Principles of General Physiology

blue, containing one part of the clye in 20,000 of physiological saline. It is then slit open longitudinally and spread upon a slide with the serous coat uppermost. A glass cover is laid upon it with slight pressure. A little thick gum at the angles will serve to keep it in place. This preparation can be viewed even with an immersion lens. The plexus of nerves will be distinctly seen. Subsequently, Ehrlich himself, followed by other workers, found that various

other dyes are taken into living cells and deposited in them. Instances will be found in other parts of this book. Special structures have been found to be stained by particular dyes, and valuable information obtained (see the book by Goldmann, 1912). In the present state of knowledge of the physics and chemistry of the cell it is impossible to make definite statements as to the meaning of this specific staining of certain structures by particular dyes. Ehrlich holds that the dyes have special affinities for certain " side-chains " of the protoplasmic molecules ; but recent work has shown that many other conditions also play a part, such as solubility, electric charge, diffusibility, and so forth.

It is difficult to see what purely chemical relationship can exist between complex, substituted diazo-sulphonates, as a large number of these specific dyes are, and the chemical constituents of cells. Moreover, although methylene blue and other thiazines are specific vital stains for nerve tissue, certain safranin azo dyes— diazingreen, for example— which have no chemical relationship to the former, are also vital nerve stains, while similar compounds of the same safranin series have no such property (Michaelis, 1902, p. 104). At the same time, one must not be too dogmatic where so little is definitely known. It will be necessary to discuss this question further in later chapters, and we shall see also that the conception of giant molecules in the chemical sense has very little evidence in its favour. For the present, it suffices to point out the fact that this specific

affinity of dyes to particular structures exists, whatever may be its explanation. . Much caution must be exercised in the interpretation of the results obtained by injection of dyes into living organisms. Very few are entirely devoid of poisonous properties, some are very toxic, so that when any one of these is found within a cell we have no means of knowing with certainty whether it found its way there while the cell was still alive, or whether it killed the cell first and then subsequently found its way to the inside, unless we have some criterion as to the vitality of the cell at the time when it is examined. This is to a certain degree possible in the case of unicellular motile organisms, but in the case of the tissues of the higher organisms the difficulty is obviously greater. Evidence may be obtained by the investigation of the permeability of the cell with respect to innocuous bodies, by methods to be referred to in Chapter V. It is clear that a dye cannot stain any constituent of a cell if unable to pass through the covering membrane, but it is not always possible to be certain that, when it does pass through, this happens without previously producing changes in the membrane itself. The dye may also be able to pass through the nonnal membrane, but may kill the cell when it reaches the internal structures. Statements are sometimes made with regard to the permeability of cells to dyes without taking due account of these possibilities. Many dyes, such as methylene blue, are reduced to colourless derivatives by certain cells while alive, but not when dead, so that reducing power in these cases may be used as a criterion of vitality (Michaelis, 1902, pp. 101 and 104). The living nucleus appears to be unstainable, so that when we see it begin to take up pigment we have warning of the death of the cell. Neutral red is one of the least toxic of intra-vital stains.

Fixed cells behave to dyes quite differently from living cells; recently dead, but unfixed, cells have also properties in this respect unlike both those of fixed cells and those of living cells. It is probable that valuable information might be obtained from more detailed study of changes in dying protoplasm. 0, reticular coagulation of cell sap, as occurs after action of osmic acid. A', paths of the "dancing" particlt-.s (Brow-Mian movement), as seen in the livingcell ; these paths are much longer than the meshes of the reticulum, so that the latter could not be present in life and must be a product of the action of the fixative.

