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Diffusion and Osmosis```
`After completing this tutorial and virtual experiments you should be able to:`
• Define: diffusion, osmosis, equilibrium, hemolysis, turgid, plasmolysis, crenation, turgor pressure, hypotonic, isotonic, and hypertonic.
• Describe how hypotonic, isotonic, and hypertonic solutions affect blood cells.
• Describe how hypotonic, isotonic, and hypertonic solutions affect plant cells.

I. Diffusion

Diffusion is the movement of molecules from a region of higher concentration to a region of lower concentration and is responsible, at least in part, for the distribution of substances from an organism's environment to all parts of the organism.

Diffusion Experiment

There will be several small petri dishes containing 2% agar available for each student. Agar is a substance extracted from seaweed that will form a gel-like solid if it is mixed with water, boiled, then cooled. Cut three wells in the agar (use a small 12 ml diameter test tube). Make certain that the wells are of equal size and are several centimeters away from each other and the sides of the dish. Do not lift the major portion of the agar away from the dish. Fill one of the wells (count the drops) with 1 M methylene blue (molecular wt. 374). Fill the second well with an equal amount of 1 M ferric chloride(mole wt. 270); and the third well with potassium permanganate (mole wt. 158). Note the diameter of the colored area at time zero and at 15 minute intervals. At the end of 90 minutes measure the final width of diffusion. Prepare a line graph of the diffusion diameter width of each of the three substances. Time of diffusion in minutes should be graphed versus diameter of diffusion in mm. Calculate the rates of diffusion per minute and per hour

Results
`Time                          Diameter of solute Diffusion`

```	Methylene Blue    Ferric              Potassium
chloride              permanganate

0 min.	 ______12______mm    ______12______mm	    ______12______mm

15 min. ______14______mm    ______13______mm	    ______20______mm

30 min. ______15______mm    ______14______mm     ______23______mm

45 min. ______16______mm    ______15______mm	    ______26______mm

60 min.	______17______mm    ______16______mm	    ______28______mm

75 min.	______18______mm    ______17______mm	    ______30______mm

90 min.	______18______mm    ______17______mm	    ______32______mm
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Discussion

1. Given the molecular weights of methylene blue, ferric chloride, and potassium permanganate, what can we conclude from the relationship of molecular weight to the rate of diffusion?

2. From the diffusion results from our experiment with ferric chloride can we conclude that lighter molecules always diffuse faster?

3. What environmental factors are there which might affect the rate of diffusion of a substance?

II. Osmosis

Osmosis is the movement of a solvent, in the case of living organisms (water), through a semipermeable membrane. Even though water will move through a membrane in both directions, there may be a net movement in one direction in response to a concentration gradient. Recall that substances will diffuse from an area of higher concentration to lower concentration. If, for instance, a cell is placed in pure distilled water, the concentration of water will be greater outside the cell and less inside the cell; thus, there will be a net movement of water into the cell. Water molecules will move toward the lower concentration of water inside the cell.The concept of the plasma membrane as a porous structure with different sized pores will explain why gasses and water penetrate quickly; why substances like glucose, amino acids, and fatty acids penetrate slowly; and why more complex molecules such as proteins, polysaccharides, lipids, and nucleic acids are unable to diffuse through the membrane at all.

Osmosis Experiments

Red blood cells (erythrocytes) are commonly used to study the permeability of living cell membranes. The plasma membrane of these cells is quite permeable to water, but relatively impermeable to salts. The bursting of red blood cells when placed in a hypotonic solution is called hemolysis. However, if erythrocytes are placed in an isotonic solution of sodium chloride (NaCl) they will maintain their size and shape. An isotonic solution has the same concentration of osmotically active particles as the cell protoplasm. Finally, if red blood cells are placed in a NaCl solution with a concentration greater than isotonic, there will be a net movement of water from the cell and the cell will crenate (shrink). In this case the external medium is said to be hypertonic to the cells. Crenated erythrocytes are characterized by an irregular and wavy border.

A. Effect of Concentration on Animal Cells (Oxblood Cells)

Red blood cells are rather opaque discs which transmit very little light when suspended in a liquid. When cells hemolyze, the suspension will become transparent. This characteristic allows one to determine the tonicity of various solutions to red blood cells. If a water suspension (distilled water ) is made of red blood cells and the test tube containing the suspension is placed against this page, the printing will become legible after a period of time. At that time (when the print is legible) approximately 75% of the cells will have hemolyzed.

Place 6 tubes, numbered one to six, in a test tube rack. Add 5 ml of each of the following solutions* to the test tubes.Working with only one test tube at a time, add 2 drops of blood, invert the tube several times and record the time to the nearest second. Observe the tubes at frequent intervals and enter your results into the following table. Use a watch to record the hemolysis time in each tube. If hemolysis does not occur within 1 minute proceed to the next tube. Be sure, however, that you record the time at which you added blood to the previous tube.

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Results

```Tube #     Solutions*         Hemolysis Time

1.         Distilled Water   _______20 secs._______

2.         0.45 % NaCl       _______10 min.________

3.         0.90 % NaCl       _______cloudy/90 min._

4.         1.80 % NaCl       _______cloudy/90 min._

5.         3.60 % NaCl       _______cloudy/90 min._

6.         7.20 % NaCl       _______cloudy/90 min._
```
Discussion

Did the cells hemolyze in all solutions?______________. Examine under the microscope a drop of any solution in which complete hemolysis did not occur. Which concentration of NaCl is nearly isotonic to the cells?____________________. Examine a drop of fresh blood to obtain the norm for cell morphology.

B. Effect of Concentration on Plant Cells (Elodea)

Plasmolysis is the shrinking of the cytoplasm of a plant cell in response to diffusion of water out of the cell and into a hypertonic solution (high salt concentration). During plasmolysis the cellular membrane pulls away from the cell wall. Hemolysis (bursting) does not occur in an hypotonic solution (low salt concentration) because of the rigid cell wall, but the organelles may be damaged. Plant cells maintain their normal size and shape in an isotonic solution.

Obtain six slides and cover slips for preparing wet mounts of Elodea. Add two or three drops of the following NaCl solutions to one edge of each of the cover slips. Wick the salt solutions under the coverslips by touching a piece of paper towel to the fluid at the opposite edge of the cover slip. Examine the cells and note the time that it takes for the cytoplasm to be no longer pressed against the cell wall. This shrinkage is plasmolysis.

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Solution	               Plasmolysis Time

Distilled Water            _____Slight change(swelling)_____

0.45 % NaCl                ___________16.5 min. ______________

0.90 % NaCl                ___________12.5 min. _____________

1.80 % NaCl                ____________7.5 min. _____________

3.60 % NaCl                ____________3.5 min. _____________

7.20 % NaCl                ____________1.5 min. _____________
```
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` Discussion`

Determine the concentration of water in an Elodea cell.__________________. What is the appearance of a plasmolyzed Elodea cell? ___________________________________________________________________________________. Prepare a graph of your data.