DETERMINATION OF THE ISOTONIC POINT OF THE CONTENTS OF A CHICKEN EGG

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1 INTRODUCTION Chicken eggs (Gallus gallus) have a membrane under the shell that regulates what type of substances can move in and out. In this investigation chicken egg membranes were subjected to various magnitudes of osmotic pressure by using fixed concentrations of sucrose in solution. It was the aim of this lab to determine what percent solution of sucrose is isotonic relative to the inside of a chicken egg. When referring to the chicken egg membrane in this investigation, it is meant as the membrane that surrounds the outer layer of albumin, and not the vitelline membrane of the yolk. In addition, the term set will refer to the number of eggs in an experimental or control group. Osmosis is a passive process where water molecules diffuse through a biological membrane. Transmembrane proteins called aquaporins facilitate this process. One key concept regarding osmosis is the difference between free and bound water molecules. Free water molecules are those that are not bound to any solute molecule or ion. When water moves across a membrane, it travels from a high concentration of free-water molecules to a low concentration. In-otherwords, water moves from a hypotonic to a hypertonic environment. Cells placed in a hypertonic solution will shrink in size and lose mass as water moves out of the cell. Conversely, cells placed in a hypotonic solution will expand in size and gain mass. Net flow of water molecules across a membrane cease when equilibrium is established. Although water molecules may still flow back and forth across the membrane, there is no net movement. At this point the environments inside and outside the membrane are said to be isotonic to one other. HYPOTHESIS When a de-shelled chicken egg is placed in an isotonic solution, it would be expected that there would be no net movement of water molecules into or out of the egg. As a consequence, the egg should not gain or lose mass. To test this hypothesis, sets of chicken eggs were placed in sucrose solutions of varying concentrations and their masses recorded at regular time intervals. The percent sucrose solution that produced the least change in average egg mass after 50 minutes soaking time approximated an isotonic equilibrium. A standard curve constructed from the results was used to determine the sucrose concentration that would result in no significant changes in average egg mass (least-squares regression, ±SE). A control set of eggs were placed in a 21% sucrose solution to establish a baseline in which to compare the other egg groups to. The 21% solution was selected because it was the half-way point between the extremes of 42% and 0%. 1

2 VARIABLES Variable Measured Method of measuring variable Dependent Variable Average mass of eggs. Each set consisted of 8 eggs. The mass of each egg was measured with an electronic scale (±0.05 g) and then averaged at the end of each 10-minute soaking period. Rate of Osmosis The rate of osmosis was calculated by the change in average egg mass (g) per minute (min) from the average baseline mass. Isotonic Point The percent sucrose solution that is isotonic to an egg s interior was determined by the point at which a line of regression crossed the baseline of zero mass change. Independent Variable Variable Measured Concentration of sucrose in solution Time eggs soak in fixedpercent solutions. Method of measuring variable A stock sucrose solution of 42% (m/v%) was used to make 1-liter diluted solutions of 36%, 30%, 24%, 18%, 12%, and 6%. Distilled water was used for 0%. To establish a baseline, all eggs were soaked for 1 day in a midpoint concentration of 21%. The sucrose was massed with an electronic scale (±0.05 g). Volumes were measured using a 600 ml beaker (±25 ml). Time was be taken using an analog clock (±0.5 min) at 10 minute intervals for a total time of 50 minutes. Variable Measured Method of controlling variable Controlled Variables De-shelling the eggs Eggs are to be completely de-shelled with 5% vinegar to ensure the entire membrane surface is available for diffusion of substances. Soaking Containers Egg sets will be placed in identical containers large enough to allow eggs to float freely thus avoiding some eggs from being totally submerged while others are not. Baseline solution In case vinegar caused egg dehydration or waterlog a point midway between 42% and 0% will be used as a baseline soaking solution. This baseline is 21%. 2