Although protoplasm shows so little structure in the living state, it might be thought that, by use of fixing and staining reagents, more could be made a, b, c, and d, forms of precipitate in 5 per cent, albumose, dissolved in 0"2 per cent, potassium hydroxide, when acted on by different reagents. Magnified about 600 diam. a, with 1 per cent, platinum chloride. 6, with Flemming's solution. d, with Altmann's mixture of potassium bichromate and osmic acid.

f, 20 per cent, albumose, faintly acid, precipitated by mercuric chloride, stained with iron haematoxylin and then differentiated. Note fusion of globules to form aggregates (a and b). 40 per cent, albumose, acted on first by 2 '5 per cent, potassium bichromate, which caused turbidity, merely, in the solution. Subsequent acidification with acetic acid caused precipitation of granules of great variety in size. In a a preparation of this mixture was stained by Flemming's method in inverse order of dyes, namely, gentian violet, acid alcohol, safranin. b was stained in the usual order with safranin, acid alcohol, gentian violet. The darker granules in the figures must be supposed to be of a violet colour ; the paler ones, red. In a the small granules are red ; the larger ones violet. In b the opposite is the case. Staining with different d.\es has, thus, no necessary relation to difference of chemical nature.

out Investigations, especially by Hardy (1899, pp. 201-210) and by Alfred Fischer (1899, pp. 1-72 and 202-336), have shown, on the contrary, that the structures obtained in this way are produced by the reagents used, and that quite different appearances are found in the same kind of cells according to the fixing substance used. A few facts will suffice to demonstrate this fact. Flemming, in 1882 (pp. 50 and 51), noticed that the cell-sap of Spirogyra, which was a clear liquid containing particles in Brownian movement during life, became a rigid network after treatment with osmic acid (Fig. 12).

Alfred Fischer (1899, p. 34) takes a clear solution of albumose and acts upon it with various fixing reagents, obtaining various kinds of structures, as shown in Fig. 13. ^Moreover, a homogeneous mixture of albumose and serum albumin, treated by Altmann s osmic and bichromate mixture, gave a structure consisting of granules embedded in a matrix of a fine reticular structure. These two structures could be stained in different colours by the usual histological methods (Fischer, op. cit., p. 53, and Fig. 5 of the plate

Again, if a mixture of different sizes of granules of the same substance, say albumose precipitated bv platinum chloride, be stained with methyl green and fuchsin, the large granules can be stained green, and the smaller ones red, or vice versa (Fig. 20 of Fischer's coloured plate. Similar figures are reproduced in monochrome in our Fig. 14). Thus, after fixation, neither the form nor the staining properties give correct information as to the relationship of the constituents of the original system.

Hardy has shown (1899, pp. 163 and 184) that when substances similar to protoplasm in many of their properties, such as gelatine or egg-white, are acted on by fixing reagents, a separation of the solid from the liquid occurs, so that the former is obtained as some kind of a framework which holds the liquid portion (or TRATE RELATION OF PHASES IN two diflerent kinds of structures which it is COLLOIDAL SYSTEMS. of some importance to distinguish from one

If the black be regarded as the solid phase, another. When a 13 per Cent. Solution of the white as the liquid phase, then A o-ploHnp is allowpd to " set, " bv roolinothere represents an ordinary hydrosol, such as gelatine IS ailOWCC t oy C .lllg, Ul that of gold, in which the solid particles is a separation of the solid from the liquid of an alveolar or honeycomb structure, phase, but the latter cannot be squeezed out in which liquid drops are imprisoned by even by a pressure of twenty-six atmospheres, more or less solid walls, such as a strong * ./ /. . uuj

solution of gelatine when cooled. Whereas, if the jelly be fixed by tormaldehyde, two kinds of structures are known as vesicular and sponge-like. The essential difference is that in the former the liquid phase is in separate droplets each surrounded by a continuous film of the solid phase; in the latter, the two phase* are reversed in position : the solid phase is in the form of a network of threads, while the liquid phase is continuous. A substance, therefore, which passes through a membrane of the former structure has to penetrate through the solid phase itself, while in the latter structure it can pass, although by a tortuous route, from one side to the other by means of the liquid phase only. In the language of colloid chemistry, one may also put it in this way : The dispersed phase in the one is in the position of the continuous phase in the other, and vice versa. The continuous phase is also called the external phase, and the dispersed phase the internal one. The relationship between the two forms of distribution may be made clearer by the diagram of Fig. 15, where the black represents the one phase and the white part the other phase. If black is solid and white is liquid, diagram B will represent the vesicular or foam structure, and A the network or reticular structure in section. The diagram A also represents an ordinary colloidal solution or an emulsion, if the black areas are supposed to be solid or immiscible liquid respectively.

