Public Financial Management

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LAB 5: DIFFUSION AND OSMOSIS

Diffusion and osmosis are important processes in all biological functions. Both diffusion and osmosis are required in

part to control the movement of ions (charged atoms or molecules), water, and organic molecules in living systems.

Thus, the proper functioning of nerves, muscles, kidneys, lungs and every organ in your body depends on diffusion

and osmosis.

The driving force of diffusion and osmosis is the random movement of particles provided by kinetic energy. KINETIC

ENERGY is the energy of motion, and occurs in all states of matter (solids, liquid s, and gases). The kinetic energy

increases as the temperature of the matter increases, and decrease s as the temperature decreases, hence the movement

of atoms and molecules changes as the temperature changes. At a very low temperature, called " absolute zero" (-273

℃ or -460℉), the kinetic energy equals zero and all motion ceases. Above absolute zero, particles " bounce" around

at random, moving in a straight path until they collide with another particle (which they " bounce" off and proceed to

travel in another direction until they bounce off another, and another, etc.).

Since biological systems always function far above absolute zero, it is safe to assume that there is always quite a bit of

kinetic energy (hence random molecular motion) within living systems. What this mean s, in a practical sense, is that

molecules are in a constant state of motion, even in material that (to the naked eye) look like it is still. A familiar

example of this motion, and the effects of temperature on the amount of motion, is the rate at which sugar dissolves in

cold versus hot tea. When the tea is cold, sugar will sit on the bottom of a glass and slowly dissolve; however, as the

temperature of the tea (hence its kinetic energy) begins to increase, the rate at which the sugar dissolves increases

dramatically. This is due in part to the different rates at which the sugar molecules are "moving away from the pile of

suga r". They still move away in cold tea, but they move much faster in hot tea due to increased level of random

motion. This molecular movement is also affected by the size of the molecules (in general, smaller molecules will

move faster than larger molecules) and the nature of the medium they are moving through (faster in gases than in

liquids, and so on). One of the first persons to observe the random motion of particles was Robert Brown (1827), so

we now call this motion BROWNIAN MOTION.

DIFFUSION

DIFFUSION is the net movement of particles from an area of high concentration to an area of low concentration. This

movement occurs via Brownian motion: the particles in the area of high concentration tend to move out to areas with

a lower concentration. Although some also move back toward the area of higher concentration, the majority (due to the

random nature of the direction of movement) tend to move to areas of lower concentration. Eventually, the particles

become equally distributed within the medium in which they are diffused; this is termed the "equilibrium" state. Since

diffusion is driven by Brownian motion, and Brownian motion increases with temperature, the rate of diffusion is also

strongly affected by temperature. Remember the example of the sugar diffusing in hot and cold tea? The diffusion of

sugar molecules in warm tea occurs much more quickly than diffusion of sugar molecules in cold tea simply because

the rates of Brownian motion are much greater in the warm tea. This means that sugar molecules are "escaping" from

the pile of sugar and moving throughout the tea at random at a much higher rate in warm than in cold tea.

As an analogy, imagine 15 pool balls clustered in the center of a pool table. If you were to hit the cue ball into the

group, the balls would move out of the "area of high concentration" to "areas of lower concentration," bouncing off

of the walls and the other balls during the process. The balls move in a straight line until they collide with something

- either the wall or another ball. Although some might, by chance, return to the center of the table, most would wind up elsewhere, " diffused across the table" in a sense. If you can imagine the balls bouncing around and never stopping,

eventually becoming equally distributed (yet still moving) across the table, you would have an idea of the result of

diffusion. In this analogy, the speed of the balls would be an indication of the temperature of the system; the higher

the temperature, the faster the balls travel, the more often they collide with each other and the walls, the faster they

spread out and reach equilibrium, etc.

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As a realistic example of diffusion, think about the effect of opening a bottle which contains a foul-smelling

liquid or gas (vinegar, rotten food, etc.) in a closed room. The gas is in high concentration in the jar, and in low

concentration in the air within the room. However, soon the " smell" (gas particles) begins to diffuse away from

the jar and out into the room. At first the smell is strongest near the bottle and diminishes as you move away

from it, but if you leave the bottle open long enough the entire room will smell the same (equilibrium has been

reached). This is diffusion.

