and rendering the remaining contents in the interior of the nucleus granular; while the darkness of the double contour of the nucleus itself is caused by an increase in density of the wall, which it representa

When the coloured blood-corpuscles of the Amphiuma are treated with a weak solution of chromic acid, and the action of the reagent continues, they are rendered colourless. At the same time, they are bordered by a distinct double contour, and their protoplasm appears finely granular. The nucleus also is distinguished by a dark double contour and by granular contents. Finally, if the action still continues, the granular contents of the corpuscle are dissolved, and the latter appears as a clear cell with a double contour.

Concerning the action of a weak solution of chromic acid upon these blood-corpuscles, I have made an interesting observation. When a fresh specimen of the blood of Amphiuma is treated with water under the microscope, and subsequently, after the coloured blood-corpuscles have become discoloured and rendered very pale by the action of the water, and a very weak solution of chromic acid is added, the following changes will be observed to take place in a number of the corpuscles: becoming almost invisible, they will reappear, bordered by decided dark double contours, while their nuclei, also distinguished by dark double contours and granular contents, are coloured yellow from the reagent. But the most interesting part of the phenomenon is a series of fine lines, radiating from the periphery of the nucleus through the protoplasm to the inner surface of the membraneous layer of the blood-corpuscle (Fig. 24). Now this picture would almost seem to corroborate the theory of Hensen, as well as that of Kollmann; the fine double lines representing the filaments, which they suppose to radiate from the nucleus to the enveloping membrane. But this is not the case. For a closer examination reveals that these lines represent nothing but fissures in the protoplasm, which appears to have assumed some form of crystallization. This becomes more evident by observing some of these fissures, deviating from their course and giving rise to subordinate branches, as is seen in Fig. 24.

What this colourless crystallizable constituent of the protoplasm, manifesting itself by the above-described reaction with a weak solution of chromic acid, may be, I must leave to the organic chemist to decide. It will be remembered, however, that a number of years ago Lehmann succeeded in discolouring the original bloodcrystals, demonstrating the albuminous body in its crystalline form, though Kuehne, who regards the coloured blood-corpuscles as consisting of a certain stroma, in the interstices of which the hæmoglobin was to be contained, subsequently declared this * O. Funke, 'Lehrbuch der Physiologie,' 4th edit., vol. i. p. 49.

*

observation to be erroneous.* If the latter investigator is right, of what do those minute colourless crystals, observed in the fresh coloured blood-corpuscle of the Amphiuma, consist?

It remains to be mentioned that I have not invariably succeeded in producing the phenomenon in question in these bloodcorpuscles. The failure, I suppose, was occasioned by not having applied the reagent soon enough, or else too late, as respected the action of the water upon the blood-corpuscles; or perhaps the strength of the solution, to ascertain which I took no care, may have been the cause. In some cases the corpuscles appear dotted over with minute granules; these belong to the liquor sanguinis surrounding them, and are produced by the action of the chromic acid.

When the blood-corpuscles of the Amphiuma are exposed to the action of chloroform vapour, they become entirely discoloured in about two minutes. When exposed somewhat longer, their outlines become very faint, but may always be discovered in the form of a double contour by close examination with a first-class objective; frequently they are hidden by the colouring matter escaped from the corpuscles into the liquor sanguinis. The nucleus is not affected by the vapour of the chloroform; on the contrary, it is rendered more distinct (Fig. 25).

In exposing the blood-corpuscles to the action of water, after they have been discoloured by the vapour of chloroform, almost the same changes will be observed as when treated with water in their fresh condition. They then appear clear, bordered by a faint double contour; they do not swell, but rather appear to diminish in size, owing to the escape of their contents by the action of this fluid. The nucleus, however, is seen to swell very considerably; in some cases to such an extent as nearly to fill the interior of the corpuscle. At the same time, by the dissolution of its granules, its contents appear homogeneous and very pale. If at this stage the preparation is subjected to the action of a weak solution of chromic acid, the outlines of the blood-corpuscles will appear more distinct, but not as dark as when in their fresh condition and when they are only acted on by water.

45

25

When a fresh specimen of blood is treated with chloroform liquid, the blood-corpuscles, besides being rendered colourless, as in the previous case, become considerably reduced in size; their diameter scarcely exceeding 150 mm. in length, and 185 mm. in breadth. Being bordered by distinct, though pale double contours, they contain numerous well-defined minute granules; their form is now more or less oval or even irregular. Their nuclei also are distinguished by very distinct dark double contours, and contain a number of larger and smaller pale granules (Fig. 26), while their * Kuehne, 'Lehrbuch der Physiologischen Chemie,' p. 207. VOL. I.

G

form is round or slightly oval. Being subsequently treated with water, the same changes are observed to take place on the bloodcorpuscles as when treated by this liquid in their fresh condition; that is, the minute granules in their interior are dissolved and the corpuscles appear clear with pale contours. The nucleus is seen to swell, while the large granules it contains are dissolved, giving to the whole a pale homogeneous appearance. In a number of cases also, the delicate membraneous layer of the corpuscles bursts, allowing the nucleus to escape as before described. If now, after the blood-corpuscles have passed through these various changes, produced by the chloroform liquid and the water, they are finally treated with a weak solution of chromic acid, nearly the same changes will be witnessed as are produced by this reagent on the fresh blood-corpuscle, namely, the size of the corpuscle becomes still more reduced, the faint delicate double contour, together with that of the nucleus, appears darker and more distinct, and the contents of the nucleus are rendered finely granular; in some cases even, its original larger granules are observed to reappear.