3 Uncontrolled Variables MATERIALS Variable Outside of Experimenter s control Membrane integrity Egg variability in volume and surface area Description It is uncertain what kind of effect 5% vinegar and the time required (2 days) to de-shell the eggs has on the integrity and permeability of the egg membrane. Some eggs that had thinner shells will de-shell quicker, exposing their raw membranes to vinegar over a longer period of time. It is an assumption, however, that since all eggs were subjected to the same de-shelling conditions, the membrane integrity will be somewhat constant. Egg sizes will vary, resulting in slight differences in surface area to volume ratios. However, it is assumed that by using 8 eggs in each experimental set and taking the average, that such differences will be mitigated. Large AAA eggs (x8) 2-Liter culture dish (20cm diameter by 7cm height) 5% vinegar solution (2-liters) 600-mL Erlenmeyer Flask (±25 ml) Electronic balance (±0.05 g) Paper towels Massing dish Analog clock with second hand sweep (±0.5 s) 1-L 21% sucrose solution (m/v%) [diluted from a 42% stock solution] METHODS 1. Eight large AAA eggs were placed in a 2- liter culture dish and covered with 1-L of 5% vinegar solution (±25 ml). 2. After 24 hours the vinegar was poured off and the eggs were rinsed in tap water. The eggs were then placed back into the culture dish and covered with 1-liter of fresh 5% vinegar for another 24-hour soaking. 3. The eggs were removed from the second 24-hour soaking and the vinegar poured off. The eggs were again rinsed in tap water and culture dish thoroughly cleaned. 4. The eggs were then placed back in the culture dish and covered with 1-L of 21% sucrose solution for 24 hours. The purpose of the 21% solution is to establish a baseline for making future measurements in egg mass. 5. After 24 hours in the baseline solution, the eight eggs were removed, rinsed, and patted dry with a paper towel. Each egg was then numbered and massed with an electronic balance (±0.05 g) and recorded to the nearest 0.1 g at time zero (Data Table 1). The eggs were then returned to the culture dish in the order they were removed to serve as the control group for the class. The masses for the egg set were then averaged (Data Table 1). 6. Every 10 minutes (for a total of 50 minutes), the masses of the eggs were again measured using the protocol outlined in step 5. 3

4 7. Other student groups soaked 8 eggs each in the baseline 21% sucrose solution for 24 hours, but then used different concentrations of sucrose solution after the baseline mass was established. The other experimental groups used sucrose solutions of 42%, 36%, 30%, 24%, 18%, 12%, 6%, and 0%. The control group used the baseline 21%. The averages for each group in the class were then collected. (Data Table 2). DISPOSAL OF CHEMICALS The used vinegar and sucrose solutions were poured down the drain with copious amounts of water. The eggs were flushed down a commode. RESULTS Data Collection CONTROL-GROUP EGG DATA (21%) Time Table 1: Individual Mass of Eggs A - H (± 0.05 g) in 21% Sucrose Solution (min) ±0.5 s A B C D E F G H Average Data Table 1: Eight eggs (A-H) were soaked in a 21% sucrose solution (control) and then massed individually at ten-minute intervals. The average mass for each time interval was then calculated. All of the eggs in the control group (21% sucrose soln.) did not experience much change in mass (Data Table 1). In fact, no egg gained or lost more than 0.21% from its baseline beginning. This small difference might have been due to small amounts of liquid remaining on the eggs during massing. It was also observed that even though eggs appeared dry, when placed on a paper towel and removed a minute later, a wet spot would be left on the towel. It seems the eggs may have been oozing water due to the weight of the egg pressing down on part of the membrane making contact with the towel. 4

5 Data Processing COMBINED-CLASS EGG DATA Table 2: Average Egg Mass of Experimental Groups (±0.05 g) Time Percent Sucrose Solutions (min) ±0.5 s 0% 6% 12% 18% 21% 24% 30% 36% 42% Data Table 2: Average egg masses for eight other experimental groups at ten-minute intervals. Since all groups were not able to record more than 30 minutes of data, only averages for the first 30 minutes will be used for further analysis. COMBINED- CLASS EGG DATA (Mass difference) Table 3: Rate of Change in Egg Mass (g/10 min) Time Percent Sucrose Solution (min) ±0.5 s 0% 6% 12% 18% 21% 24% 30% 36% 42% Data Table 3: Average change in mass (±) from the baseline measurement (time zero). The baseline measurement represents the average mass of each set after soaking 24 hours in 21% sucrose solution. COMBINED-CLASS EGG DATA (Total Mass Change) Table 4: Total Mass Difference in Class Egg Sets from Baseline (±0.05 g) Percent Sucrose Solution 0% 6% 12% 18% 21% 24% 30% 36% 42% Total mass change Data Table 4: The total mass difference (±) from the baseline measurement (time zero). The data were extracted from the final time measurement in Data Table 3. 5

6 Total Mass Change in Eggs (g) Change in Egg Mass (g) DETERMINATION OF THE ISOTONIC POINT OF THE CONTENTS OF A CHICKEN EGG Chart 1: Graph showing the rate of change in mass for all experimental and control groups (Data source: data table 3) Average Difference in Egg Mass for Various % Sucrose Solutions Time (min) 0% 6% 12% 18% Control 24% 30% 36% 42% Chart 2: Least-squares regression graph shows the total change in mass as a function of percent sucrose solution. The percent solution that is isotonic to the interior of a chicken egg is shown with the dashed line. Total Mass Change (g) of Eggs in Various Sucrose Solutions y = -21.5x R² = % 0% 5% 10% 15% 20% 25% 30% 35% 40% 45% 50% Percent Sucrose Solution 6

