Principles of General Physiology

are known as " organs," and exist as permanent structures ; whereas the FIG. 2. LEUCOCYTE OF NEWT.— Fixed by a are required. The food vacuoles, jet of steam directed on to the cover-glass. seen in Fig. 3, may be given as an Stained with hiematoxylin. Untouched instance. The water, taken in photograph Note the apparently homowith food particles, forms temporary protoplasm. (Schafer, "Essentials of stomachs, as it were, into which Histology," fig. 67, PI. 58.) digestive agents are secreted.

FIG. 3. DIXAMCEBA MIRABILIS. — Interior filled with numerous cells of an alga, Didymoprium, enclosed in drops of liquid. The vacuoles are spherical, although the organisms included have irregular shapes. Eachdivision of the scale corresix >nds to 2-8 fM. (Leidy, 1879, PI. vii. fig. 3.) This property of forming organs for temporary use, as required, is regarded by von Uexkull (1909, pp. 11-32) as demonstrating the impossibility of ever explaining protoplasmic activities on physico-chemical lines. This hopeless attitude does not seem to me to be warranted. Many of these " organs " are formed by the action of laws already known. Eor example, the Higftstivft vacuoles are produced by the water takenin__with the food, and owe their shapp to an rf «.<><* tfvnsion ; if Higp.sf.ivft en7yjrngg_fl.rf> piwipnt in thn body of the protoplasm, they wilL_na±urall^_jind theiFway into the va/Minlft. , The pseudopodial changes of form are in relation to changes of surface tension and consistency of the outer layer of the protoplasm, as will be shown later.

In this connection an interesting experiment is described by Rhumbler (1898, p. 249). If a fine bit of glass rod be pushed against a drop of chloroform under water, it cannot be made to enter the drop ; on releasing the pressure, it is^mmediately reyectectnf, on the contrary, the rod be first coated with shellac, it is at once sucked hi. As soon as the shellac is dissolved by the chloroform, the rod is thrown out again. I find it best to coat the glass with a filtered solution of shellac in chloroform, and then to dry it, since ordinary shellac is only partially soluble in chloroform. One might say that the chloroform will have nothing to do with substances which it cannot digest, and when a mixed food particle is presented to it and accepted, it digests a part and rejects the non-assimilable remainder.

My object in quoting this experiment is to call attention to the way in which quite simple combinations of well-known forces lead to the performance of complicated and apparently purposeful results. With respect to the similar process of the taking in of bacteria by leucocytes (phagocytosis), it is pointed out by Ledingham (1912, p. 324) that leucocytes, when floating freely, are spherical, and put out no pseudopodia unless in contact with some solid surface. Vigorous shaking of the mixture of serum, leucocytes, and bacteria does not affect the irigestion of the latter by the protoplasm, although there can be no pseudopodial activity. When chance contact takes place, there is taking in of the bacteria in a certain proportion of the encounters. The degree of phagocytosis is, therefore, controlled by the number of encounters in unit time. There is no indication of any kind of "seeking" on the part of the phagocytes. The process seems to be one in which surface tension is the chief factor. It is also obvious that, if the bacteria have been < aused to agglutinate into clumps, each encounter will ensure' the ingestion of a arger number of organisms at a time ; hence the " opsonic index " merely shows he presence of something that affects the surface tension of the bacteria (see also the paper by Rhumbler, 1910).

The " super-mechanical properties " of von Uexkiill are also supposed to intervene in the activities of more differentiated structures, such as the muscle cells of actinia and so forth (von Uexkull, 1909, pp. 72 and 73). Although I am unable to follow this investigator so far as to deny all possibility of future explanation, there is no doubt that simple protoplasm presents very difficult problems. It is, in fact, at present, impossible to understand how a liquid, the properties of which protoplasm presents, as we shall see in a later page, can form organs at all. At the same time, it must not be forgotten that the composition of a liquid system is not of necessity the same throughout ; a drop of oil may be floating in dilute alcohol. The various vacuoles in amoeba do not all contain the same substances in solution, as will be seen in a later chapter.

In connection with the subject of this section, it has also been pointed out that animals and plants are units in time as well as in space ; they are compared to a melody in music, whereas machines are merely units in space. It is supposed that the human mind is unable to conceive such existences (see v. Uexkiill, 1909, p. 28). But surely units in time are not wanting in the inorganic world. An atom of radium has arisen from uranium, through an intermediate element, at a certain time in past ages ; it changes again, at a definite rate, into helium and niton, while the latter subsequently disintegrates into other elements.

