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The following article will guide you to determine the osmotic quantities of plant cells.
The osmotic quantities are: (1) Osmotic Pressure (2) Suction Pressure (Diffusion Pressure Deficit, DPD) and (3) Turgour Pressure.
Osmotic Pressure:
The osmotic pressure of a solution can be determined directly by placing the solution in an osmotic chamber with a semipermeable membrane and immersing the chamber in pure water.
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The pressure developed can be easily measured by a mercury manometer attached to the open end of the chamber. It is, however, an impracticable method for determination of osmotic pressure of living plant tissues with more or less elastic cell walls.
The plasmolytic method and the purely physical cryoscopic method are in general use for determination of the osmotic pressure of plant cells. Both the methods are subject to many sources of error and neither of them is perfect.
Plasmolytic Method:
For determining the osmotic pressure of a given cell or tissue, the cell or tissue is placed in a graded series of solutions of known osmotic concentrations—-preferably in volume molar concentration of mannitol (KNO3 has been used previously as a plasmolytic agent but its use has now been discarded owing to the fact of excessive penetration of K+ and NO–3 ions into the cells which may bring about considerable errors).
Let us assume that the concentration of our known solutions fall within a range of 10 dilutions from 0.5 to 0.4 M of mannitol and pieces of vegetable tissues of more or less same size and thickness are immersed in these solutions and kept until an osmotic equilibrium is attained.
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After this, strips or sections of tissues are observed under microscope. Time required for the equilibrium between the tissue and the external solutions generally varies from 20 min. to 2 hours.
The tissue or the individual cell may show strong plasmolysis, say, at 0.5 M mannitol indicating that the concentration of the external solution is higher than the cell sap. By routine testing with the series of solutions, it is possible to find one solution, slightly stronger than the cell sap (indicated by slight plasmolysis), say, at a concentration 0.46 M and another solution, a little weaker, which just fails to plasmolyse the cell, say, at 0.44 M.
Average of the two concentrations gives us a value which is approximately equivalent to the concentration of the cell sap, i.e., at 0.45 M at incipient plasmolysis. At this stage, there is no pressure of the cell contents against the cell wall, i.e., turgour pressure is zero.
Since molar solutions of undissociated compounds like mannitol theoretically have an osmotic pressure of 22.4 atm. at 0°C., the osmotic pressure of the cell sap would be 0.45 x 22.4 or 10 atm. approximately. Corrections could easily be made for the desired temperature at which the experimental readings are taken.
Advantage of the plasmolytic method of determination of the osmotic pressure of plant cells is that the measurements of single cells are possible by this method.
Instead of working with single cell, it is better to work with an integrated tissue or groups of cells. In the series of graded solutions mentioned before, there will be a particular concentration at which about half the cells are more or less completely plasmolysed and the other half not so.
The average osmotic pressure of the cells comprising the tissue may be more or less correctly taken as equal to the osmotic pressure of the external solution.
Cryoscopic Method:
It is well known that there is a direct proportionality between osmotic pressure and the lowering of vapour pressure, the elevation of boiling point and the depression of freezing point of solutions.
Thus osmotic pressure can be calculated from the observed results of any one of these physical quantities; most frequently determinations are made of the depression of freezing point of a solution and the osmotic pressure is calculated from this.
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The presence of a solute in a solution lowers the freezing point of the solvent water and the amount of lowering is directly proportional to the number of particles in the solution, i.e., the osmotic pressure.
A 1 M solution of a non-electrolyte freeze at a temperature of — 1.86°C- and its theoretical osmotic pressure, as we know, is 22’4 atm. If an unknown solution was found to freeze, say, at — 0.46°C, the solution would have a molar concentration of 0.25 M which is equivalent to 5.6 atm.
Thus an equation relating to freezing point depression and osmotic pressure, reasonably applicable over a wide range of concentrations, can easily be derived.
If we represent by A, the freezing point depression of an unknown solution (whose osmotic pressure is to be determined), its osmotic pressure, π may be calculated as follows:
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π: 22-4 =A : 1.86
or π x 1.86=22.4 X A
π = 22.4 x A / 1.86
If we know the freezing point of an unknown solution, the osmotic pressure of such a solution is easily found. If A is —0.23°C., the osmotic pressure is 
=2.8 atm.
For actual determination of the depression of freezing point of the call sap, the cell sap at first must be extracted by killing the cells by freezing so as to destroy the protoplasmic membrane which might otherwise interfere with extraction process itself.
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The vessel containing the experimental tissue is surrounded with a mixture of ice and salt and the frozen tissue is then allowed to thaw or melt. The sap can now be easily extracted with a hand press.
It is not even necessary to separate the sap from the other insoluble residues for the freezing point of the macerated material may be determined with little error for subsequent computation of osmotic pressure values.
This cryoscopic method of determination of osmotic pressures has been extensively used in long routine analysis in the laboratory or in the field. For all practical purposes when large number of determinations is to be made, the cryoscopic method is immensely superior to the plasmolytic method.
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The relation of direct proportionality between elevation of boiling point and osmotic pressure of a solution has also sometimes been taken use of, although very rarely, for osmotic pressure determinations in routine analysis.
The boling point of water is raised by 0.52°C. by a solution of a non-electrolyte compared to pure water. For example, if a certain solution was found to raise the boiling point by 0.13°C., the solution would have an osmotic concentration equivalent to 0.25 M (5.6 atm.) (Ebulli- scopic method).
Suction Pressure (Diffusion Pressure Deficit, DPD):
Most methods of measuring the suction pressure of plant cells are based on the same principles, previously discussed, i.e., if a plant cell with inelastic walls or a tissue is immersed in a solution with an osmotic pressure equal to the suction force of the cell, an equilibrium is readily attained and there is no change in the volume of the cell or tissue.
For single cells or groups of cells, they are immersed in series of solutions of known molar concentrations as in the determination of osmotic pressure. When the equilibrium is attained, say, in about 20 minutes, the cells are measured under the microscope.
The osmotic pressure of the external solution in which the cells show no change in dimension, compared to the initial value, is taken as equal to the suction pressure of the cell or tissue.
A better method is to use thin, narrow strips of tissue cut from such structures as thin leaves or petals. Very good results are obtained with cylinders of tissue of nearly equal sizes cut from potato tuber, beet root and other tuberous organs.
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The equilibrium point can be determined by measuring changes either in weight or length or volume of the strips of tissues or cylinders of tissues. The solution in which the tissue neither gains nor loses weight (or in length or volume) is considered to have an osmotic pressure equal to the average suction force of the cells.
Determination of suction pressure by method described before should not be confused with plasmolytic determination of osmotic pressure of plant cells. In the determination of osmotic pressure, the critical measurement evidently is the osmotic pressure of the external solution with which cells or tissues come to equilibrium at incipient plasmolysis, and when the cell volume is at its relative minimum due to elimination of turgour brought about by exosmosis of water.
In suction pressure determination, it must be borne in mind, that the critical measurement is the osmotic pressure of the external solution with which cells come to equilibrium without any change in their original volume of the cells, i.e., without any change in their turgour pressure. The suction pressure values can be identical with osmotic pressure values only if the cells are initially at incipient plasmolysis.
Turgour Pressure:
The turgour pressure or actual hydrostatic pressure against the cell wall due to presence of vacuolar cell sap evidently cannot he determined by direct methods.
However, if osmotic pressure of a cell or tissue as well as its suction pressure are known, the turgour pressure can be calculated from the well-known equation, S=P—T or T=P—S.
