Regulation of Water and Solute Balance in Cells - Assignment Example

Introduction Living cells require nutrients from their surrounding and at the same time, get rid of waste products to their environment. This exchange of substances between the internal and external of a cell is very important for its life sustenance. One of the key processes that are involved in the exchange of these substances is the diffusion and osmosis processes (Gupta & Huang, 2014). Diffusion is the process that involves the movement of substances into the available space following a concentration gradient. The energy used for the movements of the molecules comes from the natural kinetic energy of the molecules, hence, the movement is said to be passive (Kent, 2000). Osmosis is a special diffusion process in which molecules and ions move through a semi-permeable membrane from a solution that is less concentrated to a more concentrated solution. A semi-permeable membrane acts like a barrier that permits specific substances to pass through and prevent others. Cells have membranes that are made of a phospholipid bilayer which is embedded with proteins (Lord, 1999). The membrane can distinguish between substances to be freely allowed through and hinder the movement of those that are not allowed through. This property of the cell membrane is called selective permeability (Conde, et al., 2011). It enables the membrane to allow smaller molecules to readily pass through, such as glucose and amino acids, while inhibiting the passage of larger molecules, such as starch and proteins (St. Rosemary Educational Institution, 2016). Dialysis is a process of separating larger molecules from smaller ones in a solution through selective diffusion across a semi-permeable membrane. Imbalance of molecules between intracellular fluid and extracellular fluid creates osmotic pressure which triggers movement of water molecules across the cell membrane. The process of maintaining an osmotic balance, i.e. water-salt balance, a cross the cell membrane is known as osmoregulation and is important in maintaining the homeostasis of the cell’s water content. The fluids are usually composed of water, and both electrolytes and non-electrolytes. Solutions on either side of the cell membrane tend to balance in solute concentration by diffusion of water and/or solute molecules across the cell membrane (Boundless, 2016). In this experiment, a dialysis tube was used to simulate a cell membrane to test for the separation of glucose, lactose, sodium chloride and starch. The tube is made of a cellulose material with selectively permeable perforated microscopic pores (Lewis, 2013). These pores are small enough to enable the tubing to be used as a model to demonstrate diffusion and osmosis across the cell membrane. Objectives The first objective of this lab experiment was to simulate diffusion of solutes and osmosis of water using living and non-living models. The second objective was to determine how different solutions affect the rate of osmosis. Finally, the sources of errors in the experiment were examined. Materials and Methods Materials: Dialysis tubes Strings Microscope Slide and cover slip Balance scale A funnel Pipette Distilled water Plastic trays Paper towels for drying Test tubes Forceps Razor Test solutions (1M glucose, 1M & 0.5M lactose and starch) and reagents (silver nitrate, Benedict's reagent and potassium iodide-iodine) Onion bulb 1.0M mannitol solution Methods: Part A A piece of dialysis 30 cm long was softened in distilled water. The last 3 cm of the tube on each end was then twisted towards the opposite end to make a knot, before pouring 15 ml of distilled water using a funnel as the first test solution. Air was squeezed out of the tube by carefully squeezing the tubing. With a lot of care to ensure that no air enters the tubing, another knot was tied to the other end, ensuring that about 15 cm of the tube are left between the knots. The entire bag was then rinsed in distilled water, rinsed by gently rolling over a paper towel and then checked to ensure that there was no leakage. The dry tube was then weighed to the nearest 0.1g, and the mass recorded in table 1. The tube was then immersed in a 250 ml distilled water in a plastic tray and the time of emersion recorded as zero. After 5 minutes, the tube was removed from the distilled water, dried gently and then weighed and the results recorded in table 1. The tube was then returned into the distilled water after weighing its weight, and the time of return recorded. After another 5 minutes, the tube was removed, dried and weighed again. This was repeated at10, 15, 30 and 60 minutes, and the results recorded in table 1, for a total period of 60 minutes (Lab Manual). The same procedure was repeated for 0.5M NaCl, 1M glucose, 0.5M lactose, 1M lactose, and starch. After the last weighing, the fluid in the plastic tray was tested to determine if there was a diffusion of the solutes from the tubing into the surrounding fluid across the membrane. This was done by taking 2 ml of test solution and another 2 ml of the fluid from the tray and putting into two separate tubes that were labeled separately. The