HHMI-TRINITY SCIENCE ACADEMY # 3 BIOLOGY MODULE
Achieving Balance.
Effects of water, salt and sugar in your blood cells.
PLEASE NOTE:
It is VERY IMPORTANT for students to understand WHY they are doing each of these
experiments. Please make sure that they are writing a hypothesis for every
question/experiment and more importantly, that they are documenting their results!
This is CRITICAL to developing their final thoughts towards a conclusion!
For all the experiments today: we will be using erythrocytes from sheep.
The erythrocyte (or red blood cell, RBC) is the largest membrane bound compartment
found in animals. These cells are also the unique home of the protein hemoglobin, and
the presence of a traceable molecule like hemoglobin has made these cells a model
system for studying transport of molecules across membranes. Movements of molecules
across membranes can be difficult to measure directly, but whenever molecules move,
water moves to maintain an osmotic equilibrium. These bulk water movements will
change the volume of any closed membrane compartment, including RBCs, and may
eventually cause them to lyse due to internal pressure (distilled water experiment) or
shrivel due to water molecules that leave the cells (salt experiment). In some instances,
the property of the cell membranes change, as they become “sticky” causing cell
aggregations called clots (sugar experiment). Thus, we can use sheep RBCs to ask
questions about finding the right balance necessary for our RBCs to stay healthy. We
will investigate questions concerning the amount of water we drink, how much sugar
should we have in our diet as well as how much salt should we ingest every day.
Basic Lab Materials:
Distilled water
Centrifuge tubes
Micropipettes
Sheep blood
Spectrophotometer
Microscope slides
Cover slips
Salt solutions
Sugar solutions
Microscope
Instructor Guidance: How much water does my body need?
For this particular module, the students will be divided into two groups with similar
tasks (as described below) and they will then compare their observations to determine if
drinking too much water can be detrimental to your health and if Gatorade (salt
balanced water) is effective in keeping the salt balance in our bodies.
1
I - Morning Session
Initiate discussion by asking how much water they drink in a day. Do you drink
enough water everyday? What happens if you don’t drink enough? What happens if
you drink too much? Do you drink a lot of water when you exercise? Do you think that
excess of water can harm your cells in some way? Why do athletes drink Gatorade?
Please have the students write their predictions/hypotheses in their lab books prior to
each set of experiments.
Water Balance
Our body has a balanced method of processing and excreting fluids, either through your
breath, urine and bowel movements or perspiration. Lack of water can lead to
dehydration, a condition that occurs when you don't have enough water in your body to
carry out its normal functions. Even mild dehydration can drain your energy and make
you tired because water flushes toxins out of vital organs and carries nutrients to your
cells. Therefore, your recommended water intake is the "8 x 8 rule" — drink eight 8ounce glasses of water a day (about 1.9 liters).
Too much water is also not very good to your body. Over-diluting mineral levels in the
blood plasma through excessive consumption of water contributes to what is called an
osmotic shift of the fluid inside and outside the cells. This process causes swelling of the
cells, often in the central nervous system, including the brain. This may result in water
intoxication (also called hyper hydration). Athletes avoid water intoxication by drinking
water with electrolytes added so that the balance of the body is maintained.
Today’s experiment we will assess how easily we can disturb this balance by using red
blood cells from sheep. We will also investigate if Gatorade is effective in keeping the
salt balance, therefore keeping the RBCs healthy.
General procedures
Explain to the students that we can determine if too much water affects our cells by
using erythrocytes from sheep. When erythrocytes burst, they release hemoglobin and
we can measure the amount of lysis by quantifying the amount of hemoglobin you have
in solution using a spectrophotometer.
First Group - Three groups of 4 students – 12 total
1. Obtain 200 μl of washed sheep RBCs in 0.15 M NaCl 0.9% saline.
2. Prepare one microcentrifuge tube with 1.5 mls of 0.15 M NaCl, and another with 1.5
mls of distilled water.
3. Add 50 µl of RBC stock to each tube and mix thoroughly.
4. Place the tubes into a microcentrifuge, being sure to balance your tubes, and spin at
7500 rpm for 6 minutes.
2
5. Record and interpret your results.
Second Group - Three groups of 4 students – 12 total
1. Obtain 200 μul of washed sheep RBCs in 0.9% saline.
2. Prepare one microcentrifuge tube with 1.5 mls of 0.15 M NaCl, and another with 1.5
mls of Gatorade.
3. Add 50 µl of RBC stock to each tube and mix thoroughly.
4. Place the tubes into a microcentrifuge, being sure to balance your tubes, and spin at
7500 rpm for 6 minutes.