Diffusion is not restricted to any one medium; it can (and does) occur in solids, liquids and gases. The rate of

movement is greatest in gases and slowest in solids, but it still occurs in all three. Also, any particle can diffuse,

however, the rate is affected by factors such as size (with large particles diffusing more slowly than small ones)

and chemical properties (charged versus uncharged, hydrophobic versus hydrophilic, etc.) of both the particles

and the medium. The following exercise is designed to give you a feel for rate of diffusion and the differences

in rates of movements between different molecules.

Watch the following video to get a better idea of how diffusion works. https://www.youtube.com/watch?v=jhszFBtBPoI&feature=youtu.be

EXERCISE 1: Effects of Temperature and Molecule Size on Diffusion

In this exercise you will examine the movement of two dyes diffusing within a semi-solid medium called agar,

which is very similar to the gelatin that you eat. You will compare the different rates of movement of two

molecules of different sizes: potassium permanganate and methylene blue. Potassium permanganate (KMn04)

is an ionic compound consisting of potassium (K+) and manganate (Mn04⁻) ions, and has a molar mass of

158.03 g/mol.

Methylene blue (C16H18ClN3S) is a complex chemical compound with a molar mass of 3l9.85 g/mol. Thus, one molecule of potassium permanganate has about half the molar mass of one molecule of methylene blue. In

addition to size, you will also examine the effect of temperature on the rate of diffusion.

Online lab modification: You will not be preparing diffusion plates in

this online lab. Instead you will be viewing demonstrations online to

answer the questions in the lab report.

https://youtu.be/EW7awHHXr0w (Molecule Size)

https://www.youtube.com/watch?v=IgbR-K1ff-w (Temperature)

You should still read through the procedures below as it is helpful to

understand how the procedure is done.

Materials:

Forceps

Paper disks

Two agar Petri dishes

1% solution of potassium permanganate (TOXIC - wear gloves and safety goggles.)

1% solution of methylene blue (TOXIC - wear gloves and safety goggles.)

Procedure:

1. Obtain two Petri dishes containing agar, one from the refrigerator and one from the incubator. Label

both plates with your initials.

2. Using the appropriately labeled forceps, pick up a paper disk and dip it into the solution of

potassium permanganate, and then place it on the surface of the agar about 1cm from the edge

of the plate. Be careful not to drip any of the solution onto the agar. Repeat for the other plate.

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3. Repeat step 2 for the methylene blue. Remember to use the appropriately labeled forceps and

place the paper disk approximately 1 cm from the opposite edge of the plate.

4. Place the plate from the refrigerator back in the refrigerator (4°C) and the plate from the incubator

back in the incubator (37°C).

5. After 90 minutes, measure the distance in millimeters each of the compounds has diffused from the

edge of its paper disk. Record your measurements and draw the plates to scale below.

6. EXERCISE CLEAN-UP a. THROW THE LIDS IN THE TRASH CAN.

b. PUT THE PLATES IN THE WASTE JAR LABELED “METHYLENE BLUE AND POTASSIUM PERMANGANATE WASTE”

Potassium

Permanganate

Methylene

Blue

Hot (37°C) mm mm

Cold (4°C) mm mm

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OSMOSIS

Both osmosis and diffusion are important biological processes because they are involved in the transport of

materials (such as oxygen and carbon dioxide) throughout your body, and also the transport of materials into

and out of individual cells. OSMOSIS is a special case of diffusion: it is the movement of water down its

concentration gradient, from an area of higher water concentration to an area of lower water concentration (in

other words, osmosis is the diffusion of water). In osmosis the concentration gradient of water (the SOLVENT)

is created by the addition of a dissolved solid (the SOLUTE (e.g., salt or sugar)) in two different areas. The

more solute present in a solution, the less room for water, and thus the lower the concentration of water.

