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(The Action of Carbonate of Ammonia on Chlorophyll Bodies Bodies.

By Charles Darwin LLD. F.R.S.

(In In my Insectivorous Plants I have described, under the term of aggregation, a phenomenon has been which has excited the surprise of all who have beheld it.* and which Pfeffer in his recent admirable work *(*Pfeffer Pflanzenphysiologie. Band ii 1881 p 248. Cohn, Die Pflanze Vorträge aus dem Gebiete der Botanik 1882 p. 361. considers as "in manifold ways respects all interesting process," & about which Cohn writes in still stronger terms.

It is best exhibited in the tentacles or so called glandular hairs of Drosera, after a minute particle of any inorganic or organic substance, or a drop of almost any nitrogenous fluid, has been placed on a gland. Under favourable circumstances the transparent purple fluid in the cells beneath nearest to the gland becomes in a few seconds or minutes slightly turbid. Soon minute spherical granules can be distinguished under a high power, which quickly coalesce or grow larger and for many hours afterwards oval or globular, or curiously shaped masses of a purple colour & of considerable size may be observed sending out processes or filaments, dividing, coalescing, and redividing in the most diversified singular manner, until

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*Pfeffer, in his recent admirable work 'Pflanzenphysiologie' (B. II. 1881 p. 248) speaks of the phenomenon as being in many respects interesting; & Cohn writes (Die Pflanze, Vorträge aus dem Gebiete der Botanik, 1882, p. 361) in still stronger terms.

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finally one or two solid spheres are formed which remain motionless. The moving masses include vacuoles which alter in change their appearance. (I append here three figures of aggregated masses copied from my son Francis's paper* (* Quart. Journal Microscop: Society vol XVI 1876, p 309) showing the forms assumed. After some aggregation has been partially effected the layer of protoplasm lining the walls of the cells may be seen with singular clearness flowing in great waves; and my son observed similarly flowing threads of interlacing protoplasm which connecting together the together the grains of chlorophyll. After some a time has elapsed the minute colourless particles which are embedded in the flowing protoplasm on the walls of the cells are drawn towards and unite with the aggregated masses; so that the flowing protoplasm on the walls being now rendered quite transparent is no longer visible, though judging from the movements of an occasional particle in the cell sap it some is still present, and still flows, as may be inferred from the occasional transport of particles in the cell-sap. Owing to this withdrawal of the colourless particles from the flowing protoplasm The withdrawn

The granules withdrawn from the walls, together probably with some matter

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derived from the flowing protoplasm & from the cell-sap, together often form a colourless, or very pale purple, well-defined layer of considerable thickness, which surrounds the aggregated & now generally spherical dark-purple masses. This surrounding layer or zone consists of solid matter, more bri brittle than the central parts of the aggregated masses, as could be seen when they were crushed beneath a cover-glass. I will only add that there is no a priori improbability in some of the protoplasm being withdrawn together with the embedded granules from the walls, for the whole of the protoplasm in within the hairs of Tradescantia contracts, when subjected to great cold into spherical several spheres; & these when afterwards when warmed again spread themselves out over the walls.*) (*Van Tieghem, Traité de Botanique 1882 p. 596. See also p. 528, on masses of protoplasm floating freely within the cavities of cells. Sachs (Physiologie Végétale p 74) & Kühne (Das Protoplasma p. 103) have likewise seen within cells small, freely-floating masses of protoplasm in the hairs of Tradescantia & Cucurbita which undergo amœboid changes of form: formed representative my same paper in Q. Journal of Microscopical Science Vol. XVI 1876 p. 318)

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and their coalescence with the adrea already aggregated and now generally spherical of a dark purple colour, these are often surrounded by a colourless or very pale purple or [insertion:] This surrounding layer or zone [2 words illeg] colourless zone of solid matter more brittle than the central part of the aggregated masses as was well could be seen when they were crushed beneath the cover-glass.)

The process of aggregation commences in the stimulated glands and slowly travels down the whole length of the tentacles and even into the disc of the leaf, but very much more slowly than the impulse which causes the basal part of the tentacle to bend inwards. It is a much more interesting fact that when the glands on the disc are stimulated they transmit some influence to the glands of the surrounding tentacles, which undergo throughout their whole length the process of aggregation, although they themselves have not been directly stimulated in any manner; and this process may be justly compared with a reflex action in

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the nervous system of an animal. After a few days or a week the solid aggregated masses in the cells of the tentacles are all re-dissolved. The process of re-dissolution commences in the cells at the bases of the tentacles and travels slowly upwards; therefore in a reversed direction to that of aggregation. Considering that the aggregated masses are solid enough to be broken into fragments, their prompt redissolution is a surprising fact; and we are led to suspect that some ferment must be generated in the disc of the leaf, and travel upbe transmitted up the tentacles. The double process of aggregation and of redissolution takes place every time that a leaf of Drosera catches an insect.

Aggregation is a vital process, that is it cannot occur in cells immediately after their death. This was shown by waving leaves in (Insectivorous Plants p 58) for a few minutes in water at a temperature of 65°.5 C. (150° F) or even at a somewhat lower temperature, and then immersing them in a rather

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strong solution of carbonate of ammonia, which does not cause in this case any aggregation, although the most powerful of all known agents.

If a tentacle is slightly crushed, so that many of the cells are ruptured, but though they still retain stil much of their purple fluid contents, no aggregation occurs in them when they are similarly immersed, notwithstanding that although in closely adjoining cells which have not been killed, as could be seen by in which the protoplasm may be seen still flowing round the walls, aggregation ensued. So that the process is quite arrested by the death of a cell, & it is much delayed if a leaf, before being immersed in the solution is kept for some time in carbonic acid; and it is this agrees with the well-known fact that protoplasm in cells moves retains its activity spontaneously only as long as it is in an oxygenated condition. (a) From these series [text pasted over]

When tentacles, including recently aggregated masses are suddenly killed or much injured by being dipped into hot water or by being irrigated with alcohol, acetic acid or a solution of iodine, the aggregated masses suddenly disintegrate and disappear, leaving only a little fine

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suddenly disintegrate and disappear, leaving only a little fine granular matter; but this disintegration does not occur with the more solid masses which have been aggregated for some time.)

