Let us make an in-depth study of the colloidal systems of plant with various experiments.
The Colloidal Systems:
The complex nature of the living substance in the cell, the protoplasm, is due to its physico-chemical properties.
Protoplasm is predominantly composed of substances in the colloidal state and certainly it is to these colloidal systems that it owes much of its characteristic properties—variation in viscosity from values not greater than that of pure water to those of jellies, pronounced capacity for imbibition of water, though itself high in water content sometimes behaving as if immiscible with water, properties of irreversible coagulation at very high or low temperatures or at high concentration of salts, etc.
Almost all the physiological processes and chemical reactions which occur in the living cells for the maintenance of life take place under the influence of organic catalysts or enzymes which are active only when they are in colloidal condition, owing many of their properties to that fact.
So it is desirable and very important that in order to properly comprehend the properties of protoplasm we should have a clear understanding of the fundamental principles regarding the colloidal systems.
The word colloid is derived from the Greek word kolla meaning glue and eidos, appearance, i.e., glue-like appearance.
If a little sucrose of NaCl is dissolved in water the solid disappears and the resulting system will be a solution. The solute particles are dispersed throughout the solvent (water) in the form of molecules or ions or both and such particles are so small that it is impossible to detect them in the system.
If instead of sucrose or NaCl, some fine river bottom silt is stirred in water, the result is a different sort of system. The silt particles are simply dispersed throughout water and not actually dissolved, for the particles of silt are of such a size that they can readily be identified under the microscope.
These systems are called suspensions for the particles of silt gradually settle down and the system becomes separated into its original components, silt and water, in a short period of time; these systems are not stable as true solutions.
Similar systems can also be prepared by vigorously shaking together two immiscible liquids such as oil and water. Such systems are known as emulsions. Like suspensions, they also are not stable as oil soon separates out from water. These emulsions can, however, be stabilised by the addition of a third component called emulsifier.
A third type of system can be prepared if a trace of sulphur be dissolved in a small volume of alcohol which then can be poured into a beaker containing a larger volume of water.
A cloudy, opalescent liquid will result which is composed of sulphur particles dispersed in water, not in the form of molecules or ions as in true solutions, but as in suspensions and emulsions in the form of aggregates of atoms or molecules, invisible under a microscope showing that the molecular aggregates here are smaller in size than those in suspensions and emulsions.
They can, however, be detected under an ultra- microscope unlike true solutions. These systems are relatively stable as the particles may remain dispersed throughout the liquid indefinitely. A simple example of stable colloidal system is given by the above sulphur in water system. Other good examples are milk and blood, which are predominantly colloidal suspensions of protein in water.
Colloidal systems are thus composed of two phases like true solutions, but unlike true solutions, the particles or molecules of the dispersed phase are either much larger as in certain dyes, proteins, etc., or as has been stated before in the form of molecular aggregates composed of hundreds or even thousands of molecules lumped together.
If the molecular aggregates are very large, the particles readily settle out of the system as in suspensions and emulsions and stability is not attained; this stability is the essential attribute of any colloidal system.
In general, if the dispersed particles fall within a range 1-100 mµ in diameter, the system is colloidal; if smaller than this it is a true solution, if larger, unstable suspensions or emulsions.
The individual molecules of some proteins and dyes are so large as to bring them within the colloidal range of dimensions and molecular dispersion. Such substances form true solutions and colloidal systems at the same time.
Thus colloids consist of two or even more substances intimately mixed only sometimes molecularly, but never ionically, dispersed as in true solutions tut forming physical systems containing a continuous or dispersion phase and a discontinuous or dispersed phase.
Another property of the dispersed particles in a colloidal system is that though the particles of the dispersed phase are invisible to the naked eye, the particles are able to diffract or scatter light giving the so-called Tyndall effect.
If a strong beam of light is passed through a colloidal solution and then observed at right angles, the path of the light is clearly seen through the medium. The visible bright spots are due to scattering of light by the large dispersed particles or molecular aggregates although the size and form of the particles in the system remain invisible.
The particles of the dispersed phase in a colloidal system are incapable of passing through a parchment membrane as was first pointed out by Graham. Graham’s original classification of substances into crystalloids (substances forming crystals and in solution readily diffusing through parchment membrane) and colloids, is no longer valid because we know now that by application of proper physical methods, theoretically at least, any substance can be brought into the colloidal state.
The particles of dispersed phase show Brownian movement, particularly in a suspension, which is an erratic, zigzag movement performed by those particles owing to their being bombarded by the moving molecules of the continuous or dispersion phase. This is partly responsible for keeping the particles of the dispersed phase from settling down and precipitating.