5. Record and interpret your results.
** The groups discuss the results. Is there anything else that you could test here? What
about different “Gatorades”? If they decide to try different Gatorades, colored ones, we
need to adjust the calibration of the spectrophotometer for the appropriate wavelength.
II - Afternoon Session
Instructor Guidance: What happens to your cells when your diet has too much salt or
too much sugar?
Initiate questions about how much sugar should be eaten daily. How much salt should
you have in your diet per day? Where do you find a lot of sugar? Where do you find a
lot of salt? What happens when you have too much salt and sugar in your blood?
First Group - Three groups of 4 students – 12 total
How much sugar should you eat?
What happens if you have too much sugar in your diet?
The American Diabetes Association (ADA) criteria for diagnosing diabetes are met
when your fasting blood glucose level is 126 mg/dL (7.0 mmol/L) or higher. Symptoms
of diabetes include increased thirst and frequent urination (especially at night),
unexplained increase in appetite, unexplained weight loss, fatigue, blurred vision, and
tingling or numbness in the hands or feet.
1. Obtain 200 μl of washed sheep RBCs in 0.9% saline.
2. Prepare one microcentrifuge tube with 1.5 mls of 0.9% saline, and another with 1.5 mls
of 4 mg/ml glucose solution.
3. Add 50 µl of RBC stock to each tube and mix thoroughly.
4. Take 25 µl of the RBC in glucose solution and put in a microscope slide and observe
the cells under the microscope. What are you going to use as a control for
comparison?
Describe what you see. Can you reverse the process? What should you do to reverse
the shrinking of the cells? Test it by adding increasing amounts of distilled water to
3
the 4 mg/ml glucose solution.
5. Place the tubes into a microcentrifuge, being sure to balance your tubes, and spin at
7500 rpm for 6 minutes.
6. Read the amount of hemoglobin in the supernatant with the spectrophotometer.
7. Record and interpret your results.
** The groups discuss the results. Is there anything else that you could test here? What
about increasing even more the amount of sugar? Would you see more cell aggregation?
Is it possible to quantify the amount of aggregation? Thus, we can provide the students
with 25%glucose solution and the students can add increasing amounts to the original 4
mg/ml glucose solution and observe if more cell aggregation will take place or if the
cells will start shriveling. Have them speculate as to how these sorts of problems might
contribute to the symptoms observed in diabetes.
Second Group - Three groups of 4 students – 12 total
Why do we need salt?
Why is too much salt bad?
How much salt should we eat?
Salt is a commonly occurring mineral, the technical name of which is sodium chloride.
Sodium helps to maintain the concentration of body fluids at correct levels. It also plays
a central role in the transmission of electrical impulses in the nerves, and helps cells to
take up nutrients. In adults, when levels of sodium are too high, the body retains too
much water and the volume of bodily fluids increases.
Many scientists are studying if salt is linked to high blood pressure, or hypertension,
which in turn is linked to a greater risk of coronary heart disease and stroke. With high
levels of fluid circulating through the brain there is a greater chance that weaknesses in
the brain's blood vessels are exposed, and that they may burst, causing a stroke.
Similarly, a greater volume of fluid passing through the heart can place additional strain
on the organ, increasing the possibility of coronary disease. Thus, how much salt should
you include in your diet?
1. Obtain 200 μl of washed sheep RBCs in 0.9% saline.
2. Prepare one microcentrifuge tube with 1.5 mls of 0.9% saline, and another with 1.5 mls
of 2% sodium chloride (table salt) solution.
3. Add 50 µl of RBC stock to each tube and mix thoroughly.
4. Take 25 µl of the RBC in salt solution and put in a microscope slide and observe the
cells under the microscope. What are you going to use as a control for comparison?
Describe what you see. Can you reverse the process? What should you do to reverse
the shrinking of the cells? Test it by adding increasing amounts of distilled water to
the sodium chloride solution.
5. Place the tubes into a microcentrifuge, being sure to balance your tubes, and spin at
4
7500 rpm for 6 minutes.
6. Read the amount of hemoglobin in the supernatant with the spectrophotometer.
7. Record and interpret your results.
** The groups discuss the results. Is there anything else that you could test here? Can
you reverse the process? What should you do to reverse the shrinking of the cells? Test
it by adding increasing amounts of distilled water to the sodium chloride solution. What
about increasing even more the amount of salt? Would you see more cell lysis? We can
provide the students with a 10% salt solution and the students can add increasing
amounts to the original 2% sodium chloride solution and observe if more cells shrivel or
if the cells start to lyse.
5