Cellular Transport Lab Report

Cellular Transport  Allawan, Giovanni B.

Department of Biology, College of Science, University of the Philippines Baguio, Baguio Baguio City 2600, Benguet

 Abstract

 This experiment experiment examined the properties properties of a cellular cellular membrane which which were validated validated through different experiments. The procedures were divided into five different experiments, each were associated with the cellular membrane’s properties. The first part of the experiment was specifically designed to determine the

structure of the plasma membrane. The membrane was found out to possess certain selectivity in order to control what goes in and out of the cell; the membrane is also regenerative in nature because even when pricked or probed with a dissecting needle, the membrane still went back to its original form. The cell membrane is also insoluble in water environment and thus proving its lipid proteins. The second portion of the experiment was to prove that there are different transport that occurs within the membrane using dyes, collodion and yeast suspensions. Utilizing such mixtures helped prove the different transport systems inside the membrane. It was found out that the membrane has the capability to diffuse through osmosis certain molecules in order to achieve equilibrium. The last portion of the experiment was designed to determine the concentration differences differences within a semi-permeable membrane. Using the blood cells and NaCl solutions, it  was found out out that the different different concentrations concentrations on the regions regions of the membrane membrane makes the cell shrivel shrivel or burst when exposed to hypertonic, hypotonic or isotonic solution.

I. Introduction

 The plasma membrane is the the edge of life, life, the boundary boundary that separates separates the living cell from its its surroundings. Like all biological membranes, the plasma membrane exhibits selective permeability; that is, it allows certain substances to cross it more easily than others (Campbell et al., 2005). Every cell is surrounded by a cell membrane. The cell membrane protects the cell and helps move substances and messages in and out of the cell. By regulating transport, the membrane helps the cell maintain consistency and order. The lipid layer that forms forms foundation of a cell’s membrane is actually a bilayer formed of two phospholipid sheets, a model also known as the fluid mosaic model (Mason et al., 2015). Cellular membranes contain (1) a phospholipid phospholipid bilayer, (2) transmembrane proteins, (3) a supporting network of internal proteins, (4) cell-surface markers composed of glycoproteins glycoproteins and glycolipids. The fluid mosaic model of the membrane and the mosaic composition of proteins floating in the phospholipid bilayer (Mason et. al., 2015).  There are two ways of transport transport that occur occur through the the plasma membrane. membrane. One method method of transport transport is called active transport which requires r equires ATP or energy to transport substances through the membrane. The second method is called the passive transport, which does not require the use of ATP or energy (Sadava et al., 2014). During the passive transport, transport, molecules are transported through through the membrane by differences in concentration or pressure between between the inside and outside of the cell. Molecules have a type of energy called thermal energy due to their constant motion. One result of this motion is diffusion, the movement of molecules of any substance so that they spread out evenly into the available space (Campbell et al., 2005). Every cell in the human body uses diffusion as an important transport process through its selectively permeable membrane. Facilitated diffusion occurs when molecules are too large to penetrate through the membrane. In this process, the carrier protein molecules located in the membrane combine with solutes and transport them down the concentration gradient. One of the most important substances that crosses membranes by passive transport is water. (Campbell et al., 2005). Osmosis is a type of diffusion of water molecules across a selectively permeable

membrane. The concentration of all solutes in a solution determines the osmotic regulation of the solution. If two solutions have unequal osmotic concentrations, the solution with the higher concentration is said to be hypertonic, and the solution with the lower concentration is said to be hypotonic. When two solutions have the same osmotic concentration, the solutions are isotonic (Mason et al., 2015).  This experiment experiment aims to validate and and establish the structure of the the plasma membrane membrane as said to to be a phospholipid bilayer that contains different proteins. In addition, this experiment also is geared to know the different transport system inside the cell and how they are associated with the membranes’ permeability and tonicity specifically the different phenomenon that happens inside a cell membrane.