The particles of the dispersed phase are electrically charged with respect to their surroundings and generally all possess the same electric charge of the same sign. Colloids have been classified as positive colloids and negative colloids in accordance with the nature of electric charge the dispersed particles in a colloidal system carry.
They behave differently, however, from electrolytes in that they migrate as a whole to one pole or another depending on the electric charge they carry and do not separate as oppositely charged ions as in electrolytic dissociation. This migration of the particles of the dispersed phase as a whole is known as cataphoresis or electrophoresis.
Since particles in the dispersed phase are similarly charged, they keep repelling each other as they move about in the dispersion phase. By varying the pH of a colloidal system the electrical charge on the particles of the dispersed phase can be neutralised.
When this is attained, the particles no longer migrate towards the poles in an electrical field and the colloidal sol is said to be at its isoelectric point. At this isoelectric point the colloidal sols are naturally and most easily precipitated by the removal of the electric charges of the dispersed particles, as stability of a colloidal solution, as we know, depends upon these charges.
Such neutralisation or removal can be effected in the laboratory by mixing colloidal sols having opposite charges—a positive colloid such as ferric hydroxide is added to a negative colloid, e.g., fine suspension of gold—in proper proportion. The charges soon neutralise each other and the particles of the dispersed phases of both systems are precipitated.
This is known as mutual precipitation. As milk, which is a colloid is treated with a few drops of an acid, there is a precipitation of protein (casein) particles. The addition, of any electrolyte—-a dissociable salt solution will also cause identical precipitation. In this case the ion having a charge opposite to that of the particles of the dispersed phase is responsible for the precipitation of the colloidal solution.
Types of Colloidal Systems:
Unlike true solutions, colloidal systems are, as we have learnt, not homogeneous, but consist of at least two distinct phases. There are eight possible types of colloidal system of which two are very uncommon, e.g., the gas-in- solid (some minerals, pumice stone) and gas-in-liquid (some foams, whipped cream).
Gases always exist as separate molecules which are distant from one another either in the dispersion phase or in the dispersed; thus it is impossible to have a gas-in-gas colloid.
The other six types are as follows:
liquid-in-gas (clouds, fog, mists, etc.), liquid-in-liquid (all emulsions; the naturally occurring important ones are milk, butter, latex, etc.), liquid-in-solid (pearls and certain other minerals), solid-in-gas (smoke, fine dust clouds, certain fumes, etc.), solid-in-liquid (all suspensions) and solid-in-solid (ice cream, certain types of coloured glass, alloys, precious stones, e.g., black diamond).
Only two of several types of colloidal systems, enumerated above, are of special interest to the biologist. These are emulsions and suspensions and though the suspensions are not commonly found in the living cells, they are present in the environment especially in the soils in which plants grow.
Colloidal systems consisting of proteins, starch, etc., when mixed with water are classed as emulsions even though the individual materials when pure are solids.
The solids, however, adsorb large amounts of water and as a result both phases have the properties of liquids and thus can only be regarded as emulsions. Emulsions are commonly met with in the living cells of plants and animals and are generally believed to be essential component of the protoplasm.
Emulsions fall naturally into two groups, generally known as oil-in-water and water- in-oil emulsions. Both types occur in the living cells but the oil-in-water type is more common. When observed under high-power microscope protoplasm often appears as an emulsion of fat or fat-like substances dispersed through a liquid phase.
Some common examples of oil-in-water emulsions are milk, cream (droplets of fats dispersed in water), emulsions of olive oil-in-water, latex, etc. One familiar example of water-in-oil is butter.
Emulsions, we know, lack stability unless stabilised by the presence of another component, emulsifier. In absence of the emulsifier, the components readily separate out, the oil being lighter rising to the top.
Emulsifiers are a group of heterogeneous substances. Some of the best-known emulsifiers are soaps, gelatine, fine suspensions of sulphur, carbon, silica, etc. Emulsions commonly occurring in nature are stabilised by proteins. Protoplasmic emulsions are also stabilised by proteins although metals or soaps may also act as emulsifier in the living cell.
A colloidal system exhibiting properties of a fluid is known as sol. Such systems can be poured more or less easily from one vessel to another. They may appear to be true solutions to the naked eye but critical examination reveals their colloidal nature.
Many sols set forming solid or nearly so, gels. Gelatine, custard, ordinary household jellies are familiar example of gels. They are fluid at first but soon set hard. Sols may also be classified as lyophobic (solvent-hating) and lyophilic (solvent-loving).
In the former, no attraction exists between the particles of the dispersed phase and the dispersion medium whereas in the latter such affinity exists appreciably. When the dispersion or the continuous medium is water, as in most biological colloidal systems, the corresponding terms are hydrophobic and hydrophilic.