Different fixing reagents produce, then, different kinds of structure in gelatine. Alcohol or mercuric chloride gives a vesicular or foam structure and formaldehyde an open network, as we have seen. In such systems as those under discussion the liquid phase corresponds to the "nonstaining " substance of histologists. FIG. 16. CELLS OF GUT OF ONISCUS. — Stained with iron hrematoxylin. Drawn with camera lucida. If we turn to cells themselves, we find that the same cell shows a different structure of its protoplasm according to the fixing reagent used. For example,

Fig. 16 shows cells of the gut of Qnuetu; A, after the action of osmic vapour; B, after mercuric chloride. Both structures cannot represent that of the living Without the necessity of further details, it may be said that if we find vesicular (Biltschli) or networkstructures in fixed protoplasm, we are not entitled to assume the pre-existence of similar structures in the living btate. What, then, are we justified in concluding from the appearances presented by fixed tissues or cells ? This much, of course, is clear, that there must have been something present in the living cell to give rise to the fixed structure ; although, without further evidence, we cannot assume that there is any similarity between the two. Moreover, when we find in cells of the same kind, fixed, stained, and treated in the same way, the presence of something in certain cells absent from others, we may reasonably draw the conclusion that something has happened in the first which has not happened in the second. On the other hand, we must not assume that what we see is the same thing as the change which had taken place in the cell before fixation.

To take an iiistuin.-, Fig. 17 represents the Purkinje cells of the dn^'s cerebellum, before and in different stages of fatigue, produced by muscular work. \W notice that there is, at first, an increase of some substance which stains 2 to 4. Progressive increase in substance staining with inethylene blue. 5. l.:ii i-r stage (fatigue), disappearance of tbe staining substance. (!. Still further stage of fatigue. With the exception of No. C, all were stained together, by Held's method, on a single slide.

deeply ("Nissl bodies"), but which afterwards disappears almost entirely. Whether this something, which appears and disappears, was originally present in the cells as aggregated masses, or uniformly diffused through the cell substance, we cannot tell. From the usual coagulating action of fixatives, especially from the separation of albumose and serum albumin in A. Fischer's experiments described above (page 14), the latter view is the more probable. Mott (1912) and Marinesco (1912, see page 470 below), in fact, have shown, by observations on living nerve cells under dark ground illumination, that there are neither Nissl bodies nor neurofibrils in the living state. Fine colloidal particles of a special nature are to be seen, but the protoplasm appears to have the uniform general nature of an "organised hydrosol. "

We may also justifiably assume that, when we find structures in the same organ or cell, stained in different colours by a method of double staining, there is some difference between them, although not necessarily of a chemical nature, and the respective structures were probably of quite a different appeal ance during life. A method has been introduced by Altmann (1894, pp. 27-29) which seems to offer possibilities for the investigation of the structure of cells without the use of fixing reagents. If a piece of tissue be allowed to dry at ordinary temperature, it is well known that it becomes so hard and horny that it is impossible to cut thin sections from it. And, even if this were possible, the structures would be altogether distorted. On the other hand, if dried over phosphorus pentoxide in vacuo, at a temperature so low that the salts of the tissue freeze out together with the water (forming a "eutectic " mixture — see the book by Nernst, 1911, p. 121), the cells are never exposed to the action of saturated salt solutions, which are formed when the tissue is dried at ordinary temperatures. A temperature of - 40° to - 30° C. is found to be low enough. The tension of water vapour at this temperature, although not absent, is very small, so that, even when accelerated by the use of a vacuum, the drying, even if very small pieces are taken, lasts for four days or so. Tissues so dried may be directly impregnated with toluene and paraffin at a temperature not exceeding 40° C. in vacua. They cut as well as the best fixed and hardened preparations.