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(From the several foregoing considerations, a from the aggregated masses being of an albuminoid nature as shown by the tests employed by my son Francis and as is admitted by Pfeffer* (* Pflanzenphysiologie B ii p 248)

— & from their incessant, long-continued amœba-like movements of the aggregated masses, I originally concluded that not only these masses, but that the minute globules which first appear in the cell-sap consist, at least in part, of living and spontaneously moving protoplasm. And I feel compelled to adhere to my original conclusion, notwithstanding that such high authorities as Cohn and Pfeffer believe that the aggregated masses consist merely of

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condensed cell-sap. Their movements of the masses, I presume, are considered by them to be of the same nature as those curious ones described by Beneke as occurring in myelin when immersed in water and in a solution of sugar* (* Archiv für wiss: Heilkunde Studien über das Vorkommen…von Gallenbestandtheil' (Giessen, 1862))

From the doubts thus thrown upon my original conclusion, it seemed to me advisable to observe the action of carbonate of ammonia on grains of chlorophyll, as every one it is generally admitted that these consist of living modified protoplasm. They not only change their positions under certain circumstances, which may be due merely to the movements of the streaming protoplasm in which they are embedded, but they likewise have the power of changing their shapes as has been recently proved to be the case by Stahl* (* See his interesting papers in the Botanische Zeitung 1880 p 298- p 413; and more especially p 361) They are, also, capable of self-division** Now if it can be shown that a solution of carbonate of ammonia tends to cause the grains of living chlorophyll to become

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** Van Tieghem, Traité de Botanique 1882 p. 493.

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confluent, a one with another and with previously aggregated masses, without killing them, this fact would support the conclusion that such the aggregated masses consist at least in part of living protoplasm, to which their incessant movements may be attributed. And it is the object if the present paper to show that chlorophyll bodies in certain cases are thus acted on by carbonate of ammonia. The fact by itself possesses some little interest, independently of the light thus which it thus throws on the remarkable process phenomenon of aggregation.)

Dionæa muscipula. — The effects of carbonate of ammonia is are best seen with shown in the case of young, small, and thin leaves produced by starved plants, as such seen to be quickly penetrated by the solution. Transverse slices sections of these such leaves and of others befor were made before they had been immersed, in the solution, and the cel cells, including those of the epidermis, could easily be seen to be packed with grains of

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chlorophyll. It is however necessary to avoid examining a leaf which has ever caught an insect; for in this case many of the cells will be found filled with aggregated yellowish matter instead of with chlorophyll grains. Several leaves were left for different lengths of time in solutions of different strengths, but it will suffice to describe a few cases. A small thin leaf was immersed for 24 hrs in a solution of 7 parts of the carbonate to 1000 of water, and transverse sections were then examined. The c cells near the margin of the leaf, throughout the its whole thickness did not now contain exhibit a single chlorophyll-grain, but in their place masses of transparent yellowish-green matter of the most diversified shapes. They resembled those of Drosera shown at fig 3, if we suppose seral several of them to be pressed lightly together. Some of the masses in the same cell were connected together by extremely fine threads. Spheres

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of more solid matter were sometimes included within the oddly shaped greenish masses. The contrast in appearance between these sections and those taken from one corner of the same leaf before it had been immersed was wonderfully great. The sections were then clarified by being left for some time in alcohol, but not a grain of chlorophyll could be seen; whereas the fresh slices similarly clarified exhibited with the utmost plainness the now colourless grains. The oddly-shaped green masses exhibited none of the movements, as is so conspicuous in the case with those in of Drosera, but this could hardly have been expected after the injury caused by slicing; and the leaves are much too opaque to be examined without the aid of sections. Some other sections from the same immersed leaf presented a rather different appearance, as they contained much extremely fine granular green matter, which became pale brown after being kept in alcohol. No chlorophyll grains could be

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seen in any of these sections. After adding iodine (dissolved in water with iodide of potassium) many particles of starch became visible by being coloured blue; but none were present in the first-described section. Some of the larger rounded aggregated masses were coated with blue particles. Others were quite free of such particles, and were turned yellow coloured by the iodine bright orange.)

(A superficial slice was taken from a fresh leaf, showing the upper epidermic and glandular surface, and all the cells abounded with large grains of chlorophyll; but with a leaf which had been immersed for 24 hrs in a solution (7 to 1000) of C of ammonia (7 to 1000) a similar section presented a wonderfully different aspect.

No chlorophyll-grains could be seen. Some of the cells contained one or two transparent yellowish spheres, which it could not hardly be doubted were had been formed by the fusing together of the fusion of previously existing chlorophyll-grains. Other cells contained very fine brownish granular matter; and this apparently had been deposited

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from the cell-sap with its colour changed. This granular matter was generally aggregated into one or two either separate distinct or more or less confluent spherical balls, having a rough surface. Sometimes a dark-brown granular sphere was surrounded by a zone of paler granular matter. In other cells brown granular spheres lay in the centre of transparent yellow spheres. In one case there were in the same cell a sphere of this latter kind, with two others consisting exclusively of the yellowish transparent matter were observed in the same cell. In other cases the brown balls were merely surrounded by an extremely very narrow border or zone of transparent matter. It appears that the granular matter is first deposited and that then had become more or less aggregated into spheres balls; and that afterwards the yellowish transparent matter, formed by the fusing together fusion of the modified chlorophyll grains, aggregates either round the granular matter or into independent spherical and oddly shaped

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masses.)

