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BIOL1408 Introductory Biology Name Lab Unit 6/7: Diffusion & Osmosis date Dr. Flo Oxley

In this lab unit, you will follow your eSciences ACC Lab Manual (posted in Blackboard: “Lab Manual”) to learn about diffusion, osmosis, and how these processes work inside cells to support life.

This document will serve as your guide, sending you to the relevant lab activities and introductory information found in the eSciences lab manual (pages for this unit are 68 - 81), or in the online replication of the eScience lab manual uploaded to Blackboard for those of you who prefer to follow along online.

NOTE: I recommend that you read from this lab guide & report document first, going to the eSciences manual materials only as directed. Students previously using the lab manual have found that the additional background information provided in this document and the step-by-step guidance through the eSciences lab materials to be beneficial.

BACKGROUND INFORMATION: DIFFUSION

Diffusion is the net movement of a solute away from an area of high concentration towards an area of lower concentration. If you have ever watched tea diffusing from a tea bag, you are familiar with the process of diffusion. You have watched the brown molecules leaving the tea bag until eventually the tea become uniformly brown. This is sometimes referred to as solute molecules moving down their concentration gradient.

Notice that I said that it is NET movement of a solute. This means that like all molecules in liquid and gas phases, solute molecules move randomly in all directions. There is no driving force for sending a solute molecule down its concentration gradient other than there is no way to prevent the random movement of molecules. Eventually, the solute molecules will become totally randomized in their distribution throughout the solvent.

What does diffusion have to do with biology? Virtually all movements of molecules into and out of, and around the interior of the cell relies on diffusion of solutes. The removal of waste products from the cell relies on the random movement of these molecules from the inside of the cell to the outside of the cell. Similarly, the uptake of vital nutrients relies on their diffusion from the outside to the inside of the cell. The circulatory system speeds these processes up by sweeping nutrients into the vicinity of cells and sweeping away waste products from the cells to be excreted elsewhere from the body.

Note that the diffusion of oxygen (a vital nutrient) and carbon dioxide (an ever-present waste product of cellular respiration) are a part of this story. Diffusion is critical in the process of providing nutrients and oxygen circulating in the bloodstream to cells. A cell must be close to a capillary, within100 microns from a capillary, in order for these metabolites to diffuse to the cell quickly enough. If it is farther than that from the cell, it will not receive the vital nutrients or be able to relieve its metabolic waste back into the blood stream.

An especially larger cell has a bigger problem with exchanging nutrients and waste products than does a smaller cell, simply because these molecules must diffuse farther in order in infuse the cell’s interior. It is believed that this is the reason that all living cells are microscopically small – they can exchange molecules more quickly with their environment if they are small, or at least have a very narrow diameter that nutrients need to traverse.

To better understand how diffusion works and how it is required to feed cells and to help cells to eliminate wastes, you may want to watch this animations on diffusion: http://www.wisc-online.com/Objects/ViewObject.aspx?ID=AP1903

Diffusion Background Questions

After reading the background information about diffusion in this lab report guide and in your eSciences lab manual (posted on Blackboard, Lab Exercises, Lab 6, Introduction), answer the following questions.

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http://www.wisc-online.com/Objects/ViewObject.aspx?ID=AP1903
1. What causes molecules to be in constant motion?

2. What are three general factors that affect diffusion rates of a solute in a solvent? Thinking about how diffusion is important in the uptake of nutrients and the discharge of waste products by cells, which of the factors affecting diffusion rates is the most operative in the health of cells in your body?

3. Describe the molecular components of the membrane that surrounds all living cells, and discuss how this membrane limits the rate of diffusion of nutrients into the cell and the diffusion of waste products out of the cell.

4. What are two chemical properties of a solute that can prevent it from freely diffusing across a biological membrane?

5. What is the one chemical property of a solute that can prevent it from freely diffusing across a dialysis membrane?

6. Go to http://highered.mcgraw-hill.com/sites/0072495855/student_view0/chapter2/ animation__how_diffusion_works.html to watch an animation about diffusion, and answer the following questions: a. At what point does the diffusion of a solute come to a complete stop?

b. Watching the diffusing molecules in this animation, are they moving in a random or in a directional manner? (Try this: pick one specific molecule and watch its motion: is it moving in one direction or randomly in all directions?)