This fact I am able to confirm. My experiments were made by the use of the calcium chloride tank of a carbon dioxide freezing machine ; the solution of calcium chloride was made of such a concentration that its freezing point \vas about - 35° C. , so that by working the compressor all day the solution froze, and, being well insulated from heat, the temperature was maintained sufficiently low until the next morning. The object aimed at by Altmann was to compare the action of different fixatives on sections of the same piece of tissue. The sections were therefore exposed to these reagents at once, and no further difficulty was met with. My object, on the other hand, was to replace the water lost in the dehydration process, in order to examine the structure when unfixed, but a great difficulty was experienced owing to the immediate disintegration of the sections when brought into contact with water. It may be found necessary to allow water to be gradually taken up from ice at the same temperature as that at which the dehydration took place, allowing the temperature to rise very slowly. In any case, the method seems deserving of more attention than it has as yet received. Most laboratories are now provided with ice-making machinery, so that opportunities should not be wanting.

It is obvious that, by ordinary methods of analysis, it is impossible to decide satisfactorily the much debated question as to whether protoplasm is a "giant molecule " in the chemical sense of the word. Even if this were so, the reagents used would certainly split it up into smaller ones. A great variety of bodies have been obtained from cells by chemical treatment, and much light has been thrown in this way on many of the activities to be described in later chapters. All the chemical elements usually found in organic compounds are present, together with salts, inorganic and organic, and water in large amount, as much as 85 per cent, to 90 per cent, or more. It is certain that the complex nitrogenous bodies known as proteins play a great part in the chemical reactions or metabolism of the cell, and it appears also that bodies of a fatty nature,

" lipoids," are essential, in addition to water and inorganic salts. Carbohydrate is probably equally important. A certain theory, that of " bioyen molecules." has attracted many investigators (Verworn, 1903). According to this view, living matter consists of large molecules, with permanent central nucleus, and a great number of " side-chains," in the chemical sense. These side-chains are supposed capable of oxidation, reduction, methylation, and so forth. Under certain conditions, parts of the biogen molecules may be split off, but the essential phenomena of life are associated with changes in which these giant molecules take part as components of chemical reactions, taking place according to the ordinary laws of mass action, equivalent combining proportion, etc. In the thoughtful address of Prof. Hopkins to the British Association (1912, p. 220), which will be read with much profit, we find the following criticism, which seems to me to be entirely justified : —

" This view conceives of the unit of living matter as a definite, if very large and very labile molecule, and conceives of a mass of living matter as consisting of a congregation of such molecules in that definite sense in which a mass of, say, sugar is a congregation of molecules, all like to one another. In my opinion, such a view is as inhibitory to productive thought as it is lacking in basis. It matters little whether in this connection we speak of a ' molecule,' or, in order to avoid the fairly obvious misuse of a word, we use the term 'biogen,' or any similar expression with the same connotation. Especially, I believe, is such a view unfortunate when, as sometimes, it is made to carry the corollary that simple molecules, such as those provided by food-stuffs, only suffer change after they have become in a vague sense a part of such a giant molecule or biogen. Such assumptions became unnecessary as soon as we learnt that a stable substance may exhibit instability after it enters the living cell, not because it loses its chemical identity, and the chemical properties inherent in its own molecular structure, by being

built into an unstable complex, but because in the cell it meets with agents (the intracellular enzymes) which catalyse certain reactions of which its molecule is normally capable." If carbohydrate utilised by the cell becomes part of the protoplasmic molecule before oxidation, it is difficult to suppose that the nitrogenous part of the molecule would escape breakdown. That this is not so is shown by some experiments of Kosmski (1902), who grew Aspergillus in water, and on sugar solution. In the case of growth on pure water, the carbon dioxide given off falls at once, but to a value which is not zero, but about one quarter of that when grown on sugar. This production may possibly be that of the protoplasmic substance itself. When transferred to solutions containing sugar, the carbon dioxide rises at owe, indicating direct utilisation without previous combination ™«-k "biogen" (see Fig. 18).