(Transverse sections of other immersed leaves presented various appearances. In one case a central transparent sphere was surrounded by a halo of brown granular matter, and this again by a zone of the transparent matter. Such matter quite filled some adjoining cells. In the cells of another leaf there were throughout its whole thickness, yellow, greenish, orange, and pale or very dark brown spheres. Some of these latter spheres had a dark centre, which as in the case of Drosera was so hard that it could be was cracked by pressure, and was distinctly the line of separation from the surrounding zone of paler matter was distinct. Two brown spheres were in one case included in within the same transparent sphere. Gradations seemed to show that the opake granular matter ultimately passed into dark coloured transparent matter. In these same sections there were some colourless or yellowish highly transparent spherical bodies spheres

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which, I believe, were merely much swollen chlorophyll-grains. One, two, or more of these sometimes clung such grains spheres grains and while still partly retaining their outlines sometimes clung to the darker granular spheres; when there were only one or two oth of them thus clinging to a sphere they assumed the sp shapes of half or quarter-moons, and It seemed that appeared as if such swollen grains when completely confluent had often hand generally given rise to the pale zones surrounding the dark centres thus originated. Thes Such The pale zones were rendered still more transparent by acetic acid. and one one occasion they quite disappeared after being left in the acid for 24 hrs; but whether the matter was dissolved or had merely disintegrated was not ascertained. We shall afterwards find that This acid produces the same effect on recently aggregated, and pale-coloured or almost colourless matter in the tentacles of Drosera.)

(In some cells In one leaf a good many unaltered chlorophyll grains could still be distinguished in some of the cells; and this occurred more frequently in the thickest part of the leaf, near the midrib, than elsewhere.

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In one section the chlorophyll grains had here run together, and formed in some of the cells formed a narrow green rims round the all four walls. In many sections, more especially in those in which the process of aggregation had not been carried very far, there was much extremely fine granular matter, which did not resemble smashed or disintegrated chlorophyll grains, such as may often be seen in ordi sections of ordinary leaves. This granular matter occasionally passed into excessively minute, transparent, more or less confluent globules. which tend to aggregate.

Finally We may therefore conclude that carbonate of ammonia first acts on the cell-sap producing a granular deposit of a pale brownish colour, and that this tends to aggregates into balls; that afterwards the grains of chlorophyll are acted on, some merely swelling up and becoming completely confluent, so that no trace of their original structure is left, and others breaking up into extremely fine greenish granular matter which appears likewise to undergo aggregation. The final result is the formation of rounded or oddly shaped masses balls of brown & sometimes reddish granular matter, often surrounded by zones, more or

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thick, of yellowish or greenish or almost colourless transparent matter. Or again spheres, ovals & oddly-shaped masses are formed, consisting exclusively of this transparent yellowish-green matter. of transparent zones surrounding the brown and sometimes reddish granular matter. As soon as the processes of aggregation has been thoroughly carried out, not a single grain of chlorophyll grain is left can be seen.)

(Drosera rotundifolia. — It is advisable to select for observation pale reddish leaves, as the dark red ones are too opaque; and the process of aggregation does not go on well in the small completely green leaves which may sometimes be found. The tentacles, which are merely delicate prolongations of the leaf, are from their transparency better well fitted from for observation. after being immersion in a solution of carbonate of ammonia, of which the best strength is 4 parts to a 1000 of water. In sections of the discs of fresh leaves, the cells of the epidermis are seen to abound with grains of chlorophyll, as well as those of the underlying parenchyma. The bases of the exterior tentacles and the part immediately

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bef beneath the glands are generally coloured pale green from the presence of chlorophyll grains in the parenchyma; and some occur throughout the whole length of the longer tentacles, but are not so easily seen on account of the purple cell-sap. Sometimes the epidermic cells of the longer tentacles include chlorophyll grains; but this is a * variable and rather a rare event. The footstalks of the short tentacles on the disc are bright green, and invariably abound with grains of chlorophyll.)

(A pale leaf, in which the other outer basal cells of the exterior tentacles two of which towards the base contained numerous grains of chlorophyll were seen, was left for 24 hrs in a solution of only 2 parts of the carbonate to 1000 of water; and now innumerable greenish spheres, resembling oil in appearance, were seen in act of present in these cells at the bases of the outer tentacle, and the ordinary chlorophyll grains had disappeared. Nevertheless in several cells some swollen grains were still distinct. Other

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cells contained fine granular or pulpy green matter collected into a f masses at one end. In a few other cells the chlorophyll grains had run together and forming a perfectly continuous green rim with a sinuous outline, attached to the walls. In fresh leaves the guard cells of the stomata include grains of chlorophyll, and these after the ammonia after the leaf has been immersed in the carbonate, these almost always become fused into a few almost nearly colourless spheres.)

(Sections of made from leaves which had been left for 22 hrs in a solution of 4 to 1000, exhibited in the upper and lower epidermic cells of the disc, and in the cells of the parenchyma cells near the bases of the oth outer exterior tentacles, greenish spheres; and in such cells there were no chlorophyll grains, but they were still present in some few of the epidermal cells which did not contain aggregated masses, and they abounded in the parenchyma in the middle of the disc, where there were only a few green spheres. These sections were irrigated with the solution of iodine, and the green spheres became yellow; and and many minute

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elliptical particles of starch, were coloured blue, could now be seen. Such particles were not visible in the sections of fresh leaves, and I believe that they had been embedded in within the chlorophyll grains, from which the enveloping protoplasm had been withdrawn to form the green spheres.)

(One of the above leaves was left in the ammonia solution for 3 days, and by which time it had become flaccid, being evidently killed. The numerous green spheres were now blackened but perfectly retained their outlines. No chlorophyll grains could now be seen in the cells but many particles of starch. When leaves were left for some time in a solution of 7 to 1000, much pulpy green matter and innumerable spheres were sometimes formed, but no large aggregated masses; so that in these cases the solution appeared to have been too strong. The degree to which the grains of chlorophyll are acted on varies much from unknown causes, for in some specimens left for 24 or 36 hrs in the stronger solution, the grains the grains could still be plainly seen in the tentacles but only after they had been when contents although they exhibited strongly marked aggregation,

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cleared by immersion in acetic acid.) although they exhibited strongly marked aggregation.)

(A leaf was laid on a glass plate kept in a damp chamber, and two or three tentacles at one end were thus covered with thin glass so as to prevent their bending, & were irrigated with the ammonia solution of 7 to 1000. After 24 hrs and 48 hrs these were these tentacles included many dark purple aggregated masses; in the tentacles, but nevertheless plenty of chlorophyll grains were still visible.

