Some recent Researches on the Continuity of the Protoplasm through the Walls of Vegetable Cells

There are several points in his paper that I should like to touch upon before giving my own conclusions.

As Dr. Elsberg at the outset admits that he is not a botanist, it is perhaps not surprising to find a want of accuracy in his botanical terminology. Thus, he talks of leaf parenchyma cells as “presenting blunt polygons separated from one another by a shining- rim of cellulose,” meaning, I suppose, polygonal cells with thin cell walls. He then proceeds to rechristen protoplasm, and proposes to substitute for it the certainly not euphonious name bioplasson. TO this it may be answered that, although the word protoplasm conveys in some ways an unsatisfactory idea, yet its persistent and wide use, would warrant its being retained, rather than that Scientific terminology should be burdened with another new word, when the value of the original word has been perfectly well defined, and has for the biological student a perfectly cleat meaning.

As far as I can judge Dr. Elsberg appears to confuse reticulate arrangement with reticulate structure, for he uses the same expression, “reticulated living matter “for both. As examples of such reticulated living matter, he gives Zygnema cruciatum; the description of which he quotes from Sachs- only altering the words “primordial utricle “for parietal sac.”¹ Other examples are: young cells of Zea mais, Fritillaria imperialis, and Vicia faba; hairs of Tradescantia virginica, and Cucúrbita. All these are of course examples of reticulate arrangement of protoplasm, and have nothing to do with the structure of the protoplasm itself.

Passing on to where he treats of the analogy between animals and plants, his terminology again becomes somewhat confused, in his endeavours to carry the analogy too far; for comparing the fact, that just as the animal cell is limited by its layer of cement substance, so is the plant-cell limited by its layer of cellulose, he proposes to commemorate “Schleiden and his cell doctrine,” by making the word cellulose subserve’ for the limiting membrane of both the animal and the plantcell. It is quite obvious, however, that this is impossible, for. the term cellulose is a name applied to a definite chemical’ substance with definite properties, and does not necessarily carry with it the idea of a limiting membrane at all. Cement substance, so far as I am aware, does not, for example, give a blue colour with iodine and sulphuric acid, nor furnish guncotton when acted upon with nitric acid.

We now come to the most important part of the paper, where Dr. Elsberg treats of the perforation of the cell wall.

The first experiments were made upon Nierembergia (printed Norembergia) gracilis.¹ He took pieces of the flower—whether calyx, corolla, stamens, or pistil does not transpire—treated them with a 2 per-cent, solution of silver nitrate for half an hour, or with a ‘5 per-cent, solution of gold chloride for forty minutes, washed, exposed to daylight, and examined. The silver nitrate preparations when seen from the surface, showed the cell walls stained dark brown, and demonstrated that every here and there were interruptions in their continuity. I have unfortunately been unable to obtain flowers of this plant, and have in consequence not had any opportunity of making this observation for myself, but I should like to point out that such pronounced and frequent interruption is quite opposed to our present knowledge, and certainly to the results I myself have obtained. It is much more probable that the walls were pitted, and that the pit membrane being thin escaped observation. The same may be said of the figure of the hair of Nierembergia, the transverse walls of which are probably pitted in a similar manner to those of the walls of Athæa hairs, a figure of which occurs in Sach’s Text-book.³ If Dr. Elsberg’s figure is drawn to scale it can scarcely be wondered at, if he has made a mistake, for his magnifying powers have not been sufficiently high. Very frequently the pit membrane is so thin that without very careful preparation, it cannot be recognised under the highest powers, and in many cases the only way to bring out such a membrane is to stain the protoplasm and leave the membrane unstained, or to stain and swell the membrane itself with Schultz solution (Chlor. Zinc Iod.).

But it was from the study of sections of the petiole of Ficus elastica, when treated with silver nitrate, that Dr. Elsberg has obtained his most conclusive results.