two solutions were then tested for chloride, glucose, lactose and starch. The data obtained for each of the tests was compared with results from the other groups to calculate the average change in tube weight and adjusted weight change (AWC), and the results recorded in table 2. Part B A thin layer of the red pigmented epidermis was peeled off from an onion bulb and placed in a drop of distilled water on a slide, and then a coverslip was added. The epidermis was then examined with high power and drawing of the cells made. In the second test, the cells were flooded with 1.0M mannitol solution before examining with high power to show the cells. After about 10 minutes, changes in the size and position of the vacuole was observed and a plasmolyzed cell was drawn. Using the same technique, distilled water was added on one side of the coverslip and the mannitol solution drawn off using a paper towel (Lab Manual). This was repeated several times, and after 10 minutes, the cells were observed and drawn. Results Part A Table 1 below shows the data for tube weight, comparing to the data obtained from different groups and obtaining the mean weight. The cumulative mean weight is also shown for each test solution. Table 1: Raw data for diffusion experiment (tube weight)   Total time (min) Test solution 0 5 10 15 30 60 water             Group 1 15.75 15.7 15.75 15.7 15.75 15.38 Group 2 15.1 15.15 15.2 15.3 15.2 15.4 Average 15.425 15.425 15.475 15.5 15.475 15.39 Cumulative 0 0 0.05 0.075 0.05 -0.035 0.5M NaCl             Group 3 15.64 15.75 15.78 15.98 16.15 16.24 Group 4 15.23 15.88 15.66 15.98 15.82   Average 15.435 15.815 15.72 15.98 15.985   Cumulative 0 0.38 0.285 0.545 0.55 0.805 1.0M glucose             Group 5 16.59 17.14 17.64 18 18.9 20.17 Group 6 16.8 17.08 17.47 17.71 18.53 19.87 Average 16.695 17.11 17.555 17.855 18.715 20.02 Cumulative 0 0.415 0.86 1.16 2.02 3.325 0.5M lactose             Group 7 16 16.46 16.81 17.12 17.91 18.82 Group 8 16.1 16.5 17.1 17.6 18.9 20.4 Group 9 16.2 16.7 17.1 17.5 18.7 20.3 Average 16.1 16.6 17.0 17.4 18.5 19.8 Cumulative 0 0.5 0.9 1.3 2.4 3.7 1.0M lactose             Group 10 16.36 16.9 17.3 17.9 19 21.5 Group 11 16.2 16.9 17.5 18 19.3 21.2 Group 12 16.2 16.5 16.5 16.6 16.7 17.2 Average 16.3 16.8 17.1 17.5 18.3 20.0 Cumulative 0 0.5 0.8 1.2 2.1 3.7 starch             Group 13 15 15.3 15.33 15.2 14.85 14.89 Group 14 15.6 15.8 15.4 15.3 15.2 15.3 Group 15 14.68 14.7 14.75 14.73 14.67 14.74 Average 15.1 15.3 15.2 15.1 14.9 15.0 Cumulative 0 0.2 0.1 0.0 -0.2 -0.1 Table 2: AWC and summary of diffusion and chemical test results Table 2 Cumulative mean change in dialysis tube weight (g) Chemical test results Test solution Total time (min) Fresh Fluid   0 5 10 15 30 60 solution in tray Water                 (Control)               wt change (g) 0 0 0.05 0.075 0.05 -0.035 - - 0.5M NaCl                 Cum. wt change (g) 0 0.38 0.285 0.545 0.55 0.805 ++ ++ AWC (g) 0 0.38 0.235 0.47 0.5 0.84     1.0M glucose             Cum. wt change (g) 0 0.415 0.86 1.16 2.02 3.325 ++ ++ AWC (g) 0 0.415 0.81 1.085 1.97 3.36     0.5M lactose                 Cum. wt change (g) 0 0.5 0.9 1.3 2.4 3.7 + + AWC (g) 0 0.5 0.85 1.225 2.35 3.735   faint 1.0 M lactose               Cum. wt change (g) 0 0.5 0.8 1.2 2.1 3.7 + + AWC (g) 0 0.5 0.75 1.125 2.05 3.735   faint Starch suspension             + - Cum. wt change (g) 0 0.2 0.1 0 -0.2 -0.1     AWC (g) 0 0.2 0.05 -0.075 -0.25 -0.065     Table 2 above gives a summary of the diffusion test, chemical test and the AWC of the tubing for all the solutions tested. Diffusion occurred for all the solutions, except for starch. The diffusion of NaCl was confirmed by the formation of a white-grey precipitate indicating the presence of Cl- when drops of silver nitrate were added into test tubes with NaCl from fresh solution and the solution in the tray. This gave a strongly positive result (++). Glucose test also showed a strong positive result, while the two lactose solutions show weak, but positive results (+). Figure 1: Graph of AWC (g) versus time (min) From the graph in figure 1 above, it can be observed that molecules of sodium chloride, glucose and lactose diffused through the tubing. There was no diffusion of the starch molecules. This is because of the larger size of the starch molecules that cannot allow them to pass through the tubing. Distilled water, which is the control in this experiment, demonstrates a state of equilibrium between the inside and the outside environment i.e. osmotic balance. Part B Figure 2(a): Onion cells as examined under high power microscope Figure 2(b): Onion cells in 1.0M mannitol hypertonic solution Figure 2(c): Plasmolysed cells Figure 2(d): Onion cells in distilled water (hypotonic solution) Figure 2(a) shows the onion cells before placing in hypertonic and hypertonic solutions. The cells appeared pink, with a red pigment. After the cells were placed in a 1.0M mannitol solution, the cells appeared to shrink due to water movement out of the tube into the surrounding. Figure 2(b) shows the onion cells after placing in a hypertonic solution. Figure 2 (c) is a diagram of cells that have lost water and become flaccid. In figure 