University of the Philippines Baguio, Gov. Pack Road

College of Science

The Characterization of the Plasma membrane Structure and its

Permeability with the Differentiation of Diffusion and Osmosis

As examples of the Modes of Membrane Transport

Albis, Adrian R.

Boydon, Abegail L.

Menorca, Maria Loraine R.

Mr. Enrico L. Jugadora

December 2013

ABSTRACT

The Plasma membrane is an essential part of the cell which acts as a barrier between the living cell and its surroundings and a regulator of molecules that enters the cell. To establish a better understanding on what its characteristics and functions are, the researchers conducted an experiment about the Structure and Selective Action of the Plasma membrane.

The plasma membrane structure displayed a regenerative and semi-permeable characteristic. Its structure is well defined and a clear difference between diffusion and osmosis; active transport and passive transport were established.

There are two general types of membrane transport. One is the Passive transport, where no process requires the use of energy and follows a downhill concentration gradient. An example of this is Diffusion and Osmosis. The other is Active transport where the processes requires energy to be performed and follows an uphill concentration gradient.

Hemolysis of red blood cells happens when the cell is put into a hypotonic solution where the water molecules tend to move inside the cell. Cell crenation is the total opposite. It happens when a cell is put into a hypertonic solution having the molecules tend to move out of the cell leaving the host cell almost drained.

Keywords: Plasma membrane, permeability, active transport, passive transport, osmosis, diffusion, isotonic, hemolysis, cell crenation

Introduction

Background of the Study

The Plasma membrane is a semi-permeable membrane that encloses the cytoplasm of a cell. It functions as a protection to the interior of the cell by being selective with the substances that it allows to enter. It is primarily composed of a mix of proteins and lipids. (Nybakken, et al, 1979)

Diffusion is a type of Passive transport where molecules move from higher concentration to lower concentration without energy requirement, down the concentration gradient. The rate of diffusion can be affected by temperature, particle size, molecular weight and particle concentration.

Osmosis, another type of Passive transport, is the diffusion of water through a selectively permeable membrane wherein water molecules move from where it is more concentrated to where it is less concentrated across the plasma membrane. (Campbell et. al, 2003)

In terms of relative concentration, there are three terms that can be used to describe the solute concentration. Isotonic is a condition where the solution inside the cell has an equal concentration with the solution outside the cell. The solution is said to Hypotonic when the solute concentration of the solution inside the cell has higher concentration than the solution outside the cell. Water molecules tend to move inside the cell which causes it to bloat and eventually, to rupture (hemolysis), whereas Hypertonic is the term used to describe the solute concentration where the solution inside the cell has lower concentration than the solution outside. Water molecules tend to move outside the cell which causes it to shrink or shrivel (cell crenation). (Taggart, 2004)

Formalin is a hazardous compound commonly used as a disinfectant and fixative or preservative in laboratories. It is an aqueous solution of formaldehyde that has a typical concentration of 37 to 40%. The most common solution for formalin is 10% v/v formalin dilution water.

Agar is a polymer made up of subunits of the monosaccharide galactose. It is a component of the cell wall that can commonly be found in several species of red algae.

Benedict’s solution is a chemical reagent used to test the presence of reducing sugars (Benedict’s test). It contains blue copper (II) ions (Cu2+) which are reduced to copper (I) ions (Cu+) and are precipitated as red copper (I) oxide which is insoluble with water. (Brook, n.d ) The color of the obtained precipitate gives an idea about the quantity of sugar present in the solution. A greenish precipitate indicates about 0.5% concentration; yellow precipitate indicates 1% concentration; orange indicates 1.5% and red indicates 2% or higher

concentration. Even more generally, Benedict’s test can detect the presence of aldehydes and apha-hydroxy-ketones. (Benedict, 1909)

Starch can be separated into two fractions--amylose and amylopectin. Natural starches are mixtures of amylose (10-20%) and amylopectin (80-90%. The amylose forms a colloidal dispersion in hot water and has a structure consisting of long polymer chains of glucose units connected by an alpha acetal linkage, whereas amylopectin is completely insoluble. (Ohardt, 2003)

Emulsion is a combination of two or more liquids that are normally immiscible with each other. It is a type of a more general class of matter called colloid.

Congo red is an acid dye used in medicines or as indicator. It has a strong, non-covalent affinity to cellulose fibers. It is usually used to stain amyloid.