In the disc of this leaf, however at however, near the bases of these tentacles, their there were some aggregated spheres of a fine green tint, and others purple in the centre surrounded by a distinctly defined green zone; & in most of the cells containing these spherical aggregated masses spheres not a grain of chlorophyll could be distinguished. That the green surrounding zones had been derived from the chlorophyll grains is I think certain, for the purple colour of the central spheres showed that the cell-sap contents had not been originally green. In most of these cells not a grain of chlorophyll could be distinguished. Other cells in these same sections included irregularly-shaped masses were of a purplish-green colour, and these were observed

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slowly to change their forms in the usual manner. When acetic acid was added to them sections, the green transparent spheres and the zones of similar green matter round the purple spheres instantly disappeared, either from being dissolved or, as seems more probable from being killed and suddenly disintegrating. by being killed; for On another cases occasion boiling water and alcohol produced a similar the same effect on another occasion on the spheres. Tentacles still retaining their chlorophyll grains, but with many very pale-coloured homogeneous aggregated masses (such masses have often been being (which were seen in movement), were irrigated with acetic acid; and it was curious to observe how instantaneously they aggregated masses became became homogenous filled with small transparent spheres. Soon In a short time, however, the outlines of the larger masses were alone left; then these disappeared, and finally the small enclosed spheres. On the other hand, Ddark-coloured solid aggregated spherical masses did not disappear when left for 24 hrs in the acetic acid.)

(The effects of the ammonia solution (4 and 7 to 1000) on the

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upper epidermal cells of the upper surface of the disc was now more especially observed. In some cases all these cells, which as already stated always invariably contain many chlorophyll grains, now included after the several immersion in the solution only one or several several green transparent spheres; but more commonly the spheres were very dark purple or brown. Sometimes the centre a central sphere which was so solid that it could be cracked consisted of a still sphere dark than the circumferential part— was surrounded by a well-defined paler zone. Numerous gradations could be traced, showing that several small spheres and irregularly sp shaped globules often coalesce so as & thus form the larger spheres rounded masses. It was repeatedly observed that when the epidermic cells contained only one or two large spheres, not a single grain of chlorophyll could be seen. It is surprising that dark purple or brown or almost black spheres should be formed as was often the case, in the epidermic cells of very green leaves; for these cells d/ before immersion the cell contents were colourless, with the exception of the chlorophyll grains; but the fact is less surprising

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when it is known that these cells turn more or less red as they grow old & if they are exposed to a bright light. In some of these leaves the basal cells of the longer exterior tentacles had become beautifully clear transparent from the aggregation of their contents into green or greenish-purple masses; and here no chlorophyll grains could be seen; but in other parts of the same tentacles, where the aggregated masses were of a fine purple tint, the chlorophyll grains w could were still x be seen. plainly visible.)

(Finally, it appears certain that in the leaves of Drosera the grains of chlorophyll if left long enough in a weak solution of the carbonate sometimes break up and form translucent translucent greenish globules, which are much smaller than the original chlorophyll-grains, and that these, by coalescing form larger masses, which again coalesce into one or a few spheres or into a single one. In other cases the chlorophyll-grains swell and coalesce without having previously broken up into globules. During

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these various changes the aggregated masses may be often become coloured by the modified cell-sap, as often occurs more especially in the case of the epidermic cells; or they may surround form a zone round the already aggregated cell-sap, in which case a dark central sphere is surrounded by a less dark or by by a light green transparent zone of matter.

It remains to be considered whether the grains of chlorophyll, after their complete fusion or aggregation, are ever reformed and reassume their normal positions on the walls of the cells. Although the purple aggregated masses within the tentacles are after so long time soon redissolved, the cells being thin becoming refilled with transparent purple fluid, it does not by no means follows that the chlorophyll-grains should be reformed; & such a capacity would be an interesting point. To ascertain whether this occurred drops of a weak solution of C. of ammonia (2 to 1000) were daily placed during 5 days on several leaves on a growing plant; but to my surprise, the tentacles remained after the first day expanded, with their glands bright red and copiously secreting, & they exhibited little aggregation.

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Large drops of a solution of 4 to 1000 were now next placed on three reddish leaves, fresh drops being added in about 18 hrs. After an interval of 41 half hrs 41 1/2 hrs half h hrs from the time when the drops were first given placed on the leaves, three short central tentacles on one leaf were examined, and the cells were seen to be filled with quickly-moving aggregated masses, and not one grain of chlorophyll could be distinguished.

In 66 hrs after the drops had been given the leaves were well syringed with water; and now the central tentacles of a second leaf were examined, in these which there was much aggregated matter and no chlorophyll grains. A third leaf was examined 5 days after the drops had been given, and the aggregated masses appeared to be now breaking up into small highly transparent spheres. In cells at the bases of & In two, however, of the short central tentacles of this leaf the cells at their bases contained no aggregated matter and plenty of chlorophyll-grains. It is therefore probable that

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if these tentacles had been examined two or three days earlier, an opposite state of things would have prevailed. In a third central tentacle from this same leaf there was still much aggregated matter in the basal cells, and here some a few irregularly shaped chlorophyll grains could be seen. Some of the aggregated masses In other tentacles from this same leaf, and from the two other two leaves which had been similarly immersed treated, some of the aggregated masses had become granular, discoloured, and opaque; and this indicates that the solution had either been too strong or that too large a quantity had been given.) (Drops of a strong filtered solution of raw meat were next tried and now placed on 7 reddish leaves, the tentacles of which all became much inflected with and their glands blackened. After 22 1/2 hrs they were syringed with water, and one leaf was cut off for examination. The contents of 5 short central tentacles from this leaf were aggregated down to their bases, and not a grain of chlorophyll

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could be seen. Some of the aggregated masses were almost white with a faint tinge of green, and were moving quickly. In the long exterior tentacles which had not at first been touched by the infusion, that is not until they had become inflected, the aggregation had not as yet travelled down to the basal cells; and here the grains of chlorophyll were quite distinct. The infusion was too strong for after 5 days, one leaf out of the six remaining leaves was dead; was killed; two others were much injured, with the outer tentacles killed, those on the disc, though longer immersed for a longer time, being still alive; the fourth leaf was considerably injured; the fifth and sixth looked fresh & vigorous, with their glands now of a red colour secreting freely. Five of the short central tentacles from one of these latter leaves were now (i.e after the 5 days) examined: in three of them there was only a trace of aggregation was left & plenty of chlorophyll grains could be seen; in a fourth tentacle there were still some aggregated masses & some only a few chlorophyll-grains; in a fifth there were many aggregated masses and some fine granular matter