BACKGROUND INFORMATION: OSMOSIS

Osmosis is similar to diffusion in that it is the random movement of molecules that has a net movement down its concentration gradient. But osmosis differs from diffusion in these ways:

1. Osmosis has to do with movement of solute molecules (water) instead of solute molecules. 2. Osmosis has to do with movement across a semipermeable membrane – one that is permeable to

the solvent molecules (water) and not the solute molecules. a. Biological membranes are permeable to water molecules, but is not permeable to large

and/or polar solute molecules. b. Dialysis membranes are permeable to all molecules that are smaller than the pore sizes of

the dialysis membrane.

In osmosis, water molecules cross the semipermeable membranes until the water content become equalized on both sides of the membrane. At this equilibrium point, the solutions on either side of the membrane are said to be “isotonic” or “isosmotic”.

Until equilibrium is reached, the solution with the greater solute content is said to be “hypertonic” or “hyperosmotic”. Conversely, the solution with the lesser solute content is said to be “Hypotonic” or “hypo-osmotic”. In the meantime, water molecules have net movement in a hypotonic to a hypertonic direction until equilibrium is met.

If you have ever soaked a twisted ankle in a solution of Epsom’s Salt, the same principles applied: the hypertonic Epsom’s Salt solution drew down your inflammation by osmosis. The rate of osmosis is increased when the concentration difference is high. Similarly, if you have ever noticed that the skin on your fingers pucker up after along swim, this is due to osmosis, too. The skin cells took on water because they were hypertonic to the swimming pool water. As the skin cells swelled, the skin puckered to accommodate their increased volume.

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http://highered.mcgraw-hill.com/sites/0072495855/student_view0/chapter2/animation__how_diffusion_works.html
If you have ever had an intravenous solution dripped into your veins, this solution was made up in a solution of sodium and potassium chloride salts and NOT in pure water. The reason for this is that unlike plant cells, your cells do NOT have a cell wall to protect it from overfilling to the point of bursting cells. Plant, fungal, and microbial cells are encased in rigid and strong cell walls to prevent the cells from bursting when exposed to hypotonic solutions.

Osmosis Background Questions

After reading the background information about diffusion in this lab report guide and in your eSciences lab manual (posted on Blackboard, Lab Exercises, Lab 6, Osmosis, Introduction), answer the following questions.

1. In diffusion, solute molecules are observed to move down their concentration gradient in a solution. What is observed to move in the process of osmosis?

2. Fill out the following table to describe solute concentration across a semipermeable membrane:

3. What chemical property might interfere with the ability of water molecules to freely diffuse across a biological membrane? What allows for this free diffusion of water across biological membranes to occur?

4. Go to http://highered.mcgraw-hill.com/sites/0072495855/student_view0/chapter2/ animation__how_osmosis_works.html and watch this animation about osmosis before answering the following questions:

a. Urea is a waste product of cellular metabolism that is excreted by the urinary system through osmosis across the kidney semipermeable membranes. What chemical property of the urea molecule prevents it from freely diffusing across a biological membrane?

b. What effect does a hypertonic solution of urea have on the movement of water across a semipermeable membrane?

c. Your kidneys selectively excrete urea molecules to rid your body of these metabolic waste compounds. Dialysis membranes also allow these small urea molecules to freely diffuse across during kidney dialysis, but not selectively. All small molecules can freely diffuse across a kidney dialysis membrane. Thinking on your own, what effects might kidney dialysis have on your body’s physiology, especially with respect to mineral balance (such as sodium and potassium ions), or circulating hormones?

5. Your cells are protected from overfilling with water to the point of bursting from osmosis by maintaining a constant level of solutes in body fluids. How do plants, fungi, and microbes avoid bursting in hypotonic solutions?

6. Go to http://highered.mcgraw-hill.com/sites/0072495855/student_view0/chapter2/ animation__how_facilitated_diffusion_works.html and after watching this animation, answer the following questions: a. How do biological membranes facilitate the diffusion of polar molecules?