He gives a drawing of one of his preparations, and it is its appearance, and the appended description of it, which, perhaps, forms the most startling part of the whole paper; for we are told that “what has been sometimes described by authors, especially in growing tissues, as ‘intercellular spaces,’ and “middle lamellæ “in the cellulose were revealed to be, in a number of instances, accumulations and filaments of living matter wedged in between the plant cells.” Since it is impossible to understand how an intercellular space (if Dr. Elsberg really means space) can be an accumulation of anything, one must proceed to deal with the question of the middle lamella being an accumulation of living matter, &c. It is an undoubted fact that the substance of the middle lamella resembles protoplasm in many of its properties. Thus, like protoplasm, it resists the action of strong sulphuric acid in cases where it has attained to any pronounced degree of development, and, like it again, dissolves in strong potash or in Schultz’s mixture; and it is very noticeable that many reagents which are used as special stains for the protoplasm will also stain the middle lamella. But whatever view be taken as to the nature of the middle lamella and the thickened cell wall, and no matter whether one accepts the intussusception theory of Nageli,¹ or the apposition theory of Schmitz² and Strasburger,³ it is quite certain that at the time of its first formation the cell wall is essentially cellulose, and is thickened by deposits of cellulose substance. If protoplasm in any way enters into the constitution of, or forms an integral part of, that structure which we recognise as cell wall, it is, to say the least of it, hard to imagine, even on the well-nigh exploded intussusception theory, that such large quantities of protoplasm should be present, not only to replace the structure which we are accustomed to regard as middle lamella, and as such consisting of altered cell wall, but even large areas on either side of it, in such proportion as to cause the wall to consist as much of protoplasm as of cellulose.

It would rather be expected that if protoplasm does perforate the substance of the cell wall, such perforations would assume the form of fine threads, of such a degree of tenuity that they could only be recognised with great difficulty, involving very careful preparation and the use of very high powers. This subject has already been ably dealt with by Strasburger,¹ whose ideas have received confirmation from Tangl’s² researches published in his work, on the Structure of the Endosperm Cells of Strychnos, Phœnix, and Areca, and from the results I myself have obtained in the pulvini of Mimosa, Robinia, and Ami ci a.³ In my later work, which will shortly be published by the Royal Society, I shall be in a position to show that, as far as my investigations have as yet progressed, there has not only not been the least suggestion of the presence of large quantities of protoplasm in the cell wall, but also that no examples of reticulate arrangement have been met with in those cases where perforation actually takes place.

I now propose to give the experiments which were made with a view of testing Dr. Elsberg’s results.

Unfortunately the name of the grass he investigated is not given. I examined in detail two grasses, viz. Poa nemoralis and Bromus maritensis. In each case it was apparent that when mounted in dilute glycerine a distinct network structure could be made out in the chlorophyll grains. The boundary line of each grain was badly defined, and it was very hard to recognise with any certainty whether the reticulate appearance was confined to the immediate substance of the grain, or whether it extended beyond these limits. Indeed, in some instances, it certainly appeared to extend from the chlorophyll grain into the general cell protoplasm.

In order to see whether any abnormal appearances had been brought about by the action of the dilute glycerine, some pieces of the blade were mounted in expressed cell sap and examined. As the thickness of the blade was, however, too great to allow of satisfactory observations being made with high powers, a small piece was teased out before mounting; and since it was found, that during the teasing process and rupture of the tissues, no observable alterations had taken place in the cells, when such a preparation was compared with an uninjured one, in subsequent experiments teased out preparations were made use of.

When such a preparation is mounted in expressed cell sap and examined, it becomes apparent that the outline of each chlorophyll corpuscle is quite defined and distinct, and that little or nothing can be made out of the reticulate structure. I was also quite unable to observe any network in the general cell protoplasm. If, however, dilute glycerine, or simply water, be run under the cover glass, the corpuscles will be seen to gradually swell up, and, in so doing, to display more and more distinctly a reticulate structure. The outline also becomes more and more diffuse, and one almost begins to make out that the network appears to extend beyond the grain into the protoplasm. I am of opinion, however, that this is not the case.

The structure of the unaltered chlorophyll grain, and the action of reagents upon it, can be much better followed in thin leaves with large grains. I found, for instance, Selaginella uncinata very good material. And just as the chlorophyll grains in uninjured cells of serial plants will swell in the way I have described when treated with water, so will those of water plants when the cell becomes broken into or otherwise injured, e. g. the chlorophyll grains of Chara, Vallisneria, and Elodia. In each case a reticulate appearance is first produced, which, upon prolonged treatment, gives way to a granulation, and is followed at length by complete disorganisation. The fact, however, appears worthy of notice, that whatever light the action of reagents may throw upon the constitution of chlorophyll corpuscles, yet that pronounced reticulation of structure and diifuseness of outline are not observable in grains which are normal and unaltered.