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& here no chlorophyll-grains were distinguishable. There can hardly be a doubt that in 4 out of these 5 tentacles the chlorophyll-grains had been reformed. The central tentacles On one of the much-injured leaves, presented a very different appearance; their glands the glands of the central tentacles were still opake, & the cells of in their foot-stalks, contained some aggregated & some brownish granular matter; & here very minute globules, could be seem just were arranged along the walls of the cells in the places where chlorophyll-grains ought to stand have stood; but whether these were remnants which had never wholly disappeared or new grains reforming could not be ascertained.

Drops of a weaker infusion of raw meat were next placed of on 7 reddish leaves, which were all greatly acted acted on; but the infusion was still rather too strong. In from 24 to 25 hrs afterwards all the leaves were well syringed; & small pieces were having been cut off two of them, & several of the short short central tentacles were examined. In one of these leaves ar a very few chlorophyll-grains could be seen in some few cells  d/ [illeg] d/ only in one of the tentacles which had not undergone so much aggregation

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as the others. In the piece from the second leaf not a single chlorophyll grain could be distinguished in any of the short central tentacles.

The sections were then immersed in alcohol, & in a few minutes all the aggregated masses were broken up into very very fine granular matter; but no chlorophyll-grains could be distinguished seen, except in the one tentacle above mentioned, tentacle.

In three days after the drops had been first given, process for four of the leaves (including one of those from which a small piece had been previously cut off) & looked vigorous, & were fully or almost fully expanded. The fifth leaf, from which a piece had likewise been cut off appeared somewhat injured. The sixth had its tentacles still inflected & seemed more much injured. The sixth was apparently almost dead.

Four of the central tentacles on the vigorous leaf from which a piece had been cut off after 24 hrs, were now (i.e. on the third day after the drop was had been given) was examined. In most of the basal cells of three of these tentacles, these three were only a trace of aggregation was left, & in most of the basal cells, & in these many many chlorophyll-grains could be seen in them; but these were not so regular in

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shape or so regularly placed as are the normal grains; as is normally the case, & an the normal grains; so that I presume they they they were in the act of reforming. Two basal cells in one of these short tentacles still contained large, quickly moving aggregated masses, & in these & not a grain of chlorophyll could be distinguished in them. When this section was irrigated with the solution of iodine, the aggregated masses in the 2 just mentioned cells instantly broke up into brownish granular matter, & the irregular & as I suppose just reformed chlorophyll-grains in the adjoining cells ran together & became confluent, forming forming narrow rims along the walls.

After intervals of, 4, 6, and 8 days from the time when the drops were given, 15 central tentacles on 3 of the leaves were examined; & in all these tentacles of these tentacles, excepting one in which there was still much aggregated matter, chlorophyll-grains could be seen. After 11 days the first mentioned leaf, the central tentacles on which in the cut-off piece one of the leaves, from which a small piece had been cut off after an interval of 24 hrs, & in which most of the central tentacles then included no chlorophyll-grains, 24 hr. after the infusion was given, was now reexamined. The central tentacles appeared perfectly healthy & were

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secreting; in 8 out of 10 of them, there were numerous the cells included chlorophyll-grains having the usual appearance; in the cells of th other 2 tentacles there was still much aggregated matter & no ordinary chlorophyll-grains chlorophyll-grains, but only a some few irregularly shaped chlorophyll-grains. ones could be seen. With respect to the second leaf from which a small piece had been cut off, & in which the central tentacles did not then (i.e. after 24 hr) contain a single chlorophyll-grain, only a very few of the central tentacles now (i.e. after 11 days) appeared healthy; but in two of them, which appeared quite uninjured, there were innumerable perfect chlorophyll-grains in all the cells from the glands down to the base.

(Considering the whole of the evidence here here given, there can hardly be a doubt that with the leaves of Drosera as soon as the aggregated masses break up & even before they are wholly redissolved wholly redissolved, grains of chlorophyll are reformed.)

Drosophyllum lusitanicum. — The footstalks of the tentacles are bright green, from the large number

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of chlorophyll-grains which they contain. Two leaves were immersed an in a solution of C. of ammonia (4 to 1000) for 23 and 24 hrs, and the cells of the foot-stalks now contained innumerable spheres, some much smaller & some much larger than the grains of chlorophyll, and some of large size, and other oddly-shaped masses, more or less confluent, of translucent bright yellow matter, which when irrigated with absolute alcohol, instantly broke up into fine granular matter. I looked in vain fo in several of these tentacles for grains of chlorophyll. Another leaf was immersed for only 16 1/2 hrs in a weaker solution of 2 to 1000, but this sufficed to produce an abundance of yellow translucent bodies, these which were seen to change their forms greatly though slowly. In many, but not in all, of the cells of this leaf the grains of chlorophyll were still quite distinct. The several leaves were left both in the stronger and weaker solutions for

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48 hrs; and now this caused the yellow spheres and masses were to disintegrated into brownish granular matter. In this respect, that is in this In the quickness of which these changes, the aggregated masses in the tentacle of Drosophyllum differ from those in Drosera and Dionæa.

Leaves were also left for 24 and 48 hrs in an infusion of raw meat, but no yellow aggregated masses were thus produced, and the grains of chlorophyll were remained perfectly distinct. This singular difference in the action of the infusion of raw meat on the tentacles, as compared with those of Drosera, may perhaps be accounted for by their serving in Drosophyllum almost exclusively for the secretion of the viscid fluid by which captures the insects are captured; the power of digestion and of absorption being chiefly confined to, as I have explained in my Insectivorous Plants (p 332–342), to the minute sessile glands on the discs of the leaves.)