Tonicity of a solution undergoing osmosis

Relative solute concentration (higher, lower, or the same)

Relative water content (higher, lower, or the same)

Hypertonic

Hypotonic

Isotonic

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http://highered.mcgraw-hill.com/sites/0072495855/student_view0/chapter2/animation__how_osmosis_works.html
http://highered.mcgraw-hill.com/sites/0072495855/student_view0/chapter2/animation__how_facilitated_diffusion_works.html
b. What direction do solute molecules diffuse?

Lab 6, Experiment 1. Diffusion Through a Liquid

In this diffusion experiment, you will study the effects of these two factors on diffusion: 1. The viscosity of the medium (Viscosity means the “thickness” of the medium, as in molasses in

January.) 2. The size of the solute molecules (The molecular weight as an indicator of molecular size.)

After following the protocol in your eScience lab manual, enter your data in the following tables and answer the questions that follow.

Table 1: Rate of Diffusion in Corn Syrup

(Pay attention to the units of measurement!)

Time (sec) Blue Dye

distance traveled (mm)

RedDye

distance traveled (mm)

Time(sec) Blue Dye

distance traveled (mm)

RedDye

distance traveled (mm)

0 70 2.4 2.7

10 1.7 2 80 2.4 2.8

20 2.1 2.3 90 2.5 2.8

30 2.2 2.5 100 2.5 2.8

40 2.3 2.6 100 ? ?

50 2.4 2.6 110 2.5 2.9

60 2.4 2.7 120 2.5 2.9

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Table 2: Speed of Diffusion of Different Molecular Weight Dyes

(Pay attention to the units of measurement!)

+ Net distance is the distance after 120 seconds minus the distance at 0 seconds. * To calculate speed, multiply the net distance diffused after 2 minutes by 30 to get the hourly diffusion rate.

Lab 6, Experiment 1 Questions

1. Examine the plot below. How well does it match the data you took in Table 1? Explain your reasoning or submit your own plot if necessary to show how your data differs from these.

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Structure Molecular Weight Net Distance Travel led after 2 minutes (mm)+

Speed of Dif fusion (mm/hr)*

Blue Dye 496g 25mm 750 mm

Red Dye 793g 29mm 870 mm

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2. Which dye diffused the fastest: the one with the larger or the smaller size? Is this the expected result? (If not, how can you explain your results?)

3. Does the rate of diffusion appear to change over time? Formulate an explanation of your observation: why or why not?

4. All your cells receive vital nutrients and rid toxic waste with the help of the circulatory system. What is the critical distance a cell must maintain from a capillary (the point of nutrient / waste exchange) in order to survive? Explain the role diffusion plays in this process.

Lab 6, Experiment 2. Diffusion – Concentration Gradients and Membrane Permeability.

A dialysis membrane is a plastic membrane with microscopic pores that allow only molecules that are smaller than the pore diameter to cross. You will measure osmosis by a net gain or loss in volume of dialysis bags.

You will also use chemical assays to detect solute molecules that cross the membrane. Notice (once again!) that whenever you do an assay, you need to have a negative control and a positive control to validate your results. The negative control would be water, or the solvent system in these assays. Without a negative result in your negative control, you would have to start your experiment all over again because it would indicate that you have started out with a contaminated solvent system. The positive control would be solutions known to have the substance that you are testing for. This would validate that you have performed the assay correctly and that you test reagents are working properly.

After following the protocol in your eScience lab manual, enter your data in the following tables and answer the questions that follow.

NOTE: Be sure to wear protective gloves when handling dialysis tubing. The oils in your skin can clog the pores of the tubing, making the osmosis process slow down. The dialysis tubing appears as a membrane in your kit, but when you soak it in water, the membrane will open up into a tube which you will seal at the ends in Experiment 2 and Experiment 3, as described in your eScience lab manual.

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Table 3: Indicator Reagent Data (Controls)

Table 4: Diffusion of Starch and Glucose in the Beaker Over Time

Lab 6, Experiment 2 Questions

1. Why is it necessary to have negative controls in this experiment? What important information do you get from the two negative controls?