Conversely, areas with lower concentrations of solutes will have a higher concentration of water. In osmosis the

solutes are prevented from diffusing by a SEMI- PERMEABLE or SELECTIVELY PERMEABLE

MEMBRANE. A semi-permeable membrane, as the name implies, is a membrane which allows certain

molecules to pass through while excluding others (in much the same way that a sieve has pores which allows

water to pass through but prevents pasta from escaping). The plasma membrane surrounding a cell is a semi-

permeable membrane: permeable to water, yet impermeable to sugar (and many other molecules).

Water diffuses from the side of the semi-permeable membrane with lower solute concentration to the side with

higher solute concentration. There are three terms that we use to compare the solute concentrations of two

solutions: HYPERTONIC, HYPOTONIC, and ISOTONIC. Simply stated, hypertonic means having a higher

concentration of solute, hypotonic means having a lower concentration of solute, and isotonic means having

equal solute concentration. If a cell is placed in a hypertonic solution (solute concentration greater, water

concentration lower outside the cell), water will move across the membrane out of the cell away from the

hypertonic solution, causing the cell to become crenated or shriveled. If a cell is placed in a hypotonic solution

(solute concentration lower, water concentration higher outside the cell), water will move across the membrane

into the cell toward the hypotonic solution, causing the cell to swell. In both cases, water always flows from a

hypotonic solution into a hypertonic solution, or from an area of high water concentration to an area of low

water concentration. If the cell is placed in an isotonic solution (solute and water concentration equal inside and

outside of the cell), there will be no net movement of water into or out of the cell, and the cell will maintain its

original shape.

Watch the following video to gain a better understanding of Osmosis

https://youtu.be/L-osEc07vMs

EXERCISE 2: Osmosis Across Living Membranes -Animal Cells

In this experiment you will be exposing mammalian red blood cells (RBCs) to different concentrations of

sodium chloride (NaCl; table salt) in deionized water. They will react in the manner of the cells described

above. The process of osmosis occurs very rapidly in RBCs. Additionally, the RBC plasma membrane is so

fragile that if it expands too much it will burst. There is a test that can be conducted with the naked eye that

can detect the bursting of red blood cells. Whole RBCs make a solution so turbid that you cannot easily see

through the solution, whereas a solution of ruptured RBCs impart a red color but no turbidity, still allowing

you to see through the solution. Thus, by holding a test tube containing a solution of RBCs against a printed

page you can quickly tell if the cells have ruptured (i.e., are in a hypotonic solution). You can also examine the

shape of the cells using the microscope (400X total magnification) to determine if the cells have crenated, burs

t, or remain unchanged. Normal RBCs in an isotonic solution appear perfectly circular.

Online modification: You will be viewing a video where they have put

red blood cells in various solutions https://youtu.be/A8cI6FkcG4c

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Materials:

0%, 0.9%, and 10% NaCl/Blood

Solutions

Three microscope slides Three cover slips

Procedure (work in groups of four):

1. Locate the three vials of blood solutions that have been prepared by your instructor.

2. Hold each tube against this page and see if you can read through the solution. Turbidity is a

measure of the cloudiness of a solution. Higher turbidity means more cloudy, lower turbidity

means clearer. Record your results in the second column of the table on the following page.

3. Place a drop of each of the blood mixtures onto the appropriate microscope slide (use a different

slide and pipette for each mixture), cover with a cover slip, and view under the microscope.

Draw the results in the third column of the table on the following page. (When viewing and

drawing RBCs, increase the magnification to 400X and reduce the light intensity for the best

image).

4. Based on the turbidity of the solution and the shape of the cells, determine whether each NaCl

solution is hypotonic, isotonic, or hypertonic to the blood cells. Record your answers in the last

column of the table on the following page.

I Solution Clear or Turbid Shape of Cells (Draw) Relative Tonicity

0% NaCl (Deionized H2O)

0.9% NaCl

10.0% NaCl

0%

NaCl

Clear

0.9%

NaCl

Turbid

10%

NaCl

Turbid

Hyper

-tonic Iso-

tonic

Hypo-

tonic

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EXERCISE 3: Osmosis Across Living Membranes -Plant Cells

Like the animal cells observed in the previous exercise, osmosis also occurs in plant cells. The behavior of plant cells

in response to osmosis, however, is remarkably different due to the cell wall. When placed in a hypertonic solution,

water moves out of the plant cell, causing the cytoplasm to shrink as the cell membrane pulls away from the cell wall,

a process called plasmolysis. When a plant cell is placed in a hypotonic solution, water moves into the plant cell. Unlike the animal cell, the plant cell does not burst, but instead becomes rigid or turgid. You may notice your house

plants beginning to wilt when you forget to water them. Once you water them, they become turgid again and stand tall.