At the second hour, the nutrient solution, containing glucose, was changed for tap water. The combustion processes immediately decreased to a low level, but were as suddenly restored, at the seventh hour, by addition of normal glucose nutrient solution. There is apparently no store of food material. The steady MM. ill respiratory exchange in the absence of food is probably due to consumption of the organised structure of the cells. The use by bacteria of energy obtained from oxidation of inorganic sulphur, etc., is further evidence of non-oxidation of protoplasm itself. In many of these cases,

the presence of sugar appears to be actually injurious to the growth of the organism. Hydrogen gas can also be used as a source of energy. When hydrogen, oxygen, and carbon dioxide are present together, it is found that the oxygen is used up to oxidise hydrogen, and the energy so obtained enables the carbon dioxide to be used as a source of carbon : the three gases disappear simultaneously. Another interesting fact is that sulphur organisms also require a supply of carbon dioxide in the form of carbonate.

Evidence will be given in Chapter XX. and elsewhere against the supposed existence of intramolecular oxygen. In fact, a view akin to that of Hofmeister (1901) is rapidly gaining ground. Hofmeister looks upon the cell rather as a laboratory, in which various operations are going on at the same time, being kept apart by membranes or partitions of some kind. Hopkins (1912, p. 220) advocates the existence of " interplasmic " reactions, in which substances formed by protoplasm are responsible for chemical changes in the cell. These reactions, then, take place in interspaces between the protoplasmic molecules, or rather molecular aggregates, themselves. Digestion in food vacuoles of Amoeba may serve as an illustration of the process on a relatively large scale. Other reactions may occur in similar spaces, too small to be visible under the microscope. It seems that living matter is a complex of association processes of various types, in which physical forces play a large part, such as the surface condensation known as "adsorption" (Chapter III.), and also electrical charges. These forces control and regulate the course of the chemical reactions (Hopkins, 1912, p. 218). In any case it is evident that protoplasm, as it presents itself in such an organism as Amoeba, is a system of many components or phases, solid and liquid, minutely subdivided and intimately mixed (see Gaidukov, 1910, pp. 61, 62, 74). In a certain sense, therefore, it may be said to have a structure, and the fact is of interest in connection with such chemical reactions as cease when the cell is ground up in a mortar. The cessation of the oxidation of lactic acid in muscle when chopped up (Fletcher and Hopkins, 1907, p. 284, and Harden and Maclean, 1911, p. 45) may be referred to. The effect produced by change of distribution of phases, as in Fig. 15, must also be borne in mind. Vernon (1912, pp.

210, 211) has been led, by his work on the effect of anaesthetics on oxidation in cells, to suggest the separation of cell constituents by membranes of a lipoid nature, a view similar to that of Hofmeister. Buchner (1903, p. 92) noticed that yeast cells containing glycogen showed no " auto-fermentation " as long as they were alive, but, when killed by acetone, this took place. Obviously, during life, the .access of zymase and other enzymes to the glycogen is not permitted to take place.

A discussion of phase relations in protoplasm with respect to equilibrium and energy will be found in the essay by Zwaardemaker (1906, pp. 137-154). As already pointed out above, protoplasm usually presents the characters of a liquid, but when dead it appears to take on a rigid structure, like melted jelly when it "sets," or egg-white when boiled. In this state it is no longer a liquid, and the Brownian movement of particles contained in it ceases, as they are held in a fixed structure.

An observation made by Gaidukov (1910, p. 58) suggests that such a change may take place temporarily during life ; if so, this may be a means of localising chemical changes in particular parts of the cell. When protoplasm presents a free surface to watery fluids it is found to exhibit a continuous movement. In vegetable cells these movements are of a circulating or streaming nature. Now Gaidukov noticed, when observing the phenomenon in Vcdlisntna, that the streaming movement occasionally ceased and only a few of the particles showed Brownian movement. Presently, the Brownian movement began to reappear, and, as it increased, the streaming recommenced. This looks very much like a reversible change from "sol" to "gel."

The " Biogen " theory is an example of the efforts of a certain school of physiologists to explain by purely chemical laws, such as mass action, facts which admit of simpler explanation, if physical phenomena are also taken into account. Forced and elaborate assumptions are sometimes necessary if chemical laws only are allowed. If a physical explanation' is forthcoming, it appears to me that it is more scientific to adopt it. It may be said indeed that it is