Practical Protocol: Observing Osmosis in Red Blood Cells

1. Title:

Observing the Effects of Osmotic Pressure on Red Blood Cell Morphology using Varying
Concentrations of Sodium Chloride Solutions.

2. Objectives:

The objectives of this practical are to visually observe and understand the phenomenon of
osmosis across a biological membrane, specifically the red blood cell membrane. Students will
learn to differentiate between hypotonic, isotonic, and hypertonic solutions based on their effects
on red blood cell morphology. This involves correlating the concentration of an extracellular
solution with the net movement of water across the cell membrane and understanding the
importance of isotonicity for biological systems. Additionally, students will practice basic
solution preparation and microscopy skills.

3. Theory:

Osmosis is defined as the net movement of solvent molecules, typically water in biological
contexts, across a selectively permeable membrane. This movement occurs from a region of
higher solvent potential, meaning lower solute concentration, to a region of lower solvent
potential, meaning higher solute concentration. The driving force behind this movement is the
tendency to equalize solute concentrations on both sides of the membrane. The red blood cell
(RBC) membrane serves as an excellent example of a selectively permeable membrane in this
context, allowing water to pass freely while restricting the passage of solutes such as sodium
chloride (NaCl).

The concept of tonicity is used to describe the relative concentration of solutes in the solution
surrounding a cell compared to the solute concentration within the cell's cytoplasm. Tonicity
dictates the direction of net water movement across the cell membrane. An isotonic solution
possesses the same solute concentration as the cell's cytoplasm. Consequently, there is no net
movement of water across the membrane, and the cell maintains its normal shape and volume.
For mammalian red blood cells, a solution of approximately 0.9% weight by volume (w/v) NaCl
is considered isotonic. A hypotonic solution has a solute concentration lower than that of the
cell's cytoplasm. In this environment, water will move from the solution into the cell, causing it
to swell. If the osmotic gradient is sufficiently large, the influx of water can exceed the cell
membrane's capacity, causing it to rupture, a process termed lysis. For red blood cells
specifically, this is called hemolysis. Conversely, a hypertonic solution has a solute
concentration higher than that of the cell's cytoplasm. In this case, water will move from the
inside of the cell out into the surrounding solution. This efflux of water causes the cell to shrink
and often develop a characteristic spiky or scalloped surface, a morphological change known as
crenation.

In this experiment, we will expose samples of red blood cells to NaCl solutions prepared at
hypotonic, isotonic, and hypertonic concentrations. By observing the cells under a light
microscope, we will directly visualize the morphological changes resulting from the osmotic
movement of water dictated by these different solution tonicities.
4. Material Required:

Chemicals needed are Sodium Chloride (NaCl) of Analytical Reagent Grade and Purified Water
(Distilled or Deionized).

The biological sample required is fresh mammalian or human blood obtained ethically, treated
with an anticoagulant like EDTA or Heparin.

Note: Handle all biological samples using appropriate Biosafety Level 1 or 2 precautions.

Glassware and Equipment include an analytical balance, weigh boats or weighing paper, a
spatula, three 100 mL volumetric flasks, three beakers (approximately 150-250 mL capacity), a
glass stirring rod, a wash bottle containing purified water, three test tubes or small Eppendorf
tubes, Pasteur pipettes or an adjustable micropipette with corresponding tips, microscope slides,
coverslips, and a light microscope.

Personal Protective Equipment (PPE) required includes a lab coat, safety glasses, and disposable
gloves.

5. Procedure:

Part A: Preparation of NaCl Solutions (Handle chemicals safely)

Step 1: Prepare 0.9% w/v NaCl (Isotonic Solution). First, calculate the required mass: 0.9 grams
of NaCl are needed for every 100 mL of solution. Accurately weigh 0.90 g of NaCl using the
analytical balance and carefully transfer this amount to a clean 150 mL beaker. Add
approximately 70-80 mL of purified water to the beaker. Dissolve the NaCl completely by
stirring with a glass stirring rod. Carefully transfer the resulting solution into a 100 mL
volumetric flask. Rinse the beaker with small portions of purified water and add these rinsings to
the volumetric flask to ensure complete transfer of the solute. Add purified water to the flask
until the bottom of the liquid's meniscus precisely aligns with the 100 mL calibration mark.
Stopper the flask securely and invert it gently several times to ensure the solution is
homogeneous. Label this flask clearly as "0.9% NaCl (Isotonic)".

Step 2: Prepare 0.2% w/v NaCl (Hypotonic Solution). Calculate the required mass: 0.2 grams of
NaCl are needed per 100 mL of solution. Repeat the procedure detailed in Step 1, but use 0.20 g
of NaCl instead of 0.90 g. Label the final solution clearly as "0.2% NaCl (Hypotonic)".

Step 3: Prepare 2.0% w/v NaCl (Hypertonic Solution). Calculate the required mass: 2.0 grams of
NaCl are needed per 100 mL of solution. Repeat the procedure detailed in Step 1, but use 2.00 g
of NaCl. Label the final solution clearly as "2.0% NaCl (Hypertonic)".

Part B: Observing Osmotic Effects on Red Blood Cells (Handle blood safely)

Step 1: Ensure you are wearing the appropriate PPE, including gloves, safety glasses, and a lab
coat.

