Membrane Lab Data - Lab Quiz #1

DIFFUSION, OSMOSIS, CELL MEMBRANE PERMEABILITY, AND TONICITY

1. Diffusion Through Agar: Influence of Temperature

Obtain 3 agar-filled petri dishes. Create a 1 cm well (circle of gel removed from the center by punching with a test tube). Mark each dish with a permanent marker for identification. Fill each well with 1.5% methylene blue solution (MW= 319.9). Mix the dyes well beforehand and fill the wells completely with approximately equal volumes of dye (you may want to count an equal number of drops to place in each well), but do not exceed the capacity of the well. Choose plates that have about the same agar thickness.

Note the time the dye was added. Place one of the dishes containing methylene blue in the refrigerator and one in the incubator. Leave the last plate at room temperature by setting it on a shelf in the back of the room. Record the temperature in the refrigerator, room, and incubator. Allow the dishes to remain undisturbed for ~24 hours. Use a metric rule to measure the distance (in cm or mm) from the edge of the well to the diffusion edge in each of the dishes. Note how long the plates were in their respective temperatures and record all measurements. How is temperature influencing the dye molecules and, thus,diffusion distance? Explain.

2. Diffusion Through Agar: Influence of Molecular Weight (MW) and/or Permeability

Obtain 4 agar-filled petri dishes. Create a 1 cm well as in Experiment 1. Mark each dish with a permanent marker for identification. Fill one well with 1.5% methylene blue solution (MW= 319.9), one well with 1.5% Azocarmine B solution (MW= 681.6), one well with 1.5% Congo red solution (MW = 696.7), and one well with 1.5% Evans Blue solution (MW= 960.8). Mix the dyes well beforehand and fill the wells completely with approximately equal volumes of dye (you may want to count an equal number of drops to place in each well), but do not exceed the capacity of the well. Choose plates that have about the same agar thickness. Note the time the dyes were added. Leave all the plates at room temperature by setting them on a shelf in the back of the room. Allow the dishes to remain undisturbed for ~24 hours. Use a metric rule to measure the distance (in cm or mm) as in Experiment 1. Note how long the plates were left at room temperature and record the diffusion distance. Are the diffusion distances the same for each dye? Is there a relationship between MW and diffusion distance? If not, what could explainthese data?

3. Diffusion Through Agar: Influence of Concentration Gradient

Obtain 3 agar-filled petri dishes. Create a 1 cm well in each dish as in Experiments 1 and 2. Mark each dish with a permanent marker for identification. Mix the dyes well beforehand and fill three wells with methylene blue solution consisting of the following concentrations: 0.5%, 1.5%, and 2.5%. Add equal volumes of dye to each well and be careful not to exceed the capacity of the well. Note the time the dye was added. Put all the plates on a shelf at room temperature. Allow the dishes to remain undisturbed for ~24 hours. Use a metric rule to measure the distance as in Experiments 1 and 2. Note how long the plates were at room temperature and record all measurements. Are the diffusion distances different for the three concentrations of methylene blue? Why or why not? Explain.

4. Osmosis in Plant Cells

a. Cut and skin two "similar" potato strips, each about half the length of the large test tubes. Each member of the group should feel the potato to get a subjective idea of its consistency and flexibility.

b. Gently blot the slices dry and weigh each.

c. Label two test tubes # 1 and #2 and place one potato strip in each tube.

d. Fill tube #1 with deionized water until the potato strip is covered.

e. Fill tube #2 with a 10% sodium chloride solution until the potato strip is covered.

f. Allow both test tubes to stand at room temperature until the end of the lab period.

g. Remove each potato from the test tube, gently blot dry, and re-weigh. Observe each potato strip for limpness or rigidity. In which potato did water loss occur and in which potato did water gain occur? How can these data be explained?