In this exercise you will examine the effects of sodium chloride solutions on the aquatic plant Anacharis.

Materials:

Three microscope slides

Three cover slips

Anacharis leaves Deionized water

l 0% NaCl solution

Pond water

Online modification: You will be viewing a video where they have put

Anacharis leaves in various solutions. https://youtu.be/OtPaPbVBMbM

Procedure:

1. Prepare three wet mounts of Anacharis leaves using pond water, deionized water, and 10% NaCl solution.

2. In the table below, draw one cell at 400X total magnification from each solution showing its appearance.

3. Based on the appearance of the cells, determine whether each solution is hypertonic, hypotonic, or isotonic, and record it in the table below.

Solution Deionized water Pond water 10% NaCl

Appearance

Relative Tonicity

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EXERCISE 4: Osmosis - Applying the Scientific Method

In the following experiment, we will be using Orbeez to simulate "cells" with semi-permeable membranes in order

to measure the relative rates of osmosis. The INDEPENDENT VARIABLE (the variable in the experiment that we

change) we will be testing in this lab is sodium chloride (salt) concentration. The DEPENDENT VARIABLE (the

variable that we measure for our results) is the rate of osmosis. The question we are asking in this experiment is how

different concentrations of sodium chloride will affect the rate of osmosis. In your group, formulate a hypothesis

based on this question.

Online modifications: You will be doing a virtual osmosis

experiment. Please visit the following website to do the

experiment.

https://video.esc4.net/video/assets/Science/Biology/Gateway%20

Resources/cell%20homeostasis%20virtual%20lab%20-

%20activity/index.html

Materials:

1000 mL Graduated cylinder

(4) sucrose solutions (0%, 5%, 10%, 15%)

(5) 1500-ml beakers

5 prefilled dialysis bags: (1) 0% sucrose control, (4) 10% sucrose

Electronic balance

Weigh boats

Stir rod

Procedure:

1. Using labeling tape and a marker, label five separate 1500 ml beakers each with a different

sucrose concentration, 0% control, 0%, 5%, 10%, 15%.

2. Each student will take one beaker and perform the following procedure using the

sucrose concentration indicated on the label.

3. Fill your beaker with 1000 ml of the indicated sucrose solution.

4. Weight each bag before beginning the experiment. This is your initial mass.

5. Drop all five dialysis bags simultaneously in your sucrose solution and allow to sit for 24 hours.

6. After 24 hours, remove dialysis bags from your beaker. Blot dry with a paper towel and weigh

each of the five dialysis bags in a dry weigh boat. This is your final mass.

7. To determine the percent change in mass, subtract the initial mass from the final mass, divide by the initial mass, and multiply by 100.

Percent change = [(final mass - initial mass) ÷ initial mass] x 100

OSMOSIS EXPERIMENT CLEAN-UP

a. RETURN YOUR DIALYSIS BAGS TO THE ORIGINAL CONTAINER. b. REMOVE THE LABELING TAPE FROM THE BEAKERS, RINSE THEM WITH WARM WATER IN THE SINK,

AND PLACE THEM BACK WHERE YOU FOUND THEM.

c. CLEAN UP ANY SPILLS IMMEDIATELY. SPRAY AND WIPE DOWN ALL WORK AREAS (FLOOR, LAB BENCH, BALANCES, ETC.) WITH DISINFECTANT/CLEANER WHERE SPILLS WERE POSSIBLE. A PAPER TOWEL DAMPENED WITH WARM WATER IS EFFECTIVE AT REMOVING DRIED SUCROSE SOLUTION.

