Finally, in a solution freezing at -.72, _i.e._ with an osmotic pressure at 15 C. of 9.176 atmospheres, we obtained the following mean values for the untired leg:--

After 8 hours -.04.

After 16 hours -.05.

After 24 hours -.05.

In this solution, freezing at -.72 C., some of the stimulated muscles showed no diminution in weight, while others showed a very small diminution, and others again a slight augmentation, the maximum increase being .085 of the initial weight. The solution is therefore practically isotonic with the stimulated muscle.

In this case the elevation of the intramuscular osmotic pressure produced by the electrical excitation and the muscular contractions was therefore 2.5 atmospheres, or more than 2.6 kilogrammes per square centimetre of surface.

I made further experiments in order to discover whether the variation in osmotic pressure depended on the duration of {55} the muscular contraction.

For this purpose I used a solution freezing at -.53 C. and immersed in it untired muscles, and muscles which had been electrically excited for two, four, and six minutes respectively. The following are the results:--

Untired muscles. Muscles stimulated once a second during 2 Minutes. 4 Minutes. 6 Minutes.

.000 +.026 +.084 +.094 +.001 +.034 +.065 +.093 +.005 +.045 +.079 +.097 .000 +.037 +.070 +.095 .000 +.032 +.072 +.096

Mean of all the observations--

+.0012 +.0348 +.074 +.095

These experiments show clearly that the osmotic intramuscular pressure rises in proportion to the duration of the electrical stimulation.

In order to determine the influence of the work accomplished by the muscle on the elevation of the osmotic pressure, I made the following experiment.

The two hind legs of a frog were submitted to the same electrical excitation, one leg being left at liberty, and the other being stretched by a hundred-gramme weight, acting by a cord and pulley. After exciting them electrically for five minutes, the legs were immersed for twenty-four hours in a saline solution freezing at .53 C. The free limb showed an augmentation of .085 of the initial weight, and the stretched limb an increase of .106 of the initial weight. It is evident, therefore, that the osmotic pressure increases with the amount of work done by a muscle.

Briefly, then, the results of our experiments are as follow:--

1. Muscular contraction electrically produced causes an increase of the osmotic pressure in a muscle.

2. The intramuscular osmotic pressure may reach, or even exceed, 2.5 atmospheres, or 2.6 kilogrammes per square centimetre of surface.

3. When a muscle is made to contract once a second, the {56} elevation of the osmotic pressure increases with the number of contractions.

4. The intramuscular osmotic pressure increases with the work done by the muscle.

5. Fatigue is caused by the increase of osmotic pressure in a contracting muscle.

[Ill.u.s.tration: FIG. 3.--Fields of diffusive force.

(_a_) Monopolar field of diffusion. A drop of blood in a saline solution of higher concentration.

(_b_) Bipolar field of diffusion. Two poles of opposite signs. On the right a grain of salt forming a hypertonic pole of concentration, on the left a drop of blood forming a hypotonic pole of dilution. ]

_The Field of Diffusion._--Just as Faraday introduced the conception of a field of magnetic force and a field of electric force to explain magnetic and electrical phenomena, so we may elucidate the phenomena of diffusion by the conception of a field of diffusion, with centres or poles of diffusive force. If we consider a solution as a field of diffusion, any point where the concentration is greater than that of the rest may be considered as a centre of force, attractive for the molecules of water, and repulsive for the molecules of the solute. In the same way any point of less concentration may be regarded as a centre of attraction for the molecules of the solute, and a centre of repulsion for the molecules of water.

A field of diffusion may be monopolar or bipolar. A bipolar field has a hypertonic pole or centre of concentration, and a hypotonic pole or centre of dilution. By a.n.a.logy with the magnetic and electric fields we may designate the hypertonic pole as the positive pole of diffusion, and the hypotonic as the negative pole. {57}

The positive and negative poles and the lines of force in the field of diffusion may be ill.u.s.trated by the following experiment. A thin layer of salt water is spread over an absolutely horizontal plate of gla.s.s. If now we take a drop of blood, or of Indian ink, and drop it carefully into the middle of the salt solution, we shall find that the coloured particles will travel along the lines of diffusive force, and thus map out for us a monopolar field of diffusion, as in Fig. 3 a. Again, if we place two similar drops side by side in a salt solution, their lines of diffusion will repel one another, as in Fig. 4.

[Ill.u.s.tration: FIG. 4.--Two drops of blood in a more concentrated solution, showing a field of diffusion between two poles of the same sign.]

