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Name: ______________________________ _________________Date: ____________________

Lab 2b. Application: Lipid Solubility of Membranes

Adapted from Cell Biology Laboratory Manual – by Dr. William H. Heidcamp, Gustavus Adolphus College

Beet cells contain a high concentration of the red pigment anthocyanin. When exposed to a compound that dissolves the cell membranes, the anthocyanin will leak out of the cells and cause a red color to occur in the surrounding media. Alcohols, based on their chemical properties have the ability to enter into and disrupt the plasma membrane. In this lab you will measure the effectiveness of different types and concentrations of alcohols on their ability to penetrate and disrupt the plasma membrane.

Materials

· Fresh beets

· Solutions of the following alcohols:

· 22M

Methanol

· 8.5M

Ethanol

· 3.0M Propanol

· 1:2 and 1:4 dilutions of each alcohol above

· Razor blades

· Depression slides

· Stopwatch/timer

· Microscope

1. Cut thin slices of a beet so that they can be placed on a microscope depression slide and viewed with the lowest power (4X). When cutting the beet ensure that there are no ragged edges, that no piece has any of the outer skin on it, all of the pieces are the same size, and the pieces do not dry out. After making the cuts, rinse the beet pieces several times using a small amount of water. Immediately drain off the water. This will wash off any pigment released during the cutting process.

2. While watching the edge of the sliced beet, add 50ul of each of the alcohols below to the slide (only one at a time), until the beet section is submerged. Be careful not to allow the alcohol to flow off the slide.

3. Immediately begin to time the dissolution of the beet cell membranes. Mark the time when a red color is first observed in the surrounding alcohol solution.

4. Repeat the entire series for 1:2 and 1:4 dilutions of each of the alcohols (see quick reference for making a simple dilution).

5. Your challenge is to plot your data to express the effectiveness of each alcohol in penetrating and disrupting the membrane. Also use the data below to express how the properties of the alcohol, either the MW or the Partition coefficient (the relative solubility of the alcohol in hypdrophobic versus hydrophilic conditions).

CHOH

CHOH

Collecting the data:

30.6

Post-lab Work:
Complete your analysis, graph your results and answer the following questions after your lab time.

QUESTIONS

Adapted from Biology with Vernier:

http://www2.vernier.com/sample_labs/BWV-08-COMPalcohol_biological_membranes

accessed October 2015

1. Explain in your own words the purpose of this lab.

2. Why do we use beets in this study?

3. How (by what method) are we measuring the effects of alcohols on membranes?

4. Plot your data. You may use a program like Microsoft Excel or Googlesheetss, or you may draw your graph by hand. Be sure to accurately label your axes, units, and samples.

5.. Which alcohol seems to disrupt membranes most effectively? How did you come to this conclusion (be thorough in your explanation)?

6. At what dilution of alcohol is the cellular damage highest for methanol? ethanol? n-propanol? Is this what you expect. Why or why not?

7. The three alcohols have the following structures:

Ethanol Methanol n-Propanol

Which alcohol would you predict would disrupt the membrane most efficiently. Explain why (relate this to the structure and properties of the alcohols). Did the results match your predictions. Explain why or why not.

Micropipette quick reference:

Precautions

Never lay a pipette down while there is fluid in the tip. Hold it vertically.

Never turn the plunger button without first pressing the lateral catch.

Never turn the plunger button below or above the working range for the instrument.

Aspirating and Dispensing

Press the plunger button to the first stop. Dip the tip into the solution to a depth of 3 mm, and slowly release the plunger button. Wait 1-2 seconds and withdraw the tip from the liquid, touching it against the edge of the reservoir to remove excess liquid.

Dispense the liquid onto the walls of the receiving vessel by gently pressing the plunger button to the first stop and then press the operating button to the second stop. This action will empty the liquid from the tip. Remove the tip from the vessel, sliding it up the wall of the vessel. Eject the tip over a waste receptacle by pressing the plunger button to the third stop. Release the plunger button to the ready position.