My results then agree with those of Dr. Elsberg in so far as the reticulate structure of chlorophyll grains is concerned, but I have been unable to trace any reticulation in the protoplasm itself. Since I was unfortunately unable to obtain any Nierembergia material it only remains for me to deal with Ficus elastica. When transverse sections of the petiole were examined in dilute glycerine I could make out the reticulation in the chlorophyll corpuscles, but, as in the case of the grass, was quite unable to see anything of the kind in the protoplasm. Thin transverse sections, treated for half an hour with a 2 per-cent, solution of silver nitrate, washed, exposed to daylight, and mounted in glycerine, exhibited a structure somewhat similar to the figure drawn by Dr. Elsberg, viz. that on the cut surfaces of the cell walls were a number of exceedingly small, darkly-stained patches, separated from one another by light and unstained narrow areas. The reduction appeared not to have taken place uniformly all over the section, being, for instance, specially pronounced just under the epidermis.

The most obvious questions that arose were: Are these dark patches confined to the surface of the section, or are they present, as one would naturally suppose, in the entire thickness of the wall, and thus admit of being seen at any focus ? Secondly, what is their nature ? Do they consist of stained cellulose.

Now, the epidermal and cortical parenchyma cells being freely pitted, it is easy to focus to any determinate depth by fixing upon any given pit. I examined carefully in this manner several thin and well-prepared sections, but was unable to see any staining whatsoever below the free surface. On the contrary, my observations led me distinctly to the conclusion that the black patches were granules resting upon the cut surface, and that the substance of the wall itself was quite free from them.

I proceeded to make several experiments to test the truth of this conclusion. When sections of the petiole are cut in water a considerable amount of latex escapes from the injured surface and runs over the section, and it seemed not impossible that the latex mechanically deposited on the cut surfaces of the cell walls had reduced the silver. In order to expel the latex as much as possible before cutting the sections, a short piece of the petiole was taken and fitted into a bored india-rubber cork, which was then tightly fastened into the shorter limb of a manometer tube. The manometer was filled with mercury, with the exception of a short length next the cork, which contained water. Then, under a pressure of about 50 inches of mercury, a current of water was rapidly driven through the petiole tissue, and the latex was almost entirely expelled. Sections of the petiole however, showed the same granular appearance as in the first instance, thus proving that the latex had not been the cause of the reduction. Indeed, direct observation of sections of fresh petiole showed that little or no reduction had taken place in the laticiferous cells.

It was still possible, however, that some of the contents of the other cells might have been smeared over the walls either in the act of cutting or of escaping when cut. Thin sections of the petiole were cut, some of which were vigorously shaken with water in a test-tube. They were both treated together with silver nitrate. In the shaken-up sections there was much less reduction than in the others. I then resolved to try alcohol material where coagulation and hardening of the protoplasm would occur, and there would be a greater propability of getting a clean surface. In order to make out whether the action of the alcohol would interfere with the reduction of the silver, sections of fresh petiole were cut in water, and then treated with absolute alcohol. Having been washed with water, and treated with silver nitrate in the usual manner, they were examined, and it was seen that they exhibited the granulation quite as well as fresh sections. Thus having established that alcohol did not interfere with the reduction, I cut sections of petiole which had been for twenty-four hours in absolute alcohol. The sections were washed and manipulated as before. However, hardly any reduction was found to have taken place on the cell walls, although the cell-contents themselves exhibited well-marked reduction. This, again, suggested that escaped cell-contents were the cause of the granulation.

To make this quite plain. I took sections which were well reduced, and were in every way satisfactory preparations, and with a camel-hair brush freely brushed their surfaces. Nearly all the granules disappeared from the surface of the cell wall. In the cell lumen they were still numerous where the contents had not fallen away, but the cut surfaces of the walls themselves were quite clean and bright, and so was the entire thickness of the cell wall.

Again, having taken sections of fresh material, I brushed some well before the treatment with silver nitrate. Others I did not brush. The former showed no reduction. The latter exhibited well-marked granulation in the usual manner.