2(d), the cells appear rigid with enlarged vacuoles after placing the cells back in distilled water. This is because of the movement of water from the surrounding into the cell. Discussion In part 1 of the experiment, the 0.5M lactose tubing gained water faster than the 0.5M NaCl. This is because the rate of diffusion depends on the size of molecules and the molecular mass of the solutes. The molecular mass of lactose is higher (342.30gmol-1) compared to that of NaCl (58.44gmol-1), and therefore, molecules of NaCl can easily slip through the membrane unlike those of lactose. This means that more molecules of NaCl will diffuse across the membrane than those of lactose. After 60 minutes, both the 0.5M and 1M lactose tubes weighed the same masses. However, it is known that the rate of diffusion increases with increase in diffusion gradient which is created by the difference in solute concentration between the internal and external environments of the membrane. If more time could have been given for the experiment, may be these results would change and the tube with 1M lactose would weigh more than the 0.5M tube. There was no significant change in weight of the tubing containing starch, and in addition, a chemical test done on the fluid in the tray gave negative results. This means that starch molecules did not diffuse across the membrane of the tubing and this is because of the larger size of the starch molecules that inhibit their movement across the membrane of the tubing. Since the tubing is a non-living model membrane, the discrimination is purely based on the size of the molecules. In the actual living cells, processes of exocytosis and endocytosis allow the passage of macromolecules through the plasma membrane (Stein, 2012). Movement of polar molecules is facilitated through active transport by proteins embedded in the membrane (Gunning & Steer, 1996). The chemical tests for the fluid on the tray gave positive result, meaning that molecules of these substances moved out from the tube into the surrounding water by diffusion. In living cells, this process normally continues until there is a balance between extracellular fluid and the intracellular fluid (Rastogi, 1997). The tubing creates a microscopic membrane with the internal fluid being the substances in this experiment and the distilled water being the extracellular matrix. The rate of osmosis differs from solute to solute and the concentration of the solute. It can be seen from the graph in figure 1 that the rate of osmosis is greater with lactose compared to glucose and sodium chloride. The rate of osmosis increases with increase in osmotic pressure (Strange, 2004). Water molecules freely crosses the membrane from the regions of low solute concentration to regions of higher solute concentration following a concentration gradient. Osmolality tends to balance the concentration of solutes outside and within a cell by movement of water molecules. The concentration of solutes is higher in the tubing than the surrounding water. This creates a diffusion gradient that makes solute molecules to move to areas of lower concentration across the membrane, out into the fluid on the tray. In part two of the experiment, onion cells were flooded with 1M mannitol solution.The cells lost water through a process known as osmosis into the surrounding and become plasmolyzed as can be seen in figure 2(c). The plasma membrane is pulled away from the cell wall, and the vacuoles shrink. The water moved from the cell, where there was a low solute concentration of Mannitol, to the outside where the concentration of mannitol was high following an osmotic gradient. When the cells were placed in a solvent (distilled water), solvent molecules moves into the cell, enlarging the cytoplasm and the vacuoles (MicrobeHunter, 2016). Hence, the cell becomes turgid and regains its shape as seen in figure (2d). Errors in this experiment could result from handling of solutions, where some spills may occur during the experiment. If the tubing is not completely submerged, it may not allow free osmosis and this could result into an error. Measuring of the weights on the balance may also be faulty if one is not keen enough to ensure that there is no dripping water. This experiment can be improved by using test solutions with a wide range of concentrations, to observe the effect of solute concentration on the rate of diffusion and osmosis. Conclusion The dialysis tubing only allows a selected kind of substances, which are smaller in molecular size, to readily pass through micro-pores of its membrane, i.e. it has a selective permeability to molecules. The tubing was permeable to NaCl, glucose and lactose, but not starch. This is because starch has larger molecular size. Greater rates of diffusion were observed with lactose and starch compared to NaCl possibly because of higher osmotic pressure created by these substances. Onion cells placed in hypertonic solution lose water and become flaccid. When placed in a hypotonic solution, the cells draw in water and become turgid. The moves in and out of the cell through osmosis, while solutes move through the process of diffusion. References Read More