Lyophilic or hydrophilic sols are thus easy to prepare but difficult to precipitate whereas the converse is true of lyophobic or hydrophobic sols. Most colloidal systems composed of metallic substances dispersed in water are familiar examples of lyophobic sols whereas gelatine, agar, starch, protein sols are typically lyophilic.
Adsorption is the condensation in the form of a film of molecules of a gas or of a solute or suspended particles upon the surface of a solid or liquid.
Specially treated charcoal is one of the best adsorbents known for both gases and solutes. Gas masks which are used as a protection against poisonous gases in warfare owe their efficiency to the adsorptive capacity of charcoal present in the gas masks.
Soluble and coloured impurities are often removed from liquids by treating them with charcoal which adsorbs them. The forces involved in adsorption are partly forces of cohesion, i.e., attraction between similar particles and adhesion, attraction between dissimilar particles, surface tension and partly also the electric charges on the surface of the adsorbent and on the particles it is adsorbing.
The phenomena of adsorption play significant and manifold role in practically all the activities of the living cells. Protoplasm and many of the constituents of the living cells are essentially colloidal in nature and adsorption phenomena are strongly exhibited by all colloidal systems.
Imbibition of water and the action of enzymes which are of tremendous importance in the water relations and metabolism of plant cells both involve to a large extent the adsorption of water. Differential adsorption of certain dyes and colouring matter by various constituents of cells, has given us an insight into the structure of living cells.
We are all familiar with the characteristic blue colour developed by starch grains when treated with iodine solution. This specific test for starch is entirely due to adsorption of iodine in potassium iodide solution on the surface of the starch grains. This is known as chemical adsorption.
The chromosomes are so named because they strongly adsorb certain stains while rejecting others and the entire technique of staining of biological matter depends on the differential adsorption of different dyes by different constituents of a living tissue.
We have seen before that colloidal systems invariably take up a large amount of water from their surrounding medium and as a result swell up considerably. Many other substances such as proteins, gums, starch, cellulose, agar, gelatine, etc., show such hydrophilic properties when placed in contact with water or lose water and shrink when devoid of water.
This taking up of water and consequent swelling of colloidal materials and other substances is commonly called imbibition. This imbibition of water by dispersed particles in a colloidal system is responsible for many of the characteristic properties shown by the functioning colloidal system and is also responsible for a number of physiological processes continually taking place on the surface of protoplasm which is, as we well know, mostly in a colloidal state.
If a few seeds with seed coats permeable to water, are soaked in water, these seeds will swell up visibly within a few hours. They will also swell similarly if placed in other solutions.
The amount of water or solution which would enter the seeds by imbibition is often very large in proportion to the dry weight of the seeds. Some seeds can take up as much as 15 times its own weight of water by imbibition.
Water is not only taken up as liquid but also as a vapour. The swelling of doors, windows and other wood-works during rains is an everyday example of the operation of the imbibitional phenomena.
The imbibitional force due to the entrance of water and solutions from medium in dry seeds can be easily demonstrated by tightly packing a bottle with dry seeds and a little water, whereupon the bottle would burst after some time due to the swelling force or pressure of the imbibing seeds. It has been estimated that this pressure of imbibing seeds may be as high as 1000 atmospheres or even more.
Imbibition is usually considered to be basically a process of diffusion (as osmosis) but capillary phenomena and surface tension are probably also involved to a considerable extent.
Imbibing substances are, as a rule, permeated with minute capillaries and it is certain that a portion of water or liquid enters the imbibant (material which is imbibing) by capillary forces through minute pores.
Over and above that, there seems to be some specific forces of attraction between the molecules of the imbibant and the water (or the liquid) it may be imbibing. It is well known that whereas dry seeds imbibe water and swell up considerably when put into water or certain other solutions, such as sugar solution, the same seeds do not swell up at all when immersed in ether or certain other organic liquids.
A piece of rubber, however, which certainly does not imbibe water, readily imbibes appreciable amounts of ether and certain other liquids. These observations certainly point to the occurrence of specific forces of attraction mentioned before.
It is probable that the force or pressure, causing the swelling of colloids and other imbibants is partly comparable to the force causing the increase in volume within an osmotic membrane in a living cell, the expansion in volume being brought about by the inward movement of water from the surrounding medium.
Difference in the diffusion pressure between the liquid in the external medium and the liquid already present in the cell (imbibant) must be there, as in osmosis, if the imbibition phenomenon is to be operative.
As long as the diffusion pressure or concentration of the liquid in the imbibant is less than the diffusion pressure or concentration of the liquid in the surrounding medium, a net movement of liquid inward into the imbibing cell will continue. A dynamic equilibrium will be established, as in osmosis, only when the diffusion pressures of liquid in the imbibant and in the external medium have attained the same value.
