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.
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.
* 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.
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