If the reduction be allowed to take place in diffused daylight the granules are small. If in sunlight they are large. Consequently, one may vary at pleasure the size of the granules, and therefore the size of the meshes of the reticulum.

I think these experiments have sufficiently established that the appearance described by Dr. Elsberg is simply due to the fact that granules of reduced silver are deposited on the cut surfaces of the cell walls, and that no staining occurs in the substance of the wall itself. The whole appearance of protoplasmic continuity can be brushed away by mere mechanical means, and the size of the granules can be varied at will. The reduction of the silver in the cell wall is caused by some of the cell contents which have escaped from the cell lumen.

The gold chloride preparations are not nearly so successful as the silver nitrate, for much less reduction occurs. I was unable to detect any network in the protoplasm, nor could I trace any perforation of the cell wall by protoplasmic filaments.

The identification of the substance causing the reduction now simply resolves itself into a micro-chemical investigation. That it was a very powerfully reducing agent was evident from the fact that gold chloride, silver nitrate, osmic acid, and chromic acid were all reduced by the cell contents of a very great number of the parenchymatous cells. When tested with alcannin it was shown that the presence of resin was confined to the laticiferous cells and to the cuticle, and there being no oil globules in any of the cell contents, with the exception of the latex, it was probable that the reaction with osmic acid had not been caused by oil or fat. The chromic acid reaction pointed to tannin. Sections were therefore treated with ferric chloride, when the cells occupying the same position as those which had especially reduced the osmic, chromic acid, &c., were turned a brown-green colour, thus proving conclusively the presence of tannin.¹

In order to examine the distribution of the tannin cells, transverse and longitudinal sections were treated with chromic acid. They are shown to be present in the tissue in very great numbers, and are especially abundant just under the epidermis (at the very place where the greatest reduction of silver nitrate occurs), and are arranged around the vascular bundle, being also dotted about irregularly in the tissue. In longitudinal section they are shown to be arranged in rows, end to end, and their cell contents exhibit a fine reticulation.

A longitudinal section of alcohol material treated with chromic acid shows that tannin has escaped over the cut surface, and thus gives confirmation to the other results. In the reduction experiments I placed along with the Ficus sections, sections of material such as the endosperm of Phœnix dactylifera, where I knew that perforation of the cell wall did occur, and obtained no satisfactory results whatever. Two years ago I made a number of experiments with gold chloride and silver nitrate, and was forced to conclude that they were unsatisfactory for botanical research. In my later work also I have tried many modifications which have met with no success as far as their use for studying the perforation of the cell wall by protoplasm is concerned, and I can only add that the experiments made in connection with Dr. Elsberg’s paper have fully confirmed my previous conclusions.

Before leaving the subject I should like to make a few remarks upon a more important paper, viz. Professor From-mann’s “Beobachtungen fiber Structur und Bewegungserscheinungen des Protoplasma der Pflanzenzellen.” ¹

The only part of his paper that I shall venture to comment upon is that which deals with the perforation of the cell wall and the subject of protoplasmic continuity. His results in this direction may be summed up in his own words, in which he claims to have established “that protoplasmic nets pass from one cell to another, and connect neighbouring cells with one another by means of either smaller or larger gaps and crevices in the membrane.” This structure was especially clearly seen in the epidermal and hypodermal cells of the leaves of Rhododendron ponticum and Dracæna Draco, but the leaves of Aloe arborescens, Crocus, Hyacinthus, and Mentha were also investigated.

I propose to give several quotations from Professor From- mann’s paper which will serve to illustrate the exact nature of his statements, and at the same time make it quite clear, which of these I wish to deal with, and to criticise.

On page 9, when treating of the epidermal and hypodermal cells of Rhododendron ponticum, we find: “The intercellular spaces contain nets and granules and further on— “The partition walls, however, do not always completely shut off neighbouring cells from one another, but are pretty frequently interrupted by gaps and crevices which are generally very narrow, so that only reticular threads or one or two series of meshes find room in them, hut they sometimes attain greater breadth.”

On page 1.0, when speaking of the mode of formation of the cell wall from the protoplasm—” And this view is also supported by the occurrence here and there observed of chlorophyll grains and coloured portions of nets, not only in the crevices, but also in the substance of the partition walls into which they appear as it were forced.”