Now, how does imbibition then differ from osmosis? Well, for one thing, there is no necessity of the presence of a differentially permeable membrane separating the imbibant molecules from the surrounding external liquid in imbibition phenomena whereas osmosis, as we know, is essentially a process of diffusion of water through a membrane which is semipermeable.
Furthermore, imbibition differs from osmosis in that when colloids and other imbibants take up liquids and swell up, large amounts of solutions and not just water as in an osmotic system, may be readily absorbed. A block of gelatine or agar may swell nearly as much in a solution of 0.5 M sucrose as in pure water.
When an imbibant is taking up liquid from external medium, it always results in the release of energy in the form of heat. This can be easily demonstrated if some dry seeds are immersed in water in a vessel containing a calorimeter and noting the change in temperature.
The imbibing molecules of water or liquid lose a large part of their kinetic energy as they are adsorbed, and this released energy reappears in the system, which is measurable by the calorimeter.
Experiments on Colloids and Colloidal Systems:
I. Preparation of Suspensoids or Suspensions
(a) Precipitate a solution of BaCl2 with H2SO4. Shake well the fine precipitate of BaS04 formed. Note the gradual settling of the fine ppt.
(b) Grind clean charcoal in a clean mortar to a very fine powder. Weigh about 0.2 g (three samples).
Shake each sample with 10 ml each of the following:
(i) Distilled water,
(ii) 1% NH4OH and
(iii) 0.5% acetic acid solution.
Allow the suspensions formed to stand and observe the rate of settling of the fine particles of charcoal.
II. Preparation of Emulsoids or Emulsions:
(a) Take a drop of olive or castor oil in a test-tube; half fill the tube with absolute alcohol. Shake well and pour in a beaker of water. A fine white emulsion of oil-in-water is formed from which particles of oil (dispersed very finely) will not separate—the oil is obtained in such small drops that a degree of stability is attained.
(b) Take about equal quantities of olive oil in two test-tubes and add an equal quantity of water to each to one tube, add a drop or two of 10% NaOH. Shake well. An emulsion is formed in both but in the tube without the alkali, the oil separates out from water on standing.
In the other, the emulsion is stabilised due to the fact that the fatty acid components of oil form soap with alkali, the soap then acting as an emulsifier, renders it permanent.
III. Preparation of Suspensoid Sols:
Take 5 ml of 1% solution of AgNO3; add dilute NH4OH until the ppt. formed disappears. Dilute with 100 ml of water. Mix equal volume of this solution with tannic acid (0.5%). A colloidal solution of silver is formed.
IV. Preparation of Emulsoid Sols:
Take 2 g of dry starch, mix well with cold distilled water. Pour the paste formed in 100 ml of boiling water in a beaker and boil further for a few minutes, stirring well. A colloidal solution of starch-in-water is obtained which remains unchanged when cooled.
V. Preparation of Gels:
Add 1 g of agar to 50 ml of water and boil for half an hour. The agar gives a thick opalescent solution (sol) which sets to a gel on cooling. This change is reversible. Take a little warm solution in two test-tubes and show that the agar-mucilage is precipitated by (a) alcohol and (b) saturation with solid (NH4)2SO4.
VI. Precipitation or ‘Salting Out’ of Suspensoid Sols with Electrolytes:
The particles of metallic Ag (expt. III) carry negative electric charge; hence they are readily precipitated by di- or tri-valent cations, e.g., barium, aluminium, etc. Add a few drops of BaCl2 to the AgNO3 solution—the dispersed silver particles are precipitated. Very little electrolyte is necessary for this ‘salting out’ of suspensoid sols as these salts are very sensitive to traces of electrolytes.
VII. Precipitation of an Emulsoid with Electrolytes:
Emulsions are less sensitive to electrolytes than suspensoid sols—large quantities of electrolytes are, therefore, necessary for ‘salting out’ of an emulsion.
(a) Take 2.5 g of wheat flour in 25 ml of distilled water; stir well; allow it to stand for some time and then filter. The extract contains a colloidal emulsion of protein-in- water. Shake again and note that it froths. Saturate the system with solid (NH4)2SO4 (as in expt. V) and note that the protein is precipitated. The ppt. now readily dissolves in water.
(b) Perform similar salting out of a starch emulsion (expt. IV).
VIII. Demonstration of Brownian Movement of Dispersed Particles in an Emulsion:
(a) A drop of milky latex from a fresh sample of Euphorbia is spread out on a slide. Cover with a cover slip and make the borders of the cover slip air tight with vaseline. Observe under microscope the erratic, zigzag movements of the particles in the colloidal state.
(b) 1 ml of olive oil is thoroughly shaken in a test-tube (about a minute) with 1 ml of 1% KOH solution and the Brownian movement is observed quickly under the microscope.