Methylene blue is a compound that consists of dark green crystals or crystalline powder, having a brown-like luster and when put into a water or alcohol solution, will show a deep blue color.It is used as a bacteriologic stain and as an indicator.

Potassium permanganate is used primarily to control taste and odors, remove color, control, biological growth in treatment plants, and removal of iron and manganese

Iodine is a trace mineral and nutrient that is found inside the body. It is needed for a normal metabolism of cells and normal thyroid function (production of thyroid hormones).

Starch is a polysaccharide that is a stored form of food in plants. It has two components—the amylose and amylopectin. The amylose forms a colloidal dispersion when put in hot water whereas the amylopectin is completely insoluble.

Glucose, a carbohydrate, is the most important simple sugar in a human’s metabolism for it is one of the primary molecules which serve as a source of energy. (Campbell et. al, 2003)

Objectives

This study is conducted to fully understand the characteristics of the plasma membrane and the difference between diffusion and osmosis; passive and active transport.

Specifically, this study sought answers to the following:

1.) How can we characterize the structure of the plasma membrane?

2.) How is the selective action of the plasma membrane works?

3.) What is hemolysis and cell crenation?

METHODOLOGY

Gathering of Materials

Structure of the Plasma Membrane (Experiment A)

For this experiment, a microscope, distilled water hot plate, test tube, glass dropper, petri dish, glass slides and a 250 ml beaker were prepared and obtained from the University’s instruments room. An egg, dissecting needle and milk were brought by the students as the additional requirements for the experiment

Selective action of the Plasma membrane (Experiment B)

In this experiment, an aqueous yeast suspension, Congo red solution, 40% formalin, an alcohol lamp, a test tube holder, a microscope, 3 test tubes and 3 glass slides were prepared and collected from the University’s instruments room.

Permeability (Experiment C)

(1) Diffusion- Experiment C.1

For this experiment, a petri dish with agar, a potassium permanganate, methylene blue crystals, 20 ml collodion, 50% glucose, starch solution, 600 ml distilled water, 50 ml 2% iodine, 100 ml Benedict’s solution, a graduated cylinder, 9 test tubes and a 250 ml beaker were prepared and obtained from the University’s instruments room.

(2) Hemolysis and Crenation of Red blood cells- Experiment C.2

A 5 ml 0.9% NaCl, 5 ml 10% NaCl, 5ml distilled water, lancet, a microscope, 3 glass slides and cover slips were collected from the University’s instruments room for this experiment.

Preparation of Experiment A setup

A 5 ml water and 5 ml cooking oil were mixed into the test tube by shaking and has been left to stand for 5 minutes for observation. The test tube was shaken again without allowing the oil and water to settle. Then, 2-3 drops of the mixture was put into a clean glass slide and was observed under the low power objectives of the compound microscope.

A beaker was filled with milk and was heated almost at its boiling point and was allowed to cool after. The formation of the skin was observed and the same procedure was repeated.

Cooking oil was poured into a petri dish until it is half-full. An egg solution was dropped into it and was then observed for the formation of a membrane.

Preparation of Experiment B setup

Three test tubes (namely: 1, 2 and 3) were equally filled with 1 ml aqueous yeast suspension and 3 drops of Congo red solution. In the test tube 1, four drops of 40% formalin was added then was shaken gently. The test tube 2 was heated using the alcohol lamp and the test tube 3 was let to stand as it is. Then a drop of the suspension of each test tube was placed into a glass slide and observed under the microscope.

Preparation of Experiment C setup

C.1

A pinch of potassium permanganate and methylene blue crystals were placed 3-4 cm apart on the surface of a Petri dish with agar. Then the spread of the colour was observed.

A clean test tube was filled with 3 ml of collodion on the entire inner surface of the test tube by slowly rotating the tube. The test tube was then placed into the test tube rack in an inverted position. After it was let dry for about 10 minutes or more, the edge of the collodion was loosen at the mouth of the test tube and was removed gently. As the collodion was removed, one part 50% glucose and two parts starch solution was poured into it, a string was tied on the edge of the collodion so the solution would not leak. The outside of the collodion was rinsed. A 100ml water and 5ml 2% iodine was poured into a 500ml beaker wherein the collodion was then soaked. It was observed for any colour change.

Test for the presence of starch: 1ml of the solution was mixed with one drop of iodine in a test tube and was observed for any colour change.