According to Rutherford (1913, p. 668), the life of uranium is about 1,000,000 years ; that of ionium, 100,000 years; that of radium, 3,000 years; that of niton, 5 '55 days; that of radium A, 4'32 minutes ; that of other intermediate products to radium F (polonium), 196 days; and it is probably finally converted into lead. Moreover, it is characteristic of matter in the colloidal state (see Chapter IV.) not to be in permanent equilibrium — it is what has been called a "nonconservative system." It will become plain in later parts of this book how large a part colloidal phenomena play in the life of the cell. Van Bemmelen (1910, pp. 230-233) showed in 1896 that, if a preparation of colloidal silica as a moist jelly be taken and exposed to air containing various percent.!-. - of water vapour, the amount of water contained in the colloid varies continuously with the tension of the aqueous vapour. But the point of importance in the present connection is that, in certain regions of the curve, the amount of water present in the colloid at a given tension of water vapour is not the same if the silicic acid has previously been exposed to a lower tension, as it is if it lia^ been exposed to a higher one. For example, if it has previously been in a drier atmosphere, and is then placed in one with a tension of water vapour of

Ordinates— tension of water vapour in millimetres of mercury. Abscissae — water content of gel : A, when exposed to increasing tensions ; B, when exposed to decreasing tensions. Showing "hysteresis." Inorganic systems have time factors and "life histories." Thus, from the water content corresponding to a tension of i; mm. Hg (mid height of figure), we have information as to whether the previous history has been one of exposure to increasing or to decreasing tension of water vapour.

6'3 mm. Hg, the water contained in the gel (A) (Fig. 4), after it has come into equilibrium with the gas phase, is less than one half of what it is if placed in the same atmosphere after previous exposure to one of a higher wain vapour tension, say of 12'7 mm. Hg (B). Accordingly, if such a gel be placed in a water vapour tension of 6'3 mm. or thereabouts, information can be obtained of its previous history. The phenomenon here described is known as " hysteresis."

Again, it has been held that an organism differs from non-living matter in that its state at any moment depends not only on its previous history, but also on itfuture history. Here, also, similar conditions are not unknown in pure chemist rv. The relative concentration of the components of a reversible reaction is determined at any time, not only by the initial state, but also by the final state, namely, that of equilibrium. The rate at which acetic acid and methyl alcohol combine to form the ester depends on the distance from the final state ; if one may use a metaphorical expression, this final or equilibrium state is foreseen from the very beginning.

There is one fact about which there can be no doubt, that is, that protoplasm behaves as a liquid. This is shown by the spherical form taken by drops of 1. Normal cell — a, cell wall; b, nucleus; c, protoplasm; rf, wave of contraction in protoplasm ; e, web-like plate arising from the fusion of two fine threads ; /, moving bridge between two stronger protoplasmic currents. Length of cell, 0'3 mm. 2. Somewhat younger cells excited by induction shocks parallel to long axis.

A, shocks of moderate strength ; 6, stronger shocks. In C the protoplasm is coagulated by rupture of cell and entrance of water. Length of cell A, 0'145 mm. watery fluid when they are enclosed in it (Figs. 1 and 3). These drops must therefore be free to take the form conditioned by surface tension, and hence no fixed or solid structures can be present to deform them, unless these structures are themselves freely movable. Further, when the fine particles, present in certain

parts of protoplasmic organisms, are examined under the microscope, they are seen to be in constant movement. This phenomenon was first noticed by the botanist, Brown (1828, p. 359), and is therefore called "Brownian movement." Its nature will be discussed in Chapter IV., but its existence shows that the particles in question are suspended in liquid, and not held in a network or other kind of fixed structure. On the death of protoplasm, as Gaidukov points out (1910, p. 62), the movements cease, and a precipitation or coagulation, like the " setting " of gelatine when it cools, occurs ; in the words of Graham, the hydrosol has become a bydrogeL

Further evidence in the same direction is afforded by the mode of respoiiM «\ protoplasm to an electric shock. When such a stimulus is sent through an amn-b.-i. it is made to draw itself together so that its surface shall be the least possible, in fact it becomes more or less spherical (Kiihne, 1864, p. 32). This would be impossible if struct uiv> incapable of movement. over one another were ]>irsi-iit. Similar chan^i •> are seen in the stamina! hairs of Tradescantia (Fig. 5).