Step 2: Label three clean test tubes or Eppendorf tubes as "Hypotonic", "Isotonic", and
"Hypertonic".
Step 3: Using a pipette, dispense approximately 1-2 mL of the corresponding NaCl solution into
each labeled tube. Add 1-2 mL of the 0.2% NaCl solution to the "Hypotonic" tube, 1-2 mL of the
0.9% NaCl solution to the "Isotonic" tube, and 1-2 mL of the 2.0% NaCl solution to the
"Hypertonic" tube.

Step 4: Gently mix the anticoagulated blood sample by inverting its container several times to
ensure the cells are uniformly suspended.

Step 5: Using a clean pipette tip for each addition, add one single drop of the mixed blood
sample to each of the three tubes containing the prepared NaCl solutions.

Step 6: Gently mix the contents of each tube immediately after adding the blood. This can be
done by gently flicking the bottom of the tube or by careful inversion. Avoid vigorous shaking,
which could mechanically damage the cells.

Step 7: Proceed either immediately or after allowing the tubes to stand undisturbed for 2-5
minutes (ensure the incubation time is consistent for all three samples). Using a clean pipette tip
for each sample, carefully withdraw one small drop of the cell suspension from the "Isotonic"
tube. Place this drop onto a clean microscope slide. Gently lower a coverslip onto the drop,
trying to avoid trapping air bubbles. Repeat this procedure for the suspensions in the
"Hypotonic" and "Hypertonic" tubes, placing each on a separate, clearly labeled microscope
slide.

Step 8: Examine each prepared slide under the light microscope. Begin observation using a low
power objective (e.g., 10x) to locate the field of cells. Then, switch to a high power objective
(e.g., 40x) to observe the detailed morphology of the individual red blood cells.

Step 9: Carefully observe and compare the shape and size of the red blood cells in the hypotonic
and hypertonic solutions relative to those in the isotonic solution. Specifically look for evidence
of cellular swelling, cell lysis (indicated by fewer intact cells or the presence of faint "ghost
cells," which are ruptured membranes), or cellular shrinkage (crenation, characterized by spiky
or scalloped edges).

Step 10: Draw pictures of red blood cells in each solution and write your observation in the
results.

6. Observations:

Record your detailed observations for each of the three prepared slides. It is helpful to draw
representative diagrams illustrating the typical appearance of the red blood cells observed in each
solution.

For the Isotonic (0.9% NaCl) solution, describe the observed morphology. Note their
characteristic biconcave disc shape, which may appear as circular forms when viewed from
directly above under the microscope. These represent the normal appearance of the cells.

For the Hypotonic (0.2% NaCl) solution, describe any changes from the isotonic condition. Note
observations such as cell swelling (cells appearing larger and more spherical), a noticeable
reduction in the number of intact cells visible in the field of view, or the presence of ghost cells.
The background solution might also appear slightly more reddish and clearer if significant
hemolysis has occurred, releasing hemoglobin.
For the Hypertonic (2.0% NaCl) solution, describe the observed changes. Note any evidence of
cell shrinkage, irregular cell shapes, or the characteristic appearance of crenation, where the cell
surface appears spiky or scalloped due to water loss.

7. Results:

Picture Drawn Observation

Explain the underlying reason for water movement into the cells when placed in the hypotonic
solution and out of the cells when placed in the hypertonic solution. Relate your explanation to
the concepts of water potential and solute concentration gradients across the selectively
permeable cell membrane.

Why is the 0.9% NaCl solution used as the "control" condition in this particular experiment?
What baseline does it establish?

Predict what morphological changes you would expect to observe if the red blood cells were
placed in distilled water (effectively 0% NaCl).

Explain why maintaining isotonicity is critically important for medical procedures such as blood
transfusions and the administration of intravenous fluids. What adverse physiological effects
would occur if pure water were administered intravenously?

Consider the effect of temperature. How might increasing or decreasing the temperature
potentially affect the rate at which osmosis occurs, even if it doesn't change the final equilibrium
state?

9. Real World Application:

The principles demonstrated in this experiment have numerous important real-world

Intravenous Fluids: The formulation of intravenous (IV) fluids, such as 0.9% saline ("Normal
Saline") or 5% dextrose solution, is based on the principle of isotonicity. Administering isotonic
solutions prevents osmotic damage to blood cells and tissues. Using hypotonic IV fluids would
cause widespread hemolysis, while hypertonic fluids would lead to cell crenation and tissue
dehydration.

Cell Culture: In biological research and biotechnology, maintaining an isotonic environment is

Contact Lens Solutions: Saline solutions used for rinsing and storing contact lenses are designed
to be isotonic with the tear fluid of the eye. This prevents osmotic stress, discomfort, or potential
damage to the cells of the cornea.

Food Preservation: The use of high concentrations of salt (for curing meats or pickling) or sugar
(for making jams and preserves) creates hypertonic environments. This draws water out of
potential spoilage microorganisms (bacteria, fungi) via osmosis, inhibiting their growth and thus
preserving the food.

Diagnostic Tests: Certain clinical laboratory tests utilize osmotic principles. For example, the
osmotic fragility test assesses the susceptibility of red blood cells to hemolysis when exposed to
a series of hypotonic solutions. Increased fragility can be indicative of certain hematological
disorders, such as hereditary spherocytosis.