DETERMINATION OF THE ISOTONIC POINT OF THE CONTENTS OF A CHICKEN EGG
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%.
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Ken Elder
IB# D0781-009
DETERMINATION OF THE ISOTONIC POINT OF THE CONTENTS OF A CHICKEN EGG
VARIABLES
Dependent Variable
Variable Measured
Method of measuring 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.
The rate of osmosis was calculated by the
change in average egg mass (g) per minute
(min) from the average baseline mass.
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.
Rate of Osmosis
Isotonic Point
Independent Variable
Variable Measured
Method of measuring variable
Concentration of sucrose
in solution
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.
Time eggs soak in fixedpercent solutions.
Controlled Variables
Variable Measured
Method of controlling variable
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.
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.
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%.
Soaking Containers
Baseline solution
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Ken Elder
IB# D0781-009
DETERMINATION OF THE ISOTONIC POINT OF THE CONTENTS OF A CHICKEN EGG
Uncontrolled
Variables
Variable Outside of
Experimenter’s control
Description
Membrane integrity
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.
Egg variability in volume
and surface area
MATERIALS
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.
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Ken Elder
IB# D0781-009
DETERMINATION OF THE ISOTONIC POINT OF THE CONTENTS OF A CHICKEN EGG
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
Time
(min)
±0.5 s
0
10
20
30
40
50
CONTROL-GROUP EGG DATA (21%)
Table 1: Individual Mass of Eggs A - H (± 0.05 g) in 21% Sucrose Solution
A
93.6
93.6
93.8
93.8
93.8
93.7
B
83.9
83.8
83.9
83.9
83.9
83.9
C
89.1
89.1
89.2
89.2
89.2
89.1
D
87.7
87.7
87.8
87.8
87.8
87.7
E
81.4
81.4
81.4
81.4
81.4
81.4
F
80.0
80.1
80.0
80.1
80.1
80.0
G
86.9
86.8
86.9
86.9
86.9
86.9
H
81.8
81.8
81.8
81.9
81.9
81.9
Average
85.6
85.5
85.6
85.6
85.6
85.6
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.
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Ken Elder
IB# D0781-009
DETERMINATION OF THE ISOTONIC POINT OF THE CONTENTS OF A CHICKEN EGG
Data Processing
Time
(min)
±0.5 s
0
10
20
30
40
50
COMBINED-CLASS EGG DATA
Table 2: Average Egg Mass of Experimental Groups (±0.05 g)
Percent Sucrose Solutions
0%
86.3
90.0
91.0
92.4
-
6%
86.5
87.8
89.3
90.8
-
12%
86.0
87.9
87.5
87.9
88.3
-
18%
86.3
86.4
86.5
86.6
-
21%
85.6
85.5
85.6
85.6
85.6
85.6
24%
87.5
87.4
87.8
87.5
86.7
-
30%
86.1
85.7
85.4
85.3
-
36%
86.5
85.8
85.0
84.4
85.1
-
42%
90.0
88.8
87.7
86.4
-
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.
Time
(min)
±0.5 s
0
10
20
30
COMBINED- CLASS EGG DATA (Mass difference)
Table 3: Rate of Change in Egg Mass (g/10 min)
Percent Sucrose Solution
0%
0.0
3.7
4.7
6.1
6%
0.0
1.3
2.8
4.3
12%
0.0
1.9
1.5
1.9
18%
0.0
0.1
0.2
0.3
21%
0.0
0.0
0.0
0.1
24%
0.0
-0.1
0.3
0.0
30%
0.0
-0.4
-0.7
-0.8
36%
0.0
-0.7
-1.5
-2.1
42%
0.0
-1.2
-2.3
-3.6
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.
0%
Total
mass
change
6.1
COMBINED-CLASS EGG DATA (Total Mass Change)
Table 4: Total Mass Difference in Class Egg Sets from Baseline (±0.05 g)
Percent Sucrose Solution
6%
12%
18%
21%
24%
30%
36%
4.3
1.9
0.3
0.1
-0.0
-0.8
-2.1
42%
-3.6
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.
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Ken Elder
IB# D0781-009
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
Change in Egg Mass (g)
8.0
6.0
0%
4.0
6%
12%
2.0
18%
0.0
Control
24%
-2.0
30%
-4.0
36%
-6.0
0
10
20
30
42%
40
Time (min)
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 in Eggs (g)
Total Mass Change (g) of Eggs in Various Sucrose
Solutions
y = -21.5x + 5.1989
R² = 0.9543
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
-1.0
-2.0
-3.0
-4.0
-5.0
-5%
0%
5%
10%
15%
20%
25%
30%
35%
40%
45%
50%
Percent Sucrose Solution
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Ken Elder
IB# D0781-009
DETERMINATION OF THE ISOTONIC POINT OF THE CONTENTS OF A CHICKEN EGG
Table 5: Determination of Isotonic Point
x = (y – 5.1989)/21.5
X-axis
Y-axis
(Percent Sucrose
Solution)
Total Mass Change
in Eggs (g)
19.5
24.2
28.8
1
0
-1
Data Table 5: Proxy data (Y-axis column) entered into the equation x = (y-5.1989)/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 (SEx) is ± 0.45 % and the standard error along the y-axis
(SEy) 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 0.9543 (or r =
0.9769). Referencing a linear correlation table (see appendix, table 6), the critical region (N=72
eggs) is between -0254 and +0.254. Since the r value is a larger positive number than the critical
region, the data indicate a significant positive correlation.
Conclusion
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 + 5.1989. 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
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Ken Elder
IB# D0781-009
DETERMINATION OF THE ISOTONIC POINT OF THE CONTENTS OF A CHICKEN EGG
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.
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Ken Elder
IB# D0781-009
DETERMINATION OF THE ISOTONIC POINT OF THE CONTENTS OF A CHICKEN EGG
Appendix
Sample Size (n)
10
11
12
13
14
15
20
30
40
60
80
Table 6: Linear Correlation Table
Critical region
Sample Size (n)
- 0.632 to + 0.632
100
- 0.602 to + 0.602
120
- 0.576 to + 0.576
140
- 0.553 to + 0.553
160
- 0.532 to + 0.532
180
- 0.514 to + 0.514
200
-0.444 to + 0.444
250
- 0.361 to + 0.361
300
- 0.312 to + 0. 312
350
- 0.254 to + 0.254
400
- 0.220 to + 0.220
450
9
Critical region
- 0.197 to + 0.197
- 0.180 to + 0.180
- 0.166 to + 0.166
- 0.155 to + 0.155
- 0.146 to + 0.146
- 0.138 to + 0.138
- 0.124 to + 0.124
- 0.113 to + 0.113
- 0.105 to + 0.105
- 0.098 to + 0.098
- 0.092 to + 0.092
Ken Elder
IB# D0781-009