Certain organisms known as mycetozoa in one stage of their life history, form masses of naked protoplasm. One of these, Badliamia, found on logs of decayed oak, was investigated \>v A. Lister (1888). It is usually full of the dark brown spores of the fungus on whieh it feeds, but it ran he made to creep through wet cotton wool, whirli filters out the spores and clarities tinpiotoplasm. It is difficult l<> understand how a sultstan'-c other than a liquid could be separated up into fine threads, which immediately run together again to form a mass like the original one, but devoid of the suspended bodies.

G. L. Kite (1913) states that Congo red and other dyes, injected into the interior of an anuvba, diffuse rapidly throughout the protoplasm. Although we are thus compelled to look upon protoplasm as a liquid, it shows under intense, oblique illumination ("ultra-microscope") that it is not homogeneous like water, or solutions of sodium chloride. On the contrary, it contains an immense number of minute particles, seen by this method (also called " dark ground illumination ") as shining points, or diffraction discs (Fig. 6). There are present, therefore, substances in what we shall learn to recognise as the colloidal state. It is to be noted that the protoplasm in the upper of the two figures given is quite clear and homogeneous when ordinary methods of illumination are used, even under the highest magnifications.

Similar conclusions are drawn by Mott (1912) from observations on living nerve cells by the same method. It is unfortunate that the study of the phenomena presented by living cells is rendered difficult by the fact that so little can be seen by microscopic observation. A few words may, therefore, be useful here as to the nature of microscopic vision. /?. under brilliant dark ground illumination ("ultra-microscope"). The protoplasm appears clear and structureless in A ; full of minute granules in B. Length of cell, O'OSC mm.

Abbe, as is well known, attempted to reduce the formation of all images by the microscope to phenomena of difiraction. There is no doubt of the importance of this point of view, but, under correct methods of illumination, diffraction may be reduced so far that other modes of vision, refraction, and absorption, are preponderant. We will first consider diffraction. This phenomenon is due to the wave form in which light is propagated. It may be roughly described as the property of waves to bend round corners. Sound can be heard from a street at an angle to that in which it is produced, and the fact is sometimes disturbing, just as the corresponding phenomenon in vision through the microscope is. Another instructive fact is shown by the following case : —

Suppose a deep bay, narrowed at its opening to the sea by two stone jetties projecting from each side, and leaving only a small passage between them (Fig. 7). Waves approaching from the open sea pass through the gap, and spread out inside the harbour somewhat as represented in the diagram. An observer at A, supposing that he did not look at the opening, would obtain no evidence of its width from the waves arriving at his feet. . On the other hand, supposing that a close fire of bullets were directed at right angles to the jetties, those that reached a cliff face at A would show the width of the opening.

In a similar manner light waves bend round the edges of objects, and diminish the sharpness with which images of these objects are formed on the retina. Blurred and incorrect definition of the boundaries of objects are only too frequently seen in published photographs of microscopic preparations. When such preparations have a regular pattern, such as the shells of diatoms, a number of totally distinct images may be formed according to the position of the objective.

Apathy (1901, p. 514) describes the following experiment. A diatom of coarse structure, such as Triceratium famis, is observed by an apochromatic objective of 16 mm. focus, and ocular 8, 12, or 18. The substage iris is narrowed to 0'5 mm. in order to give a narrow cone of light. It will be found that no less than fifteen distinctly different images can be seen, as the objective is raised and lowered by the fine adjustment. These images are situated in the course of a movement of about 250 p, whereas the total thickness of the diatom is only 4 n, so that they cannot be due to differences of structure in the depth of the diatom itself. At the

Fiu. 8. REAL IMAGE OF ONE OF THK SECTIONS OF ABBE'S DIFFRACTION PLATE. — Photographed with Leitz j^-iu. oil immersion, projection oc. 4, Powell and Lealaud condenser, full aperture of iris. FlU. 9. FOUR DIFFRACTION IMAOES OF THE SAME PART OF THE PLATE AS FIG. 8. — Photographed with narrow illuminating cone. The images M-ere taken first with Zeiss 16 mm. apochromatic lens, projection oo. 4. No condenser, plane mirror, iris aperture about 0'75 mm. The negatives were then enlarged to the same magnification as the real image in preceding figure, that is, 370 diam. Each division of the scale corresponds to 26 /t- The series of images were obtained by raising the objective through the space between real focus and one-third of a millimetre above. The lower right-hand image is that at the highest position (one-third of a millimetre) above the focus.

