimental data and your experimental observations, try to answer your original questions. Is your hypothesis correct? Now is the time to pull together what happened, and assess the experiments you did.

Related Questions & Answers:

What you have learned may allow you to answer other questions. Many questions are related. Several new questions may have occurred to you while doing experiments. You may now be able to understand or verify things that you discovered when gathering information for the project. Questions lead to more questions, which lead to additional hypothesis that need to be tested.

Possible Errors:

If you did not observe anything different than what happened with your control, the variable you changed may not affect the system you are investigating. If you did not observe a consistent, reproducible trend in your series of experimental runs there may be experimental errors affecting your results. The first thing to check is how you are making your measurements. Is the measurement method questionable or unreliable? Maybe you are reading a scale incorrectly, or maybe the measuring instrument is working erratically.

If you determine that experimental errors are influencing your results, carefully rethink the design of your experiments. Review each step of the procedure to find sources of potential errors. If possible, have a scientist review the procedure with you. Sometimes the designer of an experiment can miss the obvious.

References:

List of References

UNESCO Biology experiments

http://employees.csbsju.edu/SSAUPE/biol116/Botany/plant_transport.htm

http://members.aol.com/profchm/osmotic.html

Calculate Osmotic Pressure

Transport in and out of cell