Diluted acetic acid discolours the blood-corpuscles and causes them to swell. Their outlines are then represented by pale greenish, delicate double contours, and in their interior numerous granules of the same tint are observed, which disappear by the continued action of the acid, or when applied in a stronger form. Eventually the membraneous layer, distended by the reagent, contracts again, and the blood-corpuscle reassumes its original form, or, as observed in a number of cases, its outlines appear wrinkled or otherwise irregular. Frequently the nucleus leaves its central position, and floats about in the interior of the corpuscle, or may escape through a rupture of the membraneous layer. The granules within the nucleus are observed to swell; but while some coalesce and fuse to a certain extent with each other, assuming a worm-like or varicose form, others preserve their original shape and individuality. The double contour of the nucleus disappears, and a dark single contour takes its place, which appears as if belonging to the fused granules; for those not fused are bordered by one of the same character. Around this dark contour a slightly reddish zone in the form of a border is observed, which I suppose to represent the wall of the nucleus, altered by the reagent (Fig. 27). The granules of the nucleus, after having been once exposed to the action of the acetic acid, seem to resist the solvent action of water.

When the coloured blood-corpuscles of the Amphiuma are exposed to the action of a 2 per cent. solution of boracic acid, they are gradually rendered colourless. The double contour, which appears very distinct, is of a greenish tint. The nucleus also is very pale, but shows distinctly its granules and double contour.

On many of the blood-corpuscles a number of straight longitudinal folds are observed. These disappear again by the continued action of the acid vapour, and the wrinkled surface of the corpuscle then appears as smooth as before. But in the depth of some of the previously existing folds a fine stripe of a pink colour is left, appearing to represent a rent or fissure in the contents of the corpuscle.

When the 2 per cent. solution of boracic acid itself is applied to the blood-corpuscles, they are rendered pale, and their contents appear finely granular, while the double contour becomes very distinct. Finally, by the continued action of the acid the granular appearance is lost, and they become perfectly clear. The nucleus is rendered coarsely granular, and its outlines appear irregular and serpentine; the outer contour of its enveloping membrane is dark, while the inner one has become more indistinct (Fig. 28).

Exposed to the action of the vapour of a 4 per cent. solution of osmic acid for about five minutes, the blood-corpuscles are rendered paler, almost colourless. Their double contour appears somewhat more distinct than in their fresh condition. The nucleus remains almost unchanged. In some cases its outline and granules become more distinct. But while perhaps the greater portion of the blood-corpuscles remain unaltered in form, a number of smooth wrinkles running mostly in a longitudinal direction are observed on others. Some of these latter have assumed a dish-like form, the nucleus projecting on the convex surface (Fig. 29).

In exposing the coloured blood-corpuscles to the vapour of a solution of osmic acid of only 2 per cent. for about two or three minutes, the above-mentioned changes do not occur, but the corpuscles remain almost entirely unaltered in appearance. For this reason E. Ray Lankester, in his article "On the Red Bloodcorpuscle" mentioned in the beginning of this paper, recommended the vapour of a solution of osmic acid as the best agent for the preservation of these bodies. I can endorse his recommendation; for a number of specimens of the blood of the Amphiuma and the Frog, prepared in this manner and mounted in glycerine instead of in a nearly saturated solution of acetate of potassa more than three years ago, show hardly any alteration up to this time. Previously to employing the glycerine, I had made use of the solution of acetate of potassa, but found it rendered the blood-corpuscles very indistinct in quite a short time.

In exposing a specimen of blood of the Amphiuma to the action of the vapour of a 50 per cent. solution of the hydrate of chloral for about two minutes, a portion of the coloured corpuscles are rendered paler without alteration of their form, while others are wrinkled in a longitudinal direction. In a third portion, howover, the protoplasm is observed to contract in the form of an

irregular mass upon the nucleus, which becomes hidden from view. The rest of the body, after having considerably contracted and assumed an irregular angular form, breaks up into small fragments. After an exposure of about ten minutes, the blood-corpuscles become entirely discoloured, the nucleus before hidden reappears, showing a distinct irregular serpentine double contour, and also the granules in its interior.

The changes taking place in these blood-corpuscles when treated with the solution of the hydrate of chloral are very interesting and important, as they manifestly show the existence of the membraneous layer of these bodies, such as I have described it. Thus, after the solution has been applied, the protoplasm of the blood-corpuscle without much or any alteration of form gradually contracts upon the nucleus. As the result of this contraction, it becomes entirely separated from the membraneous layer, which manifests itself in the form of a delicate double contour (Figs. 30, 31, and 32). The interspace left between the contracted protoplasm and the double contour representing the membraneous layer is very considerable, as will be seen from the drawings, and it seems to me should be sufficient evidence to prove the existence of such a layer to an unbiassed mind.

At first the protoplasm is observed to become coarsely granular. When its contraction and simultaneous separation from the membraneous layer commences, it assumes a fibrillar appearance at its periphery (Fig. 32), and as the metamorphosis proceeds similar fibrillar rings concentrically arranged appear throughout the whole mass (Fig. 30). A number of coarse granules are observed to adhere to the contracted mass or to its periphery. In fact, it appears almost as if those concentrically arranged fibrille were produced by a fusion of granules. The contracted protoplasm retains its yellow colour, while the membraneous layer and also the vacuum left between it and the former are perfectly clear. Although the general oval form of the blood-corpuscle, especially of its membraneous layer, remains unaltered during the whole process of metamorphosis, its size is very considerably reduced; for in many instances its long diameter amounts only to 100 mm. in length, or seven less. Considering this great diminution in the size of the blood-corpuscle, it seems that when the protoplasm commences to contract the membraneous layer contracts simultaneously to a certain limit, beyond which it cannot contract any farther; if then the contraction of the protoplasm continues, a separation from the membraneous layer must be the result. The nucleus undergoes almost no change by the action of this solution; it is only rendered more distinct.

50

When the action of the solution of the hydrate of chloral is allowed to continue for some time, the contracted protoplasm is