Test for the presence of glucose: 1ml of the solution was combined with 1ml of Benedict’s solution into a test tube and was placed in a boiling water bath for 5 minutes. It was then observed for any colour change.

C.2

Three glass slides were prepared and were labelled 1 to 3. A drop of 0.9% NaCl was placed into the first slide, 10% NaCl to the second and a drop of clean water to the third. A finger of a volunteer was swabbed with 70% alcohol, was let dry and was pricked with a lancet. The blood was then added to the solution in each slide. The cells were observed under the microscope (specifically, their size and shape).

RESULTS AND DISCUSSIONS

A. Structure of the Plasma membrane

As the oil and water were put in the same container, the oil just settled at the top. Even after several times of shaking and letting it stand, the oil just settled at the top of the water. When drops of the mixture were viewed under the Low Power Objective, it is seen that oil globules are floating with water molecules but they never mixed. That is because the two liquids are not miscible with each other, called emulsion (a type of colloid).

A living cell like the amoeba remains intact in an aquatic medium because of its contractile vacuole. Inside an amoeba, bits of food molecules and other materials are floating around in its cytoplasm making the protoplasm concentrated. Water molecules are now attracted and tend to move inside the cell (following the idea of osmosis). Too much water intake can cause the amoeba to burst but this doesn’t happen because of the presence of a contractile vacuole. It regulates and sees to it that the amount of water inside the body does not exceed the amount it can hold.

In the heated beaker with milk, a skin formation was observed. After removing the skin formed and repeating the process all over again, the formation of skin is still apparent. This is because the milk has regenerative abilities like the plasma membrane and the removal of any components does not prevent further skin formation. (Or for the case of the plasma membrane, does not prevent regeneration) However, the organization of the plasma membrane differs with that of the milk. Milk is made up of proteins while the plasma membrane has a phospholipid bilayer.

After a few minutes of making the egg solution, a membrane-like structure was formed. Having the oil and egg albumen mixture as the membrane, the egg yolk served as the nucleus and the oil underneath as the cytoplasm. When ruptured, the components separated but slowly healed like what is observed to be a characteristic of the real plasma membrane.

B. Selective Action of the Plasma membrane

Test tube Contents Stain

1

1ml aqueous yeast suspension

+

4 drops of 40% formalin

None

2 Heat Red

3 None Light red

The test tube 1 displayed no stain for the cells did not accept the dye. Because of the presence of formalin, the cells are preserved. In the test tube 2, a visible red stain can be observed. Heat gave energy to the mixture. This caused the molecules to vibrate more and become unstable. Thus, the components of the plasma membrane became fragile, allowing the dye to stain the cell more. In test tube 3, the mixture was let to stand as it is, being the control group. Yet a light red stain was seen. That is because some yeast may contain amyloid which reacts with the Congo red dye that caused it to stain the cell.

C. Permeability

Diffusion

Having the distance and amount of the two compounds set as constant, it can be readily observed that the molecules of potassium permanganate (red) spread much faster than the methylene blue crystals (blue). The deviation of their rate of diffusion can be related to the difference in their molecular mass. It is observed that the lighter (potassium permanganate) the molecule is, the faster its rate of diffusion.

Osmosis

After the processes done with the collodion, a colour change of light yellow or close to light orange was observed as it was soaked into the beaker with water and 2% iodine.

Test for the presence of starch: As a drop of Iodine was added to the 1 ml solution, the change of colour to dark blue or blue black was seen—an indicator that starch is present (or specifically, amylose, a component of starch). Amylose forms a colloidal dispersion when subjected to water.

Test for the presence of glucose: As the Benedict’s solution was added to the 1 ml solution, the colour changed from light yellow-orange to orange or dirty-orange and a formation of white precipitate in the solution was noticed. These are some of the changes that indicate the presence of glucose.

Hemolysis and Cell crenation

In Test tube 1 (0.9% NaCl), the cells remain as they are. The addition of the solution and the blood cells displayed no change at all. This is because 0.9% NaCl is the normal saline content of red blood cells so placing it into a solution with the same saline content would yield nothing, meaning that the solution is isotonic or in a state of dynamic equilibrium. There is still movement among the molecules of both directions but the rate is now equal.

In Test tube 2 (10% NaCl), the cells shrieveled or shrank. Given that the concentration of the solution inside the cell is lower than that of the outside so the molecules tend to move outside the cell. This solution is said to be Hypertonic. Inside the cell, the hydrostatic pressure decreases causing the plasma membrane to collapse resulting to the shrinkage of the cell.