Now let us put into the solution, side by side, one drop of less concentration and another of greater concentration than the solution. The lines of diffusion will pa.s.s from one drop to the other, diverging from the centre of one drop and converging towards the centre of the other (Fig. 3 _b_). In this manner we are able to obtain diffusion fields a.n.a.logous to the magnetic fields between poles of the same sign and poles of opposite signs.

The conception of poles of diffusion is of the greatest importance in biology, throwing a flood of light on a number of phenomena, such as karyokinesis, which have hitherto been regarded as of a mysterious nature.

It also enables us to appreciate the role played by diffusion in many other biological phenomena. Consider, for example, a centre of anabolism in a living organism. Here the molecules of the living protoplasm are in process of construction, simpler molecules being united and built up to form larger and more complex groups. As a result of this aggregation the number of molecules in a given area is diminished, _i.e._ the concentration and the osmotic pressure fall, producing a hypotonic centre of diffusion. We may thus regard every centre of anabolism as a negative pole of diffusion. {58}

Consider, on the other hand, a centre of catabolism, where the molecules are being broken up into fragments or smaller groups. The concentration of the solution is increased, the osmotic pressure is raised, and we have a hypertonic centre of diffusion. Every centre of catabolism is therefore a positive pole of diffusion. Similar considerations as to the formation and breaking up of the molecules in anabolism and catabolism apply to polymerization.

The diffusion field has similar properties to the magnetic and the electric field. Thus there is repulsion between poles of similar sign, and attraction between poles of different signs. A simple experiment will show this. A field of diffusion is made by pouring on a horizontal gla.s.s plate a 10 per cent. solution of gelatine to which 5 per cent. of salt has been added. The gelatine being set, we place side by side on its surface two drops, one of water, and one of a salt solution of greater concentration than 5 per cent. We have thus two poles of diffusion of contrary signs, a hypotonic pole at the water drop, and a hypertonic pole at the salt drop.

Diffusion immediately begins to take place through the gelatine, the drops become elongated, advance towards one another, touch, and unite. If, on the contrary, the two neighbouring drops are both more concentrated or both less concentrated than the medium, they exhibit signs of repulsion as in Fig. 4.

Diffusion not only sets up currents in the water and in the solutes, but it also determines movements in any particles that may be in suspension, such as blood corpuscles, particles of Indian ink, and the like. These particles are drawn along with the water stream which pa.s.ses from the hypotonic centres or regions toward those which are hypertonic.

These considerations suggest a vast field of inquiry in biology, pathology, and therapeutics. Inflammation, for example, is characterized by tumefaction, turgescence of the tissues, and redness. The essence of inflammation would appear to be destructive dis-a.s.similation with intense catabolism. We have seen that a centre of catabolism is a hypertonic focus of diffusion. Hence the osmotic pressure in an inflamed region is increased, turgescence is produced, and {59} the current of water carries with it the blood globules which produce the redness.

The phenomenon of agglutination may also possibly be due to osmotic pressure, a positive centre of diffusion attracting and agglomerating the particles held in suspension.

_Tactism and Tropism._--The phenomena of tactism and tropism may also be partly explained by the action of these diffusion currents of particles in suspension, these polar attractions and repulsions. In all experiments on this subject we should take into account the possible influence of osmotic pressure, since many of the causes of tactism or tropism also modify the osmotic pressure at the point of action, and it is possible that this modification is the true cause of the phenomenon. Osmotactism and osmotropism have not as yet been sufficiently studied.

[Ill.u.s.tration: FIG. 5.--Liquid figures of diffusion.

The six negative poles of diffusion are coloured with Indian ink. The positive pole in the centre is uncoloured and is formed by a drop of KNO_3 solution.]

Thus it may be said that osmotic pressure dominates all the kinetic and dynamic phenomena of life, all those at least which are not purely mechanical, like the movements of respiration and circulation. The study of these vital phenomena is greatly facilitated by the conception of the field of diffusion and poles of diffusion, and of the lines of force, which are the trajectories of the molecules of the solutes, and the particles and globules in suspension.

_The Morphogenic Effects of Diffusion._--Many interesting experiments may be made showing variations of the lines of force in a field of diffusion, and how liquids subjected only to differences of osmotic pressure diffuse and mix with one {60} another in definite patterns. When a liquid diffuses in another undisturbed by the influence of gravity, it produces figures of geometric regularity, and we may thus obtain figures and forms of infinite variety. The following is our method of procedure. A gla.s.s plate is placed absolutely horizontal and is covered with a thin layer of water or of saline solution. Then with a pipette we introduce into the solution, in a regular pattern, a number of drops of liquid coloured with Indian ink. A wonderful variety of patterns and figures may be obtained by employing solutions of different concentration and varying the position of the drops.