II. Materials and Methods  A. Structure of the plasma plasma membrane membrane  A1.

 A 5 ml of of oil and a 5 ml of water were placed placed into a test test tube. The water was shaken shaken vigorously vigorously and the observations were recorded. The test tube was again shaken, but this time, the oil and water were not allowed to settle at the bottom. Afterwards, several drops of the mixture were transferred to a clean glass slide. The mixture was then examined under the LPO objective for the interphase between oil and water.  A2.

 A beaker was was filled with with milk and and it was heated heated to almost almost boiling point. point. After After the milk cooled, the observations were noted. The skin was removed and again, the milk was heated. The process was repeated several times.  A3.

 An oil was was poured into into a petri dish until it is half half full. A drop of egg albumen solution solution was dropped dropped into the oil solution. The solution was observed to watch the formation of delicate membrane at the protein-

lipid interphase. The lab performers wrinkled the membrane by gently probing with a dissecting needle and the membrane was also ruptured using a needle. Lastly, a small particle of nigrosine was placed on top of the membrane and was gently pushed with a needle through a membrane. B. Selective action of cell membrane B1.

One ml of aqueous yeast suspension was placed in each of the three test tubes labeled 1, 2, and 3.  Three drops of of congo red solution were were added. To test tube 1, 1, four drops of of 40% formalin formalin were added added and  were shaken gently. gently. Next, the test tube tube 2 was heated heated while test test tube 3 stood stood as it is. A drop for for each suspension was placed on a glass slide and was observed under the microscope. B2.

 A 25 ml of alkaline yeast suspension suspension was placed placed into a 100 100 ml Erlenmeyer Erlenmeyer flask. Following Following this this step, a 25 ml of neutral red solution was added and the color was immediately observed within 5 minutes. Next, the 10 ml mixture was filtered to separate yeast cells from the liquid. The color of the cells and the supernatant fluid were observed. C. Diffusion C1

 A pinch of potassium permanganate permanganate (KMnO4) (KMnO4) and methylene methylene blue crystals crystals were placed placed in a petri dish. Subsequently, the spread of the color the color was observed and recorded at regular time intervals. C2

 A 3 ml of of collodion was placed in a 10 ml test tube. The The tube was slowly filled filled and rotated rotated so that a thin film forms on the entire inner surfaces. The excess solution was drained out. Subsequently, the tube was placed in an inverted position in a test tube rack until the film dried. With the use of knife blade, the edge of the film was loosen at the mouth of the tube and water was placed in between the film and the tube. Next,

the outside of the tube was rinsed. The collodion was placed in a beaker full of water. Thereafter, a 5 ml of 2% iodine solution was added. D. Osmosis

 A water mount mount of a Boat Boat of moses moses was viewed viewed under the microscope. Then, Then, the distilled distilled water was drained out using filter paper. Thereafter, a 0.5% NaCl was added to the side of the cover slip. The changes in size of the cell were observed. Subsequently, another another water mount of a boat of moses was prepared and  was viewed under the microscope. microscope. The distilled distilled water was also drained drained out. Finally, Finally, a drop of of 1.0% NaCl NaCl was placed on the side of the cover slip. E. Hemolysis and crenation of the red blood cell

 With the use of a sterile cotton soaked soaked in 70% 70% alcohol, a finger was cleaned. cleaned. Afterwards, Afterwards, the finger finger  was pricked using using a sterile disposable blood lancet. A drop of blood blood from the finger was added to a drop of 0.9% NaCl on a clean glass slide. Another drop of blood was added to a drop of distilled water on the slide. A cover slip was secured and was examined under the microscope. The observations were recorded.  Afterwards, a drop of 10% 10% NaCl was was added to one one edge of the cover slide slide glass of the slide with with sodium chloride and a piece of blotting paper to the opposite side. Again, Again, the observations were noted.