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LAB 5 LAB REPORT QUESTIONS (10 points):

1. In the demonstration of diffusion of methylene blue and potassium permanganate in the agar plate, which

of the two compounds had a faster rate of diffusion? (0.5 pt)

2. Which of the factors that you learned about in your lab manual, affected the rate of diffusion in the

demonstration from question 1? Explain. (1.5 pt)

3. What effect did temperature have on the rate of diffusion of the potassium permanganate? Compare the

diffusion in the hot water to that in the cold water. (1 pt)

Questions 4-9: Based on the appearance of the cells, match the plant and animal cells with the environmental

tonicity in which they are submerged. Put the letter for your answer in the box next to the corresponding image. (3

pts)

A. Hypotonic Environment B. Isotonic Environment C. Hypertonic Environment

4

5

6

7

8

9

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10. In the video, you saw the human blood cells in 0% (Deionized H2O), 0.9%, and 10% NaCl solutions. The 0.9% NaCl solution was identified as an isotonic environment for the blood cells. What does that tell you

about the NaCl concentration inside your blood cells? Support your answer? (2 pt)

11. For each of the following comparisons, indicate whether the concentration of the first solution is hypotonic, isotonic, or hypertonic to the second solution? (1.5 points)

Environment Cell Environment Cell

5% is to 3% 1% is to 3%

5% is to 10% 3% is to 3%

5% is to 5% 5% is to 0.5%

12. Using any resources that you have available, give an example of one process that occurs in your cells that involves diffusion and/or osmosis. (0.5 pt)

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OSMOSIS EXPERIMENT (15 points)

Using the osmosis experiment simulation that you performed, fill in the data you recorded.

13. What is your hypothesis for this experiment? After having learned about osmosis, what do you predict will happen to the dialysis bags in each of the experimental concentrations of sucrose solutions? (3 pts)

Hypothesis: Dialysis bags with 10% solute concentration (sucrose) placed in an environment with higher solute

concentration will ____________________, dialysis bags with 10% sucrose concentration placed in an

environment with lower solute concentration will ____________________, and dialysis bags with 10% sucrose

concentration placed in an environment with equal solute concentration will ____________________.

14. Record your data in the table below, to the nearest tenth of a gram, (0.1 g). (5 pts)

• Record the initial mass of each dialysis bag in column 1 of the table. (1 pt)

• Record the final mass of each dialysis bag in column 2 of the table. (1 pt)

• Record the differences between the final and initial mass in column 3 (1 pt)

• Calculate the percent change in mass for each experimental dialysis bag, in column 4. (2 pts)

• Use the following equation to calculate your % change in mass.

Percent change = (Difference in Mass ÷ Initial Mass) x 100 Example: Initial Mass = 5.0 g; Final Mass = 1.0 g

Difference in Mass = (1.0 g – 5.0 g) = -4.0 g

% Change = (-4.0 g ÷ 5.0 g) x 100 = -0.80 or 80% decrease in mass

Initial Mass Final Mass Difference (F-I) % Change in Mass

0% Control

0% Sucrose

5% Sucrose

10% Sucrose

15% Sucrose

15. Of the four experimental solutions, which appear to be hypertonic to the "cell"? Which appear to be

isotonic? Which appear to be hypotonic? Place a check in the box to indicate your answer. (4 pts)

(Remember that your “Cell” is 10% sucrose)

Experimental

Solutions Hypertonic Isotonic Hypotonic

0% Sucrose

5% Sucrose

10% Sucrose

15% Sucrose

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Questions 16-18: Below is the graph of the results you should have gotten. Answer the questions below regarding

your data points.

16. A trendline indicates the general trend of the data. Does this graph indicate that the rate of osmosis has a positive, negative, or zero correlation with solution concentration? (0.5 pt)

17. Does the data support or refute your hypothesis? (0.5 pt)

18. Support your reasoning for your answer to question 17. Use the information in the graph as it relates to your hypothesis. (2 pts)

-20.0%

-15.0%

-10.0%

-5.0%

0.0%

5.0%

10.0%

15.0%

20.0%

25.0%

-5% 0% 5% 10% 15% 20%

% C

h an

ge in

M as

s

Sucrose Concentrations of Environments

Rate of Osmosis vs Solute Concentration