In Test tube 3 (Distilled water), the cells can hardly be seen and many of them disappeared. The blood cell is put in a solution of lower concentration than the solution inside it, so the molecules tend to move inside the cell. This condition is said to be hypotonic. As the water moving into the cell increases, the pressure towards the plasma membrane also increases. The cells become swollen and when its elastic limit is reached, rupture eventually occurs, making it look like some of the cells disappeared when really, they were just destroyed.

Conclusion

The plasma membrane is a fluid mosaic of phospholipids and proteins, acts as a barrier and controls the molecules that come. However, it exhibits a selective permeability which makes it hard for some molecules to easily pass through the cell. The modes of membrane transport include diffusion and osmosis (isotonic, hypotonic and hypertonic).

Diffusion is the movement of particles of any kind from higher to lower concentration. The rate of diffusion is affected by temperature, particle size, molecular weight and particle concentration. However, osmosis is just the diffusion of water molecules across a selectively permeable membrane. Both are an example of Passive transport where no energy is required for the reaction to start. But this is not the case for all the modes of membrane transport. In the Active transport, energy is not needed for the reaction to start and the molecules move from lower to higher concentration.

Hemolysis of red blood cells happens when the cell is put into a hypotonic solution. The water molecules tend to move inside the cell and without the presence of a cell wall, too much water intake can cause the cell to burst (hemolysis)

Cell crenation is the total opposite. It happens when a cell is put into a hypertonic solution. The molecules tend to move out of the cell leaving the host cell almost drained.

BIBLIOGRAPHY

Benedict, S. R. (1 January 1909). "A Reagent For the Detection of Reducing Sugars." J. Biol. Chem.

Cambell, N.A., Reece, J.B., Mitchell, L.G., Taylor, M.R. (2003). Biology Concepts and Connections. Pearson Education Inc.

Ohardt, C. (2003). "Starch- Coil Structure- due to alpha acetyl." Virtual Chembook. Retrieved from <http://www.elmhurst.edu/~chm/vchembook/index.html>.

Ohardt, C. (2003). "Starch-Iodine Comlex." Virtual Chembook. Retrieved from <http://www.elmhurst.edu/~chm/vchembook/index.html>.

Taggart, Starr (2004). Biology: The Unity and Diversity of Life. Thomson Books.

Nybakken, J.W., Storer, T.I., Stebbins, R.C., Usinger, R.L. (1979). General Zoology Sixth Edition, McGraw-Hill, Inc.

Yool, Brook (n.d.). "The Effects of Benedicts Solution on Glucose." <http://www.ehow.com/about_6584528_effect-benedict-solution-glucose.html>.

"Glucose". Retrieved December 1, 2013 from <http://hyperphysics.phy-astr.gsu.edu/hbase/organic/sugar.html>.

“Amoeba”. FCPS Home Page Redirect Page. Web. 29 Feb. 2012. <http://www.fcps.edu/islandcreekes/ecology/amoeba.htm>.

“The Cell Membrane”. Retrieved December 1, 2013 from http://rwsl.nic.bc.ca/biolab/103/Bio%20103%20Lab%202%20Diffusion,%20Osmosis,%20and% 20Cell%20Membrane%20Permeability.pdf

“Cell Anatomy”. Retrieved December 2, 2013 from http://biology.about.com/od/cellanatomy/ss/cell-membrane.htm

“Starch”. Retrieved December 2, 2013 from http://www.elmhurst.edu/~chm/vchembook/547starch.html

“Potassium permanganate”. Retrieved December 3, 2013 from http://www.epa.gov/ogwdw/mdbp/pdf/alter/chapt_5.pdf

APPENDIX A

Water and oil mixture Heated milk in a beaker

First skin formation Second skin formation

Formation of membrane (Egg solution)

Test tubes 1, 2 and 3 Drop of each suspension on a glass slide

(Selective action of plasma membrane experiment)

Spread of colour on a Petri dish with agar Preparation of the collodion

solution

Collodion in water and iodine solution

Test for the presence of starch

Test for the presence of glucose

Preparation of the Red blood cells

APPENDIX B

Oil globules in LPO Experiment B: 1st tube (with 40% formalin)

Experiment B: 2nd tube (Heated) Experiment B: 3rd tube (control)

Red blood cell in 0.9% NaCl Red blood cell in 10% NaCl

Red blood cell in water (H2O)