Biology 332 - Protistology Term 2 - 2002-2003
Biology 332- Protistology - Laboratory
Osmotic Pressure and permeability in Paramecium.
Protists that live in fresh water environments are subjected to a continuous influx of water. The solute concentration inside the cell is higher than that in the surrounding water so water continuously diffuses in. This inward diffusion of water does not depend on any biological activity of the cell. It is a purely physical phenomenon that depends on only the difference in solute concentration (or more specifically, the osmotic pressure difference) between the inside of the cell and the medium and the permeability of the plasma membrane. If cells are to maintain a steady internal solute concentration, water must be removed from the cell (against its concentration gradient) at the same rate at which it enters. This process is called osmoregulation. In this exercise we will examine whether Paramecium cells are capable of osmoregulating, and will determine the values for the two parameters referred to above, internal solute concentration and membrane permeability.
The strategy of this experiment is to determine the rate of water efflux under different external solute concentrations, and then to use this information to determine whether osmoregulation is occurring. You will also determine the two key parameters required for analysis of osmoregulation, a) the iso-osmotic concentration of sucrose for Paramecium and b) the specific permeability of the membrane for water.
Before you come to the lab, think about how the rate of water expulsion would have to change as a function of external osmotic pressure if the cell were osmoregulating properly. Would you expect a higher or lower rate of water expulsion is the cell were in a medium with high osmotic pressure than you would if it were in a medium with a lower osmotic pressure? How you be able to tell when the osmotic pressure of the medium was the same as the internal osmotic pressure of the cytoplasm? Review osmotic pressure before you do this lab. Remember that osmotic pressure is one of the colligitive properties of a solution. It is measured as osmolarity (usually stated in milliosmoles) or as Atmospheres of pressure. Remember that the osmotic pressure of a one osmolar solution is = 22.4 Atmospheres. What is the relation between moles and osmoles? Review some basic physical chemistry!
The approach is to place cells in different concentrations of sucrose and determine the rate of water expulsion by noting the frequency of contractile vacuole emptying.
1) Get used to estimating contractile vacuole activity.
We will use one of two procedures:
a. Place a small drop of culture onto a layer of 0.5% agar on a microscope slide. Add a coverslip and withdraw most of the fluid. Find a cell pinned between the coverslip and the agar. OR
b. Place a small drop of culture on a thin layer of petroleum jelly on a microscope slide. Add a coverslip. Look for a cell pinned between the coverslip and the petroleum jelly.
Look for contractile vacuoles. There are two of them. Note the time between contractions. Look at 4 different cells. Are the rates of water expulsion of the anterior and posterior contractile vacuoles the same? What sort of variation is there between the cells?
2) Do the experiment
You will be assigned a sucrose concentration between 0 and 50 millimolar.
Mix 1 ml of Paramecium culture with 1 ml of sucrose solution twice as concentrated as the your final concentration. That is, if you are working with 25 millimolar, Mix 1 ml of culture with 1 ml of 50 millimolar sucrose to make a final mixture that is 25 millimolar.
Immediately observe the timing of contractile vacuole concentrations for several consecutive cycles as above. Try several cells.
Analysis of class data
1) Calculate the rate of water elimination for each concentration of sucrose. Plot the rate of water expulsion (um3/min) as a function of external sucrose concentration. Calculate the volume of the contractile vacuole as a sphere:; where d is the diameter and v is the volume. The diameter of the fully distended contractile vacuole is about 7 Ám. Calculate the volume of water expelled per minute. Do not forget that the cell contains two contractile vacuoles.
2. Estimate the iso-osmotic concentration of sucrose. Do this by extrapolating the best fit line in your graph to the point of zero contractile vacuole activity. Can you determine the standard error of the isomotic sucrose concentration? If you have taken Biol 300 you should be able to do this.
Use this concentration of sucrose to estimate the osmotic pressure of the cytoplasm of Paramecium. Express the osmotic pressure in atmospheres (1 milliosmole = 0.0224 Atm). If sucrose is an ideal solute, what is the relationship between millimoles and milliosmoles?
3). Plot Rate of water expulsion as a function of external osmotic pressure. Discuss whether the cell osmorgegulates? What would you expect if it did? What would you expect if it didn't? How good is the fit of the data to your expectation?
4) Calculate the permeability of the plasma membrane. Start by calculating the area of the cell. Assume that the cell is a prolate spheroid.
Surface Area and , where a and b are the major and minor semi-axes, respectively. The dimensions of the cell are 120 x 40 Ám. Thus a is 60 Ám and b is 20 Ám. By the way - What is a prolate spheroid? Ans. Something shaped like a football or an egg. The other kind of spheroid (oblate spheroid) is shaped like an M&M candy or flying saucer. Where would you find out how to calculate the surface area of a prolate spheroid? Try the front section of the Chem. Physics Handbook.
Remember that permeability is the rate at which water crosses a unit area of membrane per unit of osmotic pressure difference. Thus, with the same osmotic pressure difference different membranes may have different permeabilities which would be reflected in different rates of water influx. What asumptions do you have to make to calculate the membrane permeability from the experimental data? Would you expect fresh water organisms to have high or low membrane permeability? Why or why not? After you have calculated the permeability of the Paramecium membrane, discuss your value in relation to those in the table below. In this analysis assume that sucrose is an ideal solute and that one millimole = one milliosmole. A 1 molar solution has an osmotic pressure of 22.4 atmospheres. Thus, one milliosmole = 0.0224 atmospheres. The units for permeability are cubic microns of water per square micron of surface area per atmosphere difference in pressure between the inside and outside of the cell, per minute. In calculating membrane permeability what additional assumption must be made? How confident are you about the validity of this assumption?
The data in the table are from Giese, A.C, Cell Physiology (any edition). This
text contains a good discussion of osmotic pressure.