III. Results and Discussion  A. Structure of the plasma plasma membrane membrane  A1.

 The structure of the plasma plasma membrane was observed through through the mixture mixture of oil and water. In the the experiment, it was evident that the mixing of the water and oil resulted to oil globules on top of the water. Furthermore, it was also observed that there is a significant barrier that separates oil and water since these substance are immiscible even when shaken vigorously. This happens because the oil is less dense than that of the water resulting the oil to settle towards the upper portion of the mixture.

Figure 1. Mixture of oil and water  water   According to Mason (2015), (2015), the phosphate phosphate groups groups of lipids lipids in the plasma plasma membrane are are charged and other molecules attached to them are also charged or polar. The strongly polar phosphate end is hydrophilic  while the fatty fatty acid end is is strongly nonpolar nonpolar and hydrophobic. hydrophobic. As As for the experiment, experiment, oil oil is hydrophobic hydrophobic region whereas the water is the hydrophilic region of the phospholipid bilayer of the cell membrane. Hydrophilic molecules dissolve dissolve easily with water because they carry charged groups or uncharged polar groups that can form either favorable interactions between hydrogen bonds with water molecules (Rabago et

al., 2003). In contrast, the nonpolar interior of a lipid bilayer impedes the passage of any water-soluble polar or charged substances through the bilayer, just as a layer of oil impedes the passage of a drop of water (Mason et al., 2003). In connection, a lipid bilayer is stable because water’s affinity for hydrogen bonding never stops (Mason et al., 2015).  A2.

 After allowing allowing the milk to cool, it is observed observed that the heating heating of the the milk resulted to skin formation formation as seen in the Figure 2.1. According to Ballam (2015), the skin formed after heating the milk was due to the loss of solids that the milk undergoes as it is being heated. The milk can be associated with plasma membrane because of its regenerative property, wherein, it has the ability to replace substantial portions of themselves (Marshal et al., 2018). Milk provides an intriguing system for examining how mass transport can drive morphological change: upon heating a pot of liquid milk, a milk ‘skin’ forms within minutes as proteins in solution denature and are driven to the air – liquid liquid interface where they aggregate to form a thin, poroelastic film (Evans et al., 2016).

Figure 2. The milk as it is being heated.

Figure 2.1. The skin formed after heating the milk.

 A3.

 The formation formation of a delicate delicate membrane at the protein-lipid protein-lipid interphase interphase is observed observed by placing placing a drop of egg albumen into a half-filled oil perti dish. The interphase between the egg albumen and the lipids represents the phospholipid layer layer of a plasma membrane. When the nigrosine dye was added on top of the membrane, the egg albumen and oil only served as a transport channel through which which the nigrosine was diffused. In addition, even when the membrane is poked and probed with dissecting needle, the membrane healed itself and absorbed the nigrosine dye, this is due to the capability of the egg-oil interphase to repair itself and in order to protect the cell from its surroundings. (Urry et al., 2017).

Figure 3. Egg albumen right after dropping into into a petri dish of oil. B. Selective action of cell membranes B1.

 Test tubes 1, 1, 2 and 3 showed different different results. For the test tube tube 1, the color color remained the same. This This indicates that the cells in that solution did not accept the dye. This is due to the stabilizing property of

formalin (Fox et al., 1985). In addition, formalin prohibits the entrance of the Congo red solution thus reducing the pigmentation.  Among the three test tubes, tubes, it was seen that the test test tube 2 which was heated heated near boiling boiling point had the most stained cells followed by test tube 1. This happened because the solution was exposed to heat which causes the membrane to disrupt and eventually denatured of the yeast and which in turn blocks the cellular activities inside the cell.  Test tube 3 on the other other hand can undergo undergo active transport transport because the yeast can facilitate facilitate active active transport thus, making the solution less pigmented since it allows the exit of some pigments.

Figure 4.1. Aqueous yeast suspension with with

Figure 4.2. Heated aqueous yeast suspension

40% formalin and congro red solution.

with congo red solution.

Figure 4.3. Aqueous yeast suspension with with congo red solution.

B2.