[Ill.u.s.tration: FIG. 6.--Pattern produced in gelatine by the diffusion of drops of concentrated solutions of nitrate of silver and bromide of ammonium.]

Instead of the water or salt solution, we may spread on the plate a 5 or 10 per cent. solution of gelatine, containing various salts in solution. If now we sow on this gelatine drops of various solutions which give colorations with the salts in the gelatine, we may obtain forms of perfect regularity, presenting most beautiful colours and contrasts. The drops, of course, must be placed in a symmetrical pattern. In this way we may obtain an endless number of ornamental figures.

In order to cover a lantern slide 8 cm. 10 cm., about 5 c.c. of gelatine is required. To this amount of gelatine we add a single drop of a saturated solution of salicylate of sodium, and spread the liquid gelatine evenly over the plate. When the gelatine has set, we put the plate over a diagram, a hexagon for instance, and place a drop of ferrous sulphate solution at each of the six angles. The drops immediately diffuse {61} through the gelatine, and the result after a time is the production of a beautiful purple rosette. The gelatine must be carefully covered to prevent its drying until the diffusion is complete. The preparation may then be dried and mounted as a lantern slide, and will give the most brilliant effect on projection. If the gelatine has been treated with a drop of pota.s.sium ferrocyanide solution instead of salicylate of sodium, a few drops of FeSO_4 will give a blue pattern. Or we may treat the gelatine with ferrocyanide of pota.s.sium and salicylate of sodium mixed, and thus obtain an intermediary colour on the addition of FeSO_4. We may, indeed, vary indefinitely the nature and concentration of the solution, as well as the number and position of the drops. The results have all the charm of the unexpected, which adds greatly to the interest of the experiment.

[Ill.u.s.tration: FIG. 7.--Pattern produced in gelatine by the diffusion of drops of silver nitrate and sodium carbonate.]

These experiments are not merely a scientific toy. They show us the possibility, hitherto unsuspected, that a vast number of the forms and colours of nature may be the result of diffusion. Thus many of the phenomena of life, hitherto so mysterious, present themselves to us as merely the consequences of the diffusion of one liquid into another. One cannot help hoping that the study of diffusion will throw still further light on the subject.

If a number of spheres, each capable of expansion and deformation, are produced simultaneously in a liquid, they will form polyhedra when they expand by growth. This is the {62} precise architecture of a vast number of living organisms and tissues, which are formed by the union of microscopic polyhedra or cells. A section of such a polyhedral structure would appear as a tissue of polygons. It is interesting to note that the simple process of diffusion will produce such structures under conditions closely allied to those which govern the development of the tissues of a living organism.

[Ill.u.s.tration: FIG. 8.--Pattern produced in gelatine by the diffusion of drops of a solution of nitrate of silver and of citrate of pota.s.sium.]

We may obtain this cellular structure by a simple experiment. On a gla.s.s plate we spread a 5 per cent. solution of pure gelatine, and when set sow on it a number of drops of a 5 to 10 per cent. solution of ferrocyanide of pota.s.sium. The drops must be placed at regular intervals of 5 mm. all over the plate. When these have been allowed to diffuse and the gelatine has dried, we obtain a preparation which exactly resembles the section of a vegetable cellular tissue (Fig. 9). The drops have by mutual pressure formed polygons, which appear in section as cells, with a membranous envelope, a {63} nucleus, and a cytoplasm, which is in many cases entirely separated from the membrane. These cells when united form a veritable tissue, in all respects similar to the cellular structure of a living organism.

[Ill.u.s.tration: FIG. 9.--Tissue of artificial cells formed by the diffusion in gelatine of drops of pota.s.sium ferrocyanide.]

In the preparation showing artificial cells the cellular structure is not directly visible until the gelatine has dried. One sees only a gelatinous ma.s.s a.n.a.logous to the protoplasm of a living organism. This ma.s.s is nevertheless organized, or at least in process of organization, as we may see by the refraction when its image is projected on the screen.

During the cell-formation, and as long as there is any difference of concentration in the gelatine, each cell is the arena of active molecular movement. There is a double current, as in the living cell, a stream of water from the periphery to the centre, and of the solute from the centre to the periphery. This molecular activity--the life of the artificial cell--may be prolonged by appropriate nourishment, {64} _i.e._ by continually repairing the loss of concentration at the centre of the cell.