2. Why is it necessary to have positive controls in this experiment? What important information do you get from the two positive controls?

3. Which substance(s) crossed the dialysis membrane? What evidence from your results proves this?

4. Which molecules remained inside of the dialysis bag? What evidence from your results proves this?

5. Did all of the solute molecules diffuse out of the glucose bag and into the beaker? Formulate an explanation for why this did or did not happen.

Indicator Starch Posit ive

Starch Negat ive

Glucose Posit ive

Glucose Negat ive

IKI Solut ion n/a n/a

Glucose Test n/a n/a 80

Indicator Dialysis Bag After

1 Hour

Beaker Water After

1 Hour

IKI Solut ion

Glucose Test Str ip

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6. Is the bag hypotonic with regards to the IKI solution (Lugol’s Iodine solution), or the beaker? What about the starch solution? What evidence from your results proves this?

7. What type of membrane does the dialysis tubing represent in a living cell? In what ways is the biological membrane different from the dialysis membrane?

Lab 7, Experiment 2. Osmosis – Tonicity and the Plant Cell

In this experiment, you will examine whether plant cells change size when soaked in water or in salt solution. After following the protocol in your eScience lab manual, (pp. 85 - 86), enter your data in the following tables and answer the questions that follow.

NOTE: Make sure that you are mixing the NaCl salt as you add water to make sure that it doesn’t lump up so that it dissolves more quickly. It may take some time to dissolve the salt, so you might want to start this process early in the experiment. (Step #10 in the eSciences protocol) Notice also that there is a 1-hour incubation time to allow for osmosis to occur (step #11 in the eSciences protocol). Your data would actually be improved if you let this osmosis step continue for 3-6 hours, so you might want to do set up this experiment before you do the other experiments in this unit.

IMPORTANT: When measuring the displacement of water by the potato strips, make sure that the strips are submerged in the water before taking your measurement – and make sure that only the potato strip is displacing water as you do so. The water displacement is your measurement of the volume of the potato strip, so these precautions are necessary to get an accurate reading.

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Table 5: Water Displacement of Potato Samples

Lab 7, Experiment 2 Questions

1. How long did you incubate your potatoes in the solutions before measuring their final displacement of water?

2. What is actually being measured when looking at the net change in water displacement of the potato samples?

3. Did you observe water flow in or out of the plant cells (potato cells) in each of the samples examined? How do you know this?

4. Different types of potatoes have varying natural sugar concentrations. Explain how this may influence the experiment.

5. Based on the data from this experiment, hypothesize which potato has the highest natural sugar concentration. Explain your reasoning. If the russet potato is put in 100% water solution, then it will grow in size and weight because it takes on water due to osmosis. My reasoning is because this potato took on more water than the others, therefore, it had less water molecules and more sugar and salt than the other potatoes did.

6. How did the physical characteristics of the potato vary before and after the experiment? Did it vary by potato type? What was the texture and rigidity of the potato tissue prior to the experiment? After the experiment? Did it vary by type of potato?

Potato Type

Potato Observat ions

Sample Ini t ia l Displacement

(mL)

Final Displacement

(mL)

Net Displacemen

t (mL)

Sweet Brown skin with br ight

orange inside

A (water)

6 ml 7 ml 1 ml

Sweet Brown skin with br ight

orange inside

B

(20% NaCl)

7 ml 7 ml 0 ml

Russet brown skin with off

white inside

A (water)

5 ml 7 ml 2 ml

Russet brown skin with off

white inside

B

(20% NaCl)

5 ml 4 ml -1 ml

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7. Would you expect this experiment work in the same way with other types of plant cells? What about animal cells? Why or why not?

8. From what you know of tonicity, which was more hypertonic: plant cells or water? Which was more hypertonic in the salt solution: plant cells or the 20% sodium chloride solution? Explain your reasoning.

9. If the potato is allowed to dehydrate by sitting in open air, would the potato cells be more likely to absorb more or less water? Explain your reasoning.

10. Osmosis is how excess salts and cellular waste that accumulate in your cells are transferred to the blood stream so that they can be removed from the body. Explain how you think this process works in terms of tonicity.