 The internal environment of yeast cells cells is slightly acidic, with with pH between between 5.5 to 6.0 (Evans (Evans et al., 2017). As soon as the 25 ml of neutral red solution was mixed with 25 ml of alkaline yeast suspension, the color of the mixture turned dark red. This was due to the diffusion of the neutral red solution into the cells, because neutral red solution is red in acidic conditions and turns yellow in a pH of about 8.0 (Evans et al., 2017)).

Figure 5. Filtered yeast from the liquid.  As an evidence evidence that the neutral red solution solution was diffused diffused into the cell, the filtered yeast cells cells containing the dye had a little or no change when it comes to color. Since the yeast are very acidic, even in basic solutions, it undergoes diffusion until the whole solution reaches the state of equilibrium (Mason et al., 2015). Additionally, Additionally, the alkaline solution went to back its original solution as soon as it was filtered. C. Diffusion C1.

In the experiment, the diameters of potassium permanganate (KMnO4) and methylene blue grew  wider over time. time. It is observed observed that for for approximately every 2 minutes, minutes, the potassium potassium permanganate permanganate grew a lager diameter compared to methylene blue as seen in Figure 6.1 and 6.2.

 According to Chang (1998), (1998), the size or or the molecular weight weight of a certain certain substance can affect the diffusion rate. Heavier molecules move slower, therefore they diffuse more slowly. The reverse is true for lighter molecules. Potassium permanganate has a molecular weight of 158 g/mol, while methylene blue has a molecular weight of 374 g/mol. Because of the size of the particle, heavier substances find it hard to move from one place to another and thus require more concentration gradient (Chang, 1998). 1998).

Figure 6.1. Potassium permanganate and methylene blue as

Figure 6.2. Potassium permanganate and

soon as it placed in a petri dish.

methylene blue after approximately 2 minutes.

C2. It is said that a cell’s membrane are selectively permeable, and substances do not cross the barrier

indiscriminately (Urry et al., 2017). The results in this experiment can be associated with that of the membrane’s property, which is semi-permeability. As soon as the mixture of collodion and iodine was

suspended into a beaker with water, the color of the solution in the bag turned into a bluish-black color. This is because the iodine was able to penetrate through the membrane into the bag. On the other hand, the solution in the beaker turned yellowish which indicates that starch did not pass through the membrane into the beaker. The results of the experiment can further be attributed to the study of Razos (2018). In his experiment, he concluded that the collodion can be compared to cell membrane’s permeability. The collodion only allows smaller molecules to pass through it like iodine, small

enough to pass freely through the membrane and at the same time prohibiting the entrance of the starch

since it has larger molecules. Furthermore, the starch turned to bluish-black color because the iodine was able to diffuse from a higher concentration outside the collodion bag.

Figure 7. The collodion bag suspended in a solution.

D. Osmosis

 As stated by by Mason (2015), (2015), osmosis osmosis is the the diffusion of water across across a membrane toward a higher higher solute concentration (p 104-105). This concept can be associated when the four specimens in the experiment are compared.  When the water mount is is placed in a slide with distilled distilled water, water, it is observed observed that cell divisions inside inside the specimen are more compact compared to the cell boundaries with water mount and NaCl solution as shown in (Figure 8.1 and 8.3). This is because the cells observed are hypotonic which means the outside of the cell is less concentrated. So in order for the cell to reach a state of equilibrium, it had to gain water. The cells swelled as water enters by osmosis. However, the relatively inelastic cell wall will expand only so much before it exerts a back pressure on the cell, called turgor pressure that opposes further water uptake (Urry et al., 2017).

In contrast, the specimens as shown in Figure 8.2 and 8.4 showed spaces in between the cells. This is due to the exposure of the specimen to a hypertonic solution, solution, which in this case is the NaCl. The cell lost  water to its surroundings surroundings and and shrinks and and as the cell shrivel, its plasma membrane membrane pulls away from the cell cell  wall at multiple multiple places. This This phenomenon phenomenon is also called called plasmolysis plasmolysis (Urry et al., al., 2017).