(one line open)

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As in the three foregoing genera the grains of chlorophyll tended to aggregate into a moving masses under the long-continued influence of a weak solution of carbonate of ammonia, I thought that this might be an attribute of they would probably be similarly acted on in all insectivorous plants; but such this did not prove to be the case. The immersion of leaves of the common Pinguicula in such a they a solution of the ammonia and in an infusion of raw meat did not cause any aggregation of the chlorophyll-grains, though numerous transparent spheres were formed within the glandular hairs. Again, the immersion in carbonate of ammonia of pieces of young and old pitchers of a Nepenthes (garden hybrid var) caused the appearance of innumerable more or less confluent spheres of various sizes; and in the glands on the inner surface of the pitcher and in the exterior epidermic cells. These were formed of translucent matter or either almost colourless or of a brown, orange, purple, or greenish tint; but the grains of chlorophyll  

35

were not specially acted on.)

(Sarracenia purpurea. —This plant which x d/ has not the power of digestion, but is evidently adapted for catching and drowning insects, and as is believed for absorbing matter from their decaying remains, x was next tried. Many observations on the pitchers of Sarracenia were made, but one case will suffice. A piece of a pitcher was left for 24 hrs in a solution of 4 parts of the carbonate of ammonia to a 1000 of water, and for 24 additional hrs in a solution of 7 to 1000. and was then examined. In the cells of the parenchyma, especially in those close to the vascular bundles, there were many spheres and aggregated masses, of bright orange transparent matter. Spheres of the same and of various other tints were present in the epidermic cells, more especially in those on the inner surface of the pitcher; and some of these spheres were of exactly the same pale greenish colour as the swollen chlorophyll-grains which

36

were still present in some places, and were being often collected together into rounded masses. In many of the epidermic cells which contained spheres no chlorophyll-grains could be seen, though they were abundantly present in these cells in sections epidermis of fresh leaves; and its it is this fact which chiefly leads me to believe that the chlorophyll grains sometimes become so completely fused together as to form spheres; being often blended with the aggregated & coloured cell-sap. When the a solution of iodine was added to these sections, the pale coloured spheres and irregularly shaped mass aggregated masses became bright orange, and they were sometimes sprinkled over with blue particles of starch. The iodine did not cause their immediate disintegration of and disappearance of the orange masses; nor did alcohol or acetic acid. In this respect they differed from the recently aggregated masses in Drosera; though in this latter plant the

37

older and more solid aggregated masses were anot thus acted on by these reagents. (a) In some cases the acetic acid sometimes caused the green granular matter which was present in many cells, being being formed by broken or disintegrated either by the chlorophyll grains having been mechanically broken down or by their disintegration instantly to turn of the same orange tint, as the aggregated masses.)

(The orange spheres and aggregated variously shaped masses were seen in many sections of pitchers which had been exposed for different lengths of time to solutions of the carbonate of different strengths; and in many of them swollen grains of chlorophyll had a become more or less confluent. Their original nature of the latter could be recognised being shown by the sinuous outlines and greenish tint. of the rounded masses. They were not seen to change their shapes spontaneously; but this could not have been expected in sections.

Portions of a pitcher, left in distilled water for nearly 3 days did not exhibit a single orange sphere or aggregated mass; but there were some colourless oil globules which were dissolved by alcohol;

[37v]

(a) (text) p 37

Many of the cells contained green granular matter, formed either by the chlorophyll grains having been mechanically mechanically smashed or by their disintegration; & this acetic acid sometimes caused this granular matter to change at once instantly into the same orange tint as that of the aggregated masses.)

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and the chlorophyll grains, though generally much swollen, were still distinct. It may therefore be concluded that in Sarracenia the chlorophyll grains often undergo aggregation under the influence of carbonate of ammonia; but that they are less easily acted on than those of Dionæa and Drosera.)

(Leaves with glandular hairs & other leaves. — I had formerly observed, as described in my 'Insectivorous Plants,' that the glandular hairs of some species plants absorbed carbonate of ammonia and animal matter, and that aggregation was is thus produced caused in them. Consequently such leaves and others without hairs were immersed in solutions of C. of ammonia (4 and 7 to 1000) generally for 24 hrs. No marked effect was produced was produced on the chlorophyll grains, with the exception of excepting their occasional displacement, was produced in certain cases, in These observations were made on the following plants, These which belong to different families and were selected almost

39

by hazard. Namely of leaves not bearing many or any glandular hairs, those of Brassica, Fumaria, Fuchsia, Robinia, Oxalis, Tropæolum, Euphorbia, Stapelia, Beta, Allium, Lemna, a fern (Nephrodium), a n Marchantia, and a moss. Nor were the grains acted on in two species of Saxifrag Saxifraga (except on one occasion, when they formed masses, shaped like a horse-shoe, presently to be described), nor in Primula Sinensis; although the leaves of these two species plants are clothed with glandular hairs, which absorb c of ammonia and undergo aggregation. Young leaves of Dipsacus sylvestris were immersed for 24 hrs in a solution of 7 to 1000, and large yellowish hy highly refracting spheres were formed in the upper epidermic cells which do not include any chlorophyll grains; and the grains were not at all aggregated in other parts of the leaf. When the sections were irrigated with acetic acid or with alcohol, the spheres in the epidermic cells disappeared quickly, in nearly the same manner as occurs with recently aggregated

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masses in the cells of Drosera.

Leaves of Cyclamen Persicum, which do not bear hardly any glandular hairs were left in a solution of 7 to 1000 for 43 hrs, and this caused the chlorophyll grains to collect into heaps; in some parts with their the grains retained outlines outlines x of the grains were distinct, but in other parts they formed perfectly homogeneous bright green masses of the shape of a horseshoe. These were cleared by alcohol; and it became was evident that the grains had become completely fused together. The leaves were left for 24 additional hrs in the solution, and now the horseshoe masses disappeared, being converted into pulpy matter. The immersion of the leaves of this Cyclamen in water for 47 hrs caused the chlorophyll grains to accumulate into heaps, as is known to follow from any injury; but there was hardly a trace of their confluence.