lowest and highest positions of the objective no real image can be formed, by refraction, within the tube of the microscope. As the iris is opened, the number of separate images diminishes. The same facts are even better shown by the use of Abbe's diffraction plate, as supplied by Zeiss. One of the figures on this plate consists of a series of rhombic clear areas, obtained by removing the silver coating by scratching a set of crossing lines and then preparing a photographic negative. The real structure is shown in the photograph of Fig. 8. With narrow iris, as in the previous case, a number of different images can be obtained, four of which I have photographed in Fig. 9. More details will be found in an article by J. W. Stephenson (1877, p. 87). The facts given here are sufficient to show that, by diffraction, structures can be seen which are quite unlike those actually present. It will be noted that the condition favouring their production is that of a narrow cone of illumination, due to a small aperture of the substage iris diaphragm. Some interesting photographs of diffraction images will be found in Edser's "Light" (p. 433). In these cases the images are more or less similar to the real objects.

The presence of different structures in a cell, even supposing that they are colourless, can be detected if they have refractive indices differing from that of the surrounding substance. Light rays will be. deflected and give rise to darker and lighter spaces. Colourless glass beads in air, observed under transmitted light by a low power lens, show dark and light rings ; if immersed in oil of the same refractive index as themselves, they become invisible. Ordinary immersion oil is very nearly correct for this purpose.

Now most of the various structures in living cells possess very nearly the same refractive index, a fact which renders this mode of microscopic vision of limited use. Moreover, even when images are seen, they have only an indirect relation to the forms of the objects themselves, as is evident from the appearance of beads in air by transmitted light. Suppose, however, that in the above experiment we take coloured beads. It will be found that, when immersed in oil, a beautifully clear and distinct image is obtained, whereas in air it is obscured by refraction. This shows what is to be aimed at in microscopic observation. Put shortly, we desire coloured objects, mounted in 'a medium of the same refractive index as themselves, and, to avoid diffraction, illuminated by a wide angled cone of light. This latter is obtained in " critical illumination" by which an image of the source of light is produced, in or very close to the plane of the object, by a substage condenser with iris opened as widely as the numerical aperture of the objective will permit. For details the textbooks must be consulted (for example, Spitta's " Microscopy," pp. 209-226). It is sufficient here to emphasise the fact that if, in a particular case, the light of "critical" illumination is too brilliant, it must not be reduced by narrowing the iris, nor by putting the condenser out of focus, but by the interposition of a screen of the necessary degree of opacity.

The mode of vision by absorption of certain components of light by coloured objects is therefore, par excellence, the method to be aimed at. Unfortunately, it is of but limited application to living cells, where so many of the constituents are colourless. There are, however, two cases where it can be used for such objects, and it is, of course, the aim of all histological staining processes. The two cases referred to are, firstly, photography by ultra-violet light, and secondly, intra-vital staining.

Certain structures in the cell, although transparent to all visible wave lengths of light, and therefore colourless, are more or less opaque to ultra-violet light. So that if our eyes were sensitive to this light the objects in question would appear coloured. Now the photographic plate is sensitive to ultra-violet light, and Kohler (1904, pp. 129-165 and 273-304) has shown the possibility of photographing cells by this means. Fig. 10 gives photographs illustrating the fact. It will be noted that, although transparent and colourless to ordinary light, the nucleus is particularly opaque to light of the wave length of the ultra-violet. Unfortunately, the method has not as yet been made much use of, owing to the necessarily elaborate nature of the apparatus required.

Related to the method described above is that in which the fluorescence produced by ultraviolet light when impinging on various substances is observed by the micros, ope. The colour of the light emitted by these fluorescent substances differs according to their composition, so that it may be possible to detect the presence in the living cell of substances otherwise invisible. Further information will be found in Chapter XIX., and in the papers by Stiibel (1911), and by Heimstadt (1911).

1 and -1. Dividing nuclei from (fill plate of Salamander larva. Unstained, in glycerol. Photographed with ultra-violet light of 280 >XM- The chromatic substance appears as if stained. 3. Kdge of sternal cartilage of Newt. Living. Photographed with ultra-violet light. The 4. The same. Photographed with ordinary light. The nuclei are transparent. .1. Red blood corpuscles of the Newt. Living. Photographed with ordinary light. Although oblii|ue illumination was used, the nuclei are almost invisible. Traces of diffraction are seen around the corpuscles.

«. Amoeba. Living. Photographed with ordinary light. The nucleus is just visible, but transparent. The second method, that of staining the living cell, has been of much service. Ehrlich (1886) was the first to show that methylene blue stains living nervous structures. A simple way of observing this fact is given by Michaelis (1902, p. 99). A short piece of the intestine of a mouse is placed for about half an hour to one hour in a solution of methylene