Figure 8.1. Water mount of boat of moses

Figure 8.2. Water mount of boat of moses

using distilled water.

exposed to 0.5% NaCl

Figure 8.3. Second water mount of boat of moses using distilled water.

Figure 8.4. Water mount of boat of moses exposed to 1.0% NaCl

E. Hemolysis and crenation of the red blood cell

 The first glass slide, which which contains contains a drop of of blood with with 0.9% NaCl are exposed exposed in an isotonic isotonic environment where there will be no net movement of water across the plasma membrane. Water diffuses across the membrane but at the same rate in both directions (Urry et al., 2017). In the experiment, the first slide is in isotonic condition because the blood cells have almost the same levels of solute to solvent ratio as the NaCl solution thus no collapsing or bursting happened because the volume is stable. The blood cells retained their normal size and shape. In the next step, the blood cells were submerged to the water thus putting the cell in a hypotonic environment since there are more solvent than solute in the environment. Supposedly, as the water enters the cell from a hypotonic solution, the cell should have collapsed due to pressure applied to the plasma membrane allowing the escape of the hemoglobin found in the blood cells, also called hemolysis (Urry et al., 2017) However, in the experiment, the cells seemed to be round. The result showed otherwise probably because the cell membrane is enclosed by a lipid bilayer which then is responsible for the slowing of the movement of water across the membrane. For the final slide, the blood cells were added to 10% NaCl which has a higher concentration of salt.  The exposure of the blood blood cell to 10% NaCl caused a hypertonic hypertonic environment environment to the cell. This is is because the solute is more concentrated than the solvent in the environment inside the cell. In a hypertonic solution, the cells shrivelled due to the exit of the water from the cell (Urry, et al., 2017). As observed in the experiment, the cells lost their round shape which indicates that cells in a hypertonic environment did underwent osmotic pressure.

Figure 9.1. Blood cells with 0.9% NaCl.

Figure 9.2. Blood cells in distilled water.

Figure 9.3. Blood cells with 10% NaCl.

IV. References

Ballam, R. (1999). Food for thought . Why does a skin form over hot milk?. British Nutrition Foundation. Campbell, N., Reece, J., Urry, L., Cain, M., Wasserman, S. & Minorsky, P., (2005). Campbell Biology 9 th  ed . San Francisco: Pearson Education. Campbell, N., Reece, J. & Simon, E. (2004). Essential Biology Biology with physiology  physiology . United States of America: Pearson Education. Campbell, N., Reece, J., Taylor & M., Simon, E. (2015). Biology: concepts and connections 5 th  ed. United States of  America: Pearson Pearson Education. Chang, R. (n.d.). Physical Chemisty for the Bioscience . California: University Science Books. Evans, A., Cheung, E., Nyberg, K. & Rowat, A. (2016). Wrinkling of milk skin is mediated by evaporation . [PDF File]. Retrieved on September 14, 2018 at https://www.ibp.ucla.edu.re https://www.ibp.ucla.edu.research.rowat/Publ search.rowat/Publications_f ications_files/Evans.Sof iles/Evans.SoftMatter.201 tMatter.2017.pdf. 7.pdf. pp  pp 02. Marshal, W.F. & Tang, S.K. (2017). Self-repairing cells. [PDF File]. Science journal , 1022-1025 DOI: 10.1126/science.aam6496. Mason, K., Johnson, G., Losos, J. & Singer, S. (2015). Understanding Biology. New York: McGraw-Hill. Rabago, L., Joaquin, C. & Lagunzad, J., (2003). Functional Biology; modular approach . Vibal Publishing House, Inc.: Quezon City. Razos, S. (2018). Diffusion activities: starch and iodine . Evolving Sciences. Retrieved on September 14, 2018 from https://www.evolvingsciences.com/Diffusion%20starch%20and%20iodine.html.