(a) (text) Portions cut from a leaf of Mirabilis jalapa were left for 16 1/2 hours in solutions of 4 and of 7 to 1000, and the chlorophyll-grains in

[40v]

(a) (text)

Similar horse-shoe masses were seen, but only on one occasion, in the leaves of Nicotiana tabacum, after their immersion in the solution; & so it was with the grains in the stems of Euphorbia peplus.

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many of the cells became perfectly completely confluent, forming horseshoe masses or complete rings; and they were sufficiently solid to project when the cells were torn open. When these were irrigated with acetic acid they became so transparent that even their outlines could hardly be distinguished. If in these plants & more especially in Cyclamen and Mirabilis, the confluent chlorophyll-grains, forming the horse-shoe masses, are still alive and this is rendered probable by their bright green colour, and in the former plant by their breaking up when kept for an additional day in the solution, and in the latter plant by the action of acetic acid on them, we have in these cases a first step towar in the process which in some plants leads to the formation of spontaneously moving masses suspended lying free in the cell-sap.)

Pelargonium zonale. — The leaves are clothed with glandular hairs which absorb carbonate of

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ammonia and undergo aggregation. The effects produced by their immersion of the leaves of this plant for 24 or 48 hrs in solutions of 4 or 7 parts of C. of ammonia to 1000 of water are not a little perplexing. They are clothed with glandular hairs which absorb the ammonia & undergo aggregation. In nu Moreover numerous almost colourless shining translucent spheres generally, but not invariably, appear in most of the epidermic cells, in which there are no grains of chlorophyll and in the palissade cells, in which they abound, with chlorophyll, and likewise in the parenchyma. The smaller spheres blend together and thus form large ones. A solution of only 2 to 1000 sometimes sufficed to produce the spheres. Usually the spheres are not acted on by alcohol, but occasionally they were dissolved by it. If after immersion in alcohol they were are subjected to the iodine solution, they soon almost disappeared but this does not invariably occur. Acetic acid always caused their rapid disappearance & without any apparent effervescence, a slight granular residue being

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sometimes left; and this occurred with leaves which had been left kept so long in the solution that they were dead. The acid dissolved, of course with effervescence, the crystalline balls of carbonate of lime which occupy many of the palissade-cells. When sulphuric ether was added the smaller spheres of transparent matter disappeared in the course of a few minutes; while the larger ones became brownish & granular and brown in their centres; but this granular matter disappeared after a time, and empty transparent bag-like membranes being left. Traces of a similar membranes could sometimes be detected after the administration of acetic acid. Caustic potash did not act very quickly on the spheres but sometimes caused them to swell up. I do not know what to ought to be inferred from the action of from the action of these several reagents with respect to the nature of on the spheres and aggregated masses, wh (in which I never saw any movement.))

(With leaves after immersed ion in the solution that

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palissade cells On two or three occasions the palissade cells of leaves which had been immersed in the solutions were gorged with innumerable, often irregularly shaped, more or less confluent, globules, many of them being being much smaller than the chlorophyll grains, instead of containing the large transparent spheres. This occurred with a leaf which had been immersed for only 18 1/2 hrs in a solution of 4 to 1000. After sections of this leaf had been cleared with alcohol, it was irrigated with the solution of iodine, and the globules rapidly ran together and or became confluent, forming irregular amorphous masses.)

(It was difficult to ascertain whether the grains of chlorophyll-grains if ever or often became blended with other matter, so as to & thus aided in the forming the transparent spheres. The difficulty was partly due to the grains being easily acted on by water. Thus, in some cases in sections made and placed in water and then cleared in alcohol, no grains could sometimes d/ be distinguished; while they were distinct in sections of the same leaf

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if which had not been wetted before being placed in alcohol.

Many grains were also found in a disintegrated condition in x meat uninjured leaves which had been kept for 47 hrs in water before the sections were made at. It may be here added that not a single sphere could be seen in these leaves. Nor were they present in leaves slightly injured by being kept for 24 hrs in a very weak solution of osmic acid. Nor again in a leaf which had been immersed in an infusion of raw meat for 24 and for 50 hrs; and in this leaf the chlorophyll-grains were still visible in many places, being but were sometimes heaped together. not Notwithstanding the difficulty owing to the action of water in the chlorophyll-grains in observing ascertaining the effects of on them of C. of ammonia on the chlorophyll-grains, chiefly owing to the action of water on them yet I am led to believe from the gradations which could be followed, and from the absence of chlorophyll grains in the cells in which where one or two large spheres were present, in a cell, that in the case of the palissade and

46

parenchyma cells, granular matter produced by the disintegration of the grains first aggregates, together with other matter derived from the cell-sap, first into minute globules, and that these aggregate into the larger spheres. I will give a single instance: a leaf was immersed for 22 1/2 hours in a solution of the carbonate of 4 to 1000 and sections after being cleared in alcohol exhibited in many places distinct chlorophyll grains, and in other places only very fine granular matter, and in a f very few cells minute transparent spheres globules. The leaf was left for 24 additional hrs in the solution; and now sections cleared in alcohol exhibited numerous minute shining translucent spheres globules, many of which were smaller than the few still remaining chlorophyll-grains; and there were also other much larger transparent spheres, more or less confluent, which when irrigated with acetic acid, instantly disappeared.)

(A leaf was immersed in a solution of 4 parts

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phosphate of ammonia to 1000 of water, and after 23 hrs there was no trace of aggregation; it was left for 24 1/2 additional hours in the solution; & now sections cleared in alcohol exhibited not only minute spining shining colourless spheres globules, smaller than the few still remaining chlorophyll-grains, but plenty of large spheres, more or less aggregated together; and in the cells with containing such spheres no chlorophyll-grains could be seen. The spheres, both large and small, disappeared instantly when acetic acid was added, as in the case of those produced by the carbonate. It appears therefore that these two salts act in the same manner, but that the phosphate acts more slowly than the carbonate, as is likewise occurs the case with Drosera. A leaf immersed for 45 hrs in a solution of 2 pts of nitrate of ammonia to 1000 of water was a good deal infiltrated and darkened in colour, but no spheres were formed; but some of the chlorophyll-grains had, however, become

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confluent while still adhering to the walls of the cells.

