Osmosis in Erythrocytes

INTRODUCTION.

The water present within an organism’s body is divided into intracellular and extracellular fluids (inside and outside the cell respectively). The extracellular fluid that bathes the cells is either found in the blood plasma or the interstitial fluid (the fluid found between cells).  Since red blood cells (RBC’s) are found only within the confines of blood vessels, water balance in these cells, via osmosis, occurs between the intracellular fluid of the RBC’s and plasma component of the blood.

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Like all other cells, RBC’s are surrounded by a selectively permeable membrane.  Certain material pass through easily such as water, and other substances either need to use receptors to move across or cannot cross at all.  Using the principles of osmosis, we know that water will cross the membrane to try and equalize the concentrations of substances inside and outside of the cell.  This water movement has an effect on the size of the cell. If more water is entering the cell than leaving, the volume of the cell will increase.  Likewise, the volume of the cell will diminish should the amount of water leaving the cell exceed the amount of water entering.  It is therefore possible to determine the net flow of water across the membrane of cells by monitoring the dimensions of the cell.

Although it is possible to measure the dimensions of a cell using a micrometer and a microscope, this tends to be a long and tedious process.  With erythrocytes it is possible to indirectly determine their volume using a spectrophotometer.  The plasma membrane will reflect light with the wavelength of 510nm.  As the volume of a cell increases there will be a decrease in the amount of light scattered by the plasma membrane that will be observed as an increase in the percent transmittance (%T) detected by the spectrophotometer.  Once a RBC expands maximally it will lyse, a process referred to as hemolysis.  If all of the RBCs in solution have lysed there will be 100%T recorded by the spectrophotometer as the particles of exploded cells settle to the bottom of the fluid and light passes easily through the solution..

During the course of this lab you will examine how bathing the red blood cells with a solutions of either non-permeating or a permeating substances influences the osmosis occurring across the plasma membrane of erythrocytes.  This will be tracked as an increasing transmittance as the cell grows culminating in the 100 percent transmittance if the cells lyse.

 

 

 

GENERAL PROCEDURE

  1. Turn on the spectrophotometer and set wavelength to 510 nm.
  2. Connect the spec to the PowerLab Channel 1 directly (no amplifier) using red and black banana wires with a banana/BNC converter at the PowerLab end.
  3. Double click the CHART program and then open the file “Osmosis.”
  4. Place 4.5 ml of distilled water in a clean cuvette. Add 0.5 ml of blood and mix by inversion.  This tube is your BLANK.  Keep it throughout the lab.
  5. Place the cuvette into the spec and adjust the reading on the spec to 100% transmittance.
  6. Press “Start” on the PowerLab and let run until the reading is stable.
    1. Adjust the scale and optimize the trace
    2. Stop the reading and use the “Units Conversion” to set the scale from 0 to 100% transmittance (use “0” and “0” for both the mV and % readings for Point 1; and the reading of the blank for Point 2 and “100%” in the conversion)
    3. B. The experimental solutions are not necessarily 0% transmittance.

 

EXPERIMENTAL SOLUTIONS

N.B.  re-zero spec before each solution for most accurate results.

*  Each solution will be tested by adding 0.5 ml of the blood to 4.5 ml of the prepared solution, inverting and immediately placing the solution in the spec for reading.

*  You may have to adjust the chart speed if the cells lyse too fast and repeat.

 

  1. Prepared each solution first (as listed below) and then add 4.5 ml of the solution to a clean cuvette.
  2. Start the PowerLab reading.
  3. Enter a NOTE that identifies the solution (Do not hit “Return” yet)
    1. You can only type a note while the PowerLab is running.
  4. Add the 0.5 ml of blood, invert (Now hit “Return”), and place in spec QUICKLY.
  5. Wait for the reading to EITHER reach 100% (or slightly above) OR to stabilize.
    1. Note BOTH the time the solution reached its stabile transmittance AND what the value of its last % transmittance
    2. B. Time is from when “Return” is hit until reading stabilizes.
  6. Complete these five steps for the following non-permeable solutions (you will make these dilutions yourself from a 1M stock solution of each):
    1. Sucrose: 500, 400, 300, 200 and 100 mM
    2. NaCl: 300, 200, 150, 100 and 50 mM
  7. Complete these five steps for the all of the permeable solutions on the stock table (All are at 500 mM. Do not dilute!)
  8. For Glycerol ONLY, set-up as before, but read directly from the spec every 5 minutes until the reading stabilizes or 90 minutes has passed.
    1. B. You will not read this on the PowerLab
    2. Set-up first before starting any other solution…it takes a LONG time.

 

 

 

DATA  ANALYSIS

  1. Make a table that includes each solution, its time to stabilization, and its final percent transmittance.
    1. These values can be gotten by highlighting the pertinent area, choosing “Zoom Window” under “Window,” setting the “M” on your labeled line and reading the Y value (final percent transmittance) and X value (hemolysis time) where the line first stabilizes.
    2. Some solutions may cause the % transmittance to go down (think about why).
  2. Plot the relationship between solute concentration and final %T for the non-permeable solutes: sucrose and NaCl.
    1. Plot both sucrose and NaCl on the same graph but make regression lines for each separately.
    2. Note the isotonic point (this point will show little variance in %T from the initial value).
  3. Plot the permeable solutes’ hemolysis times as a function of their molecular weight (You will have to look these up). Is there a good fit line?
  4. Include a labeled printout of the results obtained from one of the permeable solutes by highlighting the reading, zooming the window, and then using “Print Zoom.”
    1. B. Do NOT Print all pages of your data or you will be in SO much trouble!

 

POINTS FOR DISCUSSION

  1. Compare your results for the different concentrations of sucrose and NaCl. Do your results match what you expect?  Describe what biological reason accounts for the different isotonic points for the two different solutes.
  2. All solutions will not reach hemolysis. Explain why not.  Support your answer with examples from your results.
  3. Based on your results, what conclusions can you draw about the influence of molecular weight on hemolysis for permeable solutes? Explain your reasoning.

 

 

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