5. Osmosis

Soak 4 pieces of dialysis tubing, about 15 cm in length, in deionized water for a few minutes until it is pliable. Tie a tight knot in one end of the 4 pieces of tubing with heavy string and fill a dialysis bag with about 10 milliliters of the following solutions:

Dialysis Bag contents

Bag 1 - Deionized water

Bag 2 - 5% sucrose

Bag 3 - 10% sucrose

Bag 4 - Deionized water

Beaker contents

Beaker 1 - Deionized water

Beaker 2 - Deionized water

Beaker 3 - Deionized water

Beaker 4 - 10% sucrose

Note: Make bags in the following order so as not to contaminate the pipette: Bag 1, 4, 2, and 3.

Tie a tight knot all the way at the other end of the tubing. When you are finished making all the dialysis bags, blot them dry and weigh them on the balance to the nearest O .1 grams. Immerse bag 1 in beaker 1, bag 2 in beaker 2, etc (this represents time 0). At 30 minutes remove the bags from the beakers, gently blot them dry, and re-weigh. Return the bags to the proper beaker. Repeat previous procedure at 60 minutes and 90 minutes and record the mass of the bags. How can these data be explained?

6. Cell Permeability

a. Arrange in order a series oflabeled test tubes containing equal amounts of the following solutions(all~ 0.3 M): urea (MW= 60), glycerol (MW= 92), and glucose (MW= 180).

b. Add 1 drop ofred blood cells to each of the three solutions and mix thoroughly. Determine the time required for hemolysis of the cells (time when the membranes of all red blood cells have been ruptured) by observing the clearing of the initial murky solution. Observe each solution carefully for the first several minutes after adding the blood and as frequently as possible until the end of the laboratory period. For the end point of complete hemolysis, use the time when you can clearly see the blue lines on a piece of ruled notebook paper held behind the test tube. When the red blood cells are intact the solution has an opaque, milky appearance; when the red blood cells have undergone hemolysis the solution becomes transparent.

Because time requirements for different substances vary, start the experiment relatively early in the laboratory period. How can these data be explained?

Note: Do test tube 1 first and then set up test tubes 2 and 3 at the same time and monitor throughout lab.

7. Tonicity

a. Arrange in order a series of labeled test tubes containing equal amounts of each of the following

solutions shown below. Note: Fill each test tube only about one-third full.

Test tube 1: 0% NaCl (Deionized water)

Test tube 2: 0.225% NaCl

Test tube 3: 0.45% NaCl

Test tube 4: 0.9% NaCl

Test tube 5: 1.35% NaCl.

Do test tubes I and 2, one at a time.

Set up test tubes 3, 4, and 5 at the same time and monitor throughout lab.

b. Add 1 drop of red blood cells to each of the test tubes and mix thoroughly. Record the time to hemolysis as you did in Experiment 6. For the end point of hemolysis, use the time when you can clearly see the blue lines on a piece of ruled notebook paper held behind the test tube. How can these data be explained? What is the relationship between the blood and the surrounding solutions? t is important to note that the sodium chloride concentration of the blood of humans is 0.9%.

Diffusion, Osmosis, Cell Membrane Permeability, and Tonicity

Results:

Experiment 1- Diffusion Through Agar: Influence of Temperature

Experiment 1

Experiment 2- Diffusion Through Agar: Influence of Molecular Weight (MW) and/orPermeability

Experiment 2

Methylene Blue Azocarmine B Congo Red Evans Blue

Substance

Experiment 3- Diffusion Through Agar: Influence of Concentration Gradient

Experiment 3

Concentration Gradient of Methylene Blue

Experiment 4: Osmosis in Plant Cells

Potato 1 (Deionized H20}

Experiment 4

Potato 2 (10% NaCl)

Solution

Iii Before Soaking

After Soaking

Experiment 5- Osmosis

Experiment 5

30 60

Diffusion Time (minutes)

Experiment 6- Cell Permeability

Experiment 6

Urea (60) Glycerol {92) Glucose (180)

Substance Molecular Weight

Note: The glucose group does not show a column as complete hemolysis did not occur in that group.

Experiment 7- Tonicity

Experiment 7

NaCl Solutions

Note: The 0.45%, 0.9%, and 1.35% groups do not show columns as complete hemolysis did not occur in those groups.