Spirogyra (crassa?). — When filaments of this Alga were placed in a solution of carbonate of ammonia (4 to 1000), the cell-sap became in a few minutes cloudy from the formation of innumerable granules; and the green spiral band of chlorophyll-band soon soon began to contract. A filament was irrigated with under a cover-glass at 11.10 am (Oct 4th) with the solution; and by 11.25 the cell sap had everywhere become granular; two of the cells the pointed ends of the chlorophyll-band and the irregular lateral projections were retracted, so that these bands now appeared much smoother and blunter than before.

In two neighbouring cells the bands now formed had become converted rounded circular or spherical into circular rounded masses round about surrounding the nuclei.) (At 12.50 two cells were selected for further & special observation; in one of them the original spiral band now formed a layer of nearly

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uniform thickness, except in three of the corners where there were rounded lumps, which adhered closely to the two transverse and to one of the longitudinal walls of the cell. with rounded lumps in three of the corners

By 4 pm the layer on the longitudinal wall had become in the middle so thin that it consisted of a mere thread, which at 4.15 broke and disappeared; the upper part end (with reference to the observer) of the layer then rapidly contracted into a pear-shaped mass. The layer at the bottom lower end of the cell had by this time assumed a dumb-bell shape, which, however, soon afterwards became expanded into cylindrical. At 7.10 pm the appearance of the cell was utterly different; for there were now at the upper end two ill-defined masses, and at the lower end two somewhat irregular balls of green matter connected together by a thin band. At 8 am on the following morning there was a large oval mass lying obliquely across the upper end of the cell, with its two quads extremities connected by bands with two spheres in the lower corners. The changes in the

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other cell, which was observed at the same time which were almost equally great. The spiral band was first converted into a two layers, lining the lower and upper both the transverse walls, & these which were connected together by a sinuous longitudinal band. At 4 pm there was in one of the x upper corners a large pear-shaped mass, which I saw contracted while it was watched into an oval mass; and at the opposite lower corner a small dark green sphere. By 7.10 pm there were two spherical masses and an one oval one mass, which latter by the next morning formed had changed into a straight a much elongated straight band; mass together with viz instead of two there was now only a single separate sphere. At this latter date same time two adjoining cells included 4 and 5 oval or spherical chlorophyll-balls; but a one cell still retained a spiral-band. Alcohol and acetic acid produced only the same clarifying effect on these masses, as in the case of ordinary chlorophyll-grains.)

(Filaments of this alga were left for 26 hrs in a solution of only one pt part of the carbonate to 1000 of water; but

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this sufficed to cause some granular deposition in the cell-sap, and many of the cells included, instead of the spiral band, spherical or oval or pear-shaped masses, and in one instance a half-moon-shaped mass, many of these masses were often connected together by the finest threads of green matter, one of which was seen to break, and the pear-shaped mass quickly became almost spherical. this (a) (text) The above weak solution seemed to be favourable to the health of the plants, for after six days immersion they looked greener and more vigorous than the other plants of the same lot which had been kept in plain water. The cell-sap still contained brownish granular matter, and many of the cells oval and or spherical masses.)

(With respect to) The brownish granular matter thus is always precipitated quickly; and when three young cells, which were as transparent as glass, were irrigated with a solution of 7 to 1000, the precipitation

[51v]

(a) text

The man changes of form & movements of the chlorophyll in the foregoing several cases, under the influence of the solutions closely resemble in most respects those which may be seen within the tentacles of Drosera.

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seemed to be instantaneous. After a time the granules are either deposited on the protoplasm lining the walls of the cells, or they collect into one or two spherical masses in the middle of the cell.

These spheres apparently consisted of a delicate membrane lined with granules and enclosing cell-sap. They distinctly lay lie within the spiral band of chlorophyll. Their appearance remind x ed x me of the bag-like masses sometimes produced in within the cells of dark-red leaves of Drosera. In one instance the granules became collected into a spiral band. They were not acted on by alcohol, sulphuric ether, acetic acid, or a solution of iodine. Alcohol caused the protoplasm lining the walls to contract, by which means the granular matter and chlorophyll bodies were all carried to be together towards the centre of the cell.)

(Three other kinds of conferva were immersed in a solution of the carbonate, and casually

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observed. In the first, in which the cell-walls were dotted over with chlorophyll-grains, there was at first some slight degree of aggregation, and then the grains all became disintegrated. In a second the second species, the filaments of which were having extremely thin, filaments the solution produced no effect. In a third the chlorophyll-bodies became aggregated into spheres. If the species in this family are difficult to distinguish, systematists might probably derive some aid by observing the differences in actions of a solution of carbonate of ammonia on them.)

(Conclusion. — Finally, from the facts given in this paper we see that certain certain some some of the salts of ammonia and, more especially the carbonate, quickly cause the cell-sap of certain in certain various plants, to belonging to widely different groups, to deposit granules apparently of the nature of protein, which These sometimes become blended become aggregated into rounded masses. These same salts, & in the case of Drosera an infusion of raw meat also tend to act in a few cases on the

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chlorophyll bodies, causing them in some few species to become completely fused to blend together, either in union with the aggregated cell-sap or separately from it. Aggregation seems to be a vital process, as it does not occur in recently killed cells; and any form the masses cause which kills a cell causes the already aggregated masses instantly to disintegrate thus formed sometimes display in some cases. These masses, moreover, display in some cases incessant movements. and anything that kills them all causes their instant disintegration. The process of aggregation however is sometimes occ not rarely carried so far that the masses can no longer move lose the power of movement; nor do they then readily disintegrate when subjected to some any deadly influence. From these facts, from other considerations, & more especially from the action of carbonate of ammonia on the chlorophyll-bodies, I am led to believe that they aggregated masses the aggregated masses include some living protoplasm, to which they were their power of movement may be attributed is due. The most remarkable point in the whole phenomenon is that with the Droseraceæ the most diverse stimuli, ─ even a stimulus transmitted from a distant part of the sameleaf induces the process of aggregation, which in the case of Drosera may be seen slowly travelling down the tentacles.

[54v]

The redissolution in the course of a few days of the solid aggregated masses, and especially, the regeneration of the chlorophyll-grains are likewise remarkable phenomena.)