7 Data Table 5: Proxy data (Y-axis column) entered into the equation x = (y )/21.5 yields the data in the X-axis column. A 24.2% sucrose solution is predicted to bring about a zero change in total egg mass. The equation in table 5 is a rearrangement of the trend-line equation in chart 2. Discussion Chart 1 shows the percent sucrose solution that resulted in the greatest change in average egg mass was the 0% solution, while the 42% solution resulted in the least change. The data also suggest that the percent sucrose solution that is isotonic to an egg s interior may be between 18% and 24%, since the average masses of the eggs did not change beyond 0.35% over a 30 minute time period. In fact, the control group displayed the least amount of change in mass (0.12%) over a 50 minute period of time. The relative rate of change in the average masses of all egg sets were as predicted with the exception of the 12% set. To be in agreement with osmotic theory, the 12% line should have stayed between the 6% and 18% lines at all times. It is at the 10-minute mark that the average change in mass of the 12% set deviated above the 6% set, a mass difference of 15%. Since these measurements were taken by a different group, it may have been a recording or calculation error. Chart 2 shows a regression graph of the data from table 4 and trend line formula. The standard error for the data points along the x-axis (SE x ) is ± 0.45 % and the standard error along the y-axis (SE y ) is ± 1.0 g. The standard-error bars for all data points in chart 2 make contact with the trend line, indicating strong correlation. The data in chart 2 also show an R 2 value of (or r = ). Referencing a linear correlation table (see appendix, table 6), the critical region (N=72 eggs) is between and Since the r value is a larger positive number than the critical region, the data indicate a significant positive correlation. Conclusion Table 5: Determination of Isotonic Point x = (y )/21.5 X-axis (Percent Sucrose Solution) Y-axis Total Mass Change in Eggs (g) The proposed hypothesis postulated that the egg set which displayed the least change in total mass would be closest to the isotonic point relative to an egg s interior. This investigation produced three candidate percent sucrose solutions (18%, 21%, and 24%) that produced very little change in total egg mass over a 30 minute period. Data from table 4, when graphed as a scatter plot, produced a regression line exhibiting strong, positive correlation and the least-squares regression formula, y = -21.5x To determine the isotonic point, proxy data were entered into the regression formula in data table 5. The resulting calculation indicated that a zero-change in total egg mass should occur in a sucrose solution of 24.2 %. This value is very close to one of 7

8 the three sucrose solutions (24%) that produced little change in total egg mass. Being that the theoretical 24.2% and the experimental, 24.0% solutions have only a 0.8% difference between them, the hypothesis is considered confirmed. Evaluation One way of improving this investigation would be to have a device that would keep the eggs submerged during the de-shelling process. This may eliminate the problem where some eggs still had portions of their membranes covered with a very thin coating of shell, which may hinder the osmotic process. Another improvement would be to increase the number of eggs in each set to attain a more accurate average. Some groups broke two out of the eight eggs during the massing process. This 25% loss can cause large discrepancies in the set average, especially if the largest or smallest eggs of the set are the ones which were broken. The problem with increasing the set size is that it will take longer for groups to mass the eggs. But this can be alleviated by staggering the times groups mass their eggs so groups could team up to help each other. A third way to improve the investigation would be to have more massing times. As is there were only four massing times, 0 min, 10 min, 20 min, and 30 min. This was primarily due to classes being only 50 minutes long. By shortening the massing interval from 10 minutes to 6 minutes, two additional massings could be obtained. However, this could very well pressure groups to work at a faster pace, which may result in more eggs being broken. Prior to being massed, eggs were dried and then sat waiting on paper towels. By the wet spots left on the towels, it became apparent that water may have been leaking out of the eggs. This may have been caused by pressure on the egg membrane due to the weight of the egg. If a given egg sits for 30 seconds on the first massing, 1 minute during the second massing, and 10 seconds for the third massing, it could account for the up-and-down weight discrepancies found in table 1 in some eggs of the control group. During the waiting time, each egg should be placed in a small plastic Petri dish. Each egg, its Petri dish, and any liquid that leaks can then be massed together. It was observed that some groups were slower in removing, rinsing, drying, and massing their eggs. Because of this, it is doubtful all egg sets had equal time soaking in solution. This may have affected the precision of the measurements. Since large volumes of sucrose were needed, using volumetric flasks would have produced a more precise dilution from the stock solution. The data suggests (see chart 2) that the isotonic point for eggs is 24.2 % ± 0.45% sucrose solution. An investigation could be designed to see if eggs don t change in total mass at this concentration. In addition, the isotonic point for eggs of other avian species (duck, goose) could be compared to the eggs of chickens. 8

9 Appendix Table 6: Linear Correlation Table Sample Size (n) Critical region Sample Size (n) Critical region to to to to to to to to to to to to to to to to to to to to to to