BIO 101L

Instructor: L. Hauser

Introduction

Diffusion is the process in which a substance moves from an area of high concentration to an area of lower concentration.  It is important for membranes to be semi-permeable.  If membranes were universally permeable, they would not legitimately serve their purpose as membranes; certain substances need to be kept out of a cell, and others kept in.  If membranes were not at all permeable, there would be no interface between the cell and its environment – effectively starving the cell.  Membranes, being selectively permeable, allow in nutrients and other necessary substances, and also provide for the purging of cell waste. 

This experiment investigates the permeability of cell membranes to various types of sugars: polysaccharides, disaccharides, and polysaccharides.  Dialysis tubing is used to simulate a cell membrane; it is permeable to small molecules and water, but not to larger molecules.

Given the generally larger size of polysaccharides, it is hypothesized that starch will not pass through the dialysis tubing, and that iodine will pass through the membrane due to the small size of its molecules.  Based on the trouble that some people have digesting lactose, it is predicted that it is a polysaccharide or disaccharide and will yield diffusion results similar to starch. 

Methods

I) Permeability of cell model to starch

The 100ml graduated cylinder was used to measure out 50ml of tap water; the water was poured into a 250ml beaker.  One teaspoon of corn starch was added to the beaker and stirred with a spoon.  A 50ml beaker was filled with 50ml of tap water.  A piece of dialysis tubing was placed into the beaker of water until it became soft and pliable.  The tubing was then extracted from the beaker; one end was tied closed with dental floss, using a double-knot.  The other end of the dialysis tubing was opened; a pipette was used to fill the dialysis tubing with the starch solution.  Another piece of dental floss was used to tie the end of the dialysis tubing closed.  A second 250ml beaker was filled halfway with tap water.  Another pipette was used to add 15 drops of iodine to the beaker; the solution was mixed with a spoon.  The filled dialysis tube was placed into the 250ml beaker so that the cornstarch mixture was submerged in the iodine water mixture.  After 15 minutes had passed, results were observed and recorded.

II) Permeability of cell model to lactose

Part I: Determining the type of sugar being tested

The 100ml graduated cylinder was used to measure out 100ml of tap water; the water was poured into a 250ml beaker.  Two teaspoons of the lactose was added to the water, and the solution was stirred with a spoon for thorough mixing.  50ml of the resultant solution was measured out using the 100ml graduated cylinder, and was reserved for Part II.

Twenty drops of Benedict’s reagent were placed in a clean, empty test tube.  Twenty drops of the lactose solution were added to the same test tube, and the solution was heated in a boiling water bath for 2 minutes.  The results were then interpreted.

Twenty drops of Barfoed’s reagent were placed in a clean, empty test tube.  Twenty drops of lactose solution were added to the same test tube.  The resultant solution was headed in a boiling water bath for 3.5 minutes.  The results were then interpreted.

Part II: Permeability of cell model

This method is based on the premise of the unknown sugar being a disaccharide.

A 50ml beaker was filled with 50 ml tap water.  A piece of dialysis tubing was placed in the beaker of water and left to soak until it became soft and pliable.  The dialysis tubing was then removed from the beaker, and one end tied closed with a double-knotted piece of dental floss.  The other end of the dialysis tubing was opened.  The tubing was filled with lactose solution (set aside from Part I); a pipette was used to transfer the solution from the graduated cylinder to the tubing.  A second piece of dental floss was used to tie the other end of the dialysis tubing closed.  A second 250ml beaker was filled halfway full with tap water.

15 drops of iodine were added to the tap water in the beaker.  The resulting solution was swirled with a spoon to mix it; the colors of the baggie solution and the beaker solution were noted.  The dialysis tubing baggie was placed in the 250ml beaker so that the lactose solution was submerged in the beaker solution, and left to sit undisturbed for 15 minutes.  The color of the baggie solution was noted.

The baggie was removed from the beaker and samples of the beaker solution were transferred to separate, appropriately marked test tubes.  20 drops of Bendict’s reagent were added to one test tube, and the tube heated for 2 minutes.  The color of the resulting solution was noted.  20 drops of Barfoed’s reagent were added to the second test tube, and the tube heated for 3.5 minutes.  The color of the resulting solution was noted.

Results

In the starch experiment as seen in Table 1, the starch solution inside of the dialysis baggie was initially a murky white color.  The solution in the beaker, external to the baggie was a clear yellow color.  After 15 minutes of submersion in the beaker solution, the baggie had turned a dark purple color.  The beaker solution remained clear and yellow.

In Part I of the lactose experiment, the lactose solution was initially a dark brown color.  Benedict’s reagent is pale blue in color.  Lactose, mixed and heated with the Benedict’s reagent, yielded a solution of a murky yellow-brown color.  Barfoed’s reagent, like Benedict’s reagent, is pale blue in color.  Lactose, mixed and heated with the Barfoed’s reagent, yielded a pale blue solution.

In Part II of the lactose experiment, as seen in Table 2, the lactose solution inside of the dialysis baggie was initially dark brown in coloration.  The iodine and water solution in the beaker was a clear yellow color.  A Benedict’s test on the beaker solution after the experiment yielded a dark brown liquid; a Barfoed’s test on the beaker solution after the experiment resulted in a clear blue liquid.

Discussion

Permeability of cell model to starch

The starch solution inside of the dialysis baggie went from a murky white color to dark purple; iodine from the beaker solution must have diffused into the dialysis baggie, reacting with the starch solution and producing the “positive” dark-purple result, confirming the presence of a polysaccharide inside of the baggie.  The beaker solution remained a clear yellow color throughout the experiment; it can hence be inferred that no polysaccharide was present in the beaker solution at the end of the experiment, and in turn, that no starch diffused out of the baggie and into the beaker solution during the 15-minute soaking.

The experimental hypothesis for this section was correct; starch was unable to diffuse through the cell model, however, iodine was able to diffuse through the cell model.  The discrepancy in permeability is due to the difference in the sizes of iodine and starch molecules. 

Permeability of cell model to lactose

The Benedict’s test control on lactose yielded a solution that was a murky yellow-brown color; this indicated the presence of a mono- or di- saccharide.  The Barfoed’s test control on lactose yielded a solution that was pale blue in color, without any red precipitate; this indicates that no monosaccharaides were present, and in turn, that lactose is a disaccharide.  The solution inside of the dialysis tubing changed color in the course of the experiment; this implies that iodine diffused into the dialysis tubing and reacted with the lactose solution.  The resulting clear yellow color indicates that there were no polysaccharides present inside of the dialysis tubing.

A negative Benedict’s test is of blue coloration; a test on the beaker solution after the experiment is a very dark red-orange-brown color that looks similar to the original lactose in the tubing.  A Barfoed’s test on the post-experiment beaker solution was a clear light blue; no monosaccharaides diffused out into the beaker solution, but this result was irrelevant.  The Benedict’s test revealed that lactose was able to diffuse out of the dialysis baggie, into the beaker solution.  If the cell model is reliable, it appears that lactose is able to diffuse in and out of cells.

The experimental hypothesis for this section appears to have been wrong; the cell model was permeable to lactose.   

Overall, the cell model has demonstrated impermeability to large molecules such as polysaccharides, and permeability to smaller molecules such as disaccharides and iodine molecules.  Since the model was permeable to a disaccharide, it would be reasonable to infer that the model will be permeable to monosaccharaides, as they are even smaller in size than disaccharides.  Further testing with a variety of disaccharides should be done, to determine whether lactose is unique or whether the cell model is permeable to all disaccharides.