On page 11 the following statement occurs:—” In the latter (i. e. the cuticle) chlorophyll corpuscles are deposited here and there.”

The next quotation, on page 17, refers to Dracæna Draco: “The partition walls separating the epidermal cells from one another and from the subjacent cells may, for a short distance, lose their brilliancy, but interruptions of continuity are more frequent which either appear isolated, or three or five of them on one partition wall; are partly very narrow, partly wider; may reach the diameter of a chlorophyll grain, and are traversed by isolated threads, or by narrow reticular bands, frequently showing isolated thickened threads and nodes, by means of which neighbouring cells are in connection.”

Again, page 21: “Narrow cracks and wider crevices, reaching the diameter of a chlorophyll granule, or of a nucleus, may occur with varying frequency.”

On page 29, referring to the epidermis of Crocus and Hyacinthus: “The threads of the neighbouring reticular layers are sunk into the membrane, while in places where cracks and crevices appear on the partition walls, the threads extend through them, and connect the nets of neighbouring cells with one another.”

Lastly, on page 38: “But continuous roundish or bandshaped lamellæ are also deposited in those layers of membrane which shut off the epidermis cells towards the outside, and may appear on their surface, and be enclosed in their texture, and as further in them as well as in the thicker partition walls chlorophyll grains also occur, it can admit of no doubt that reticular protoplasm may enter into the structure of cell mem- rane to a greater or less extent.”

Briefly stated, the principal facts involved in these statements are: that open passages of a very appreciable size are of very frequent occurrence in the common cell wall. That chlorophyl corpuscles and protoplasmic reticula occur embedded in its substance. That the intercellular spaces may contain granules and nets. That these nets and reticula of protoplasm may be traced into the cell wall, and are particularly clearly defined in the case of epidermal cells, running from the cell lumen out into the cuticle.

With all deference to Professor Frommann, I cannot but think that every one of these statements would be received with some surprise by almost any botanist who is at all acquainted with the histology of tissues.

I have investigated in as careful a manner as possible the leaves of Rhododendron ponticuin and Dracæna Draco, in order to give Professor Frommann’s results a fair test. Transverse and longitudinal sections, as well as sections parallel to the leaf surface, were examined in water, in cell sap, and in dilute glycerine. Both fresh material and that preserved in picric acid and absolute alcohol, were made use of. Iodine, Chlor. Zinc. Iod., and hæmatoxylin, which latter Schmitz¹ so successfully employed in his researches on the structure of protoplasm, and the nucleus, were used as staining reagents. Professor Frommann used expressed cell sap, sugar solution, and dilute glycerine as fluids for mounting his preparations, and employed methyl green as a stain; but since he expressly states that the staining due to this reagent was confined to the nucleus, and did not affect the nets and reticula, one must conclude that most of his observations were made upon preparations which were simply mounted in the fluids before mentioned.

1 do not intend to enter into detail with regard to the subject of the intimate structure of the protoplasm. Suffice it to say that in a great measure my results agree with those of Professor Frommann, although I am unable to see the nets and reticula with anything like the clearness with which he describes them in his paper, and figures them in his drawings. There is, however, a distinct reticulation in the cells of both Rhododendron and Dracæua, especially in the former. This reticulation is shown much more clearly by staining with haematoxylin, tissue that has been preserved in picric acid, and afterwards washed with alcohol. In Rhododendron very many of the cells, and especially the pallisade-parenchyma cells, contain tannin, and in these the reticulation is especially evident. I have frequently found this to be the case with tannin cells in general. Treatment with chromic acid, osmic acid, dilute potash, or dilute nitric acid will generally bring out a reticulate structure, although at present I am unable to give any explanation of the phenomenon. My attention was first drawn to the fact when investigating the tannin cells which occur in the pulvinus of Robinia pseudacacia.

As far as regards the reticulate structure, I can, therefore, in the main bear out the statements of Professor Frommann, whose results certainly accord with those of Schmitz¹ and Strasburger;¹ both these investigators having established that a reticulation can be observed in the protoplasm and the nucleus. The chlorophyll grains in the same way exhibit reticulation, but I should like to point out, as I did in the case of Dr. Elsberg’s research, that they are much swollen and somewhat disorganised by the action of dilute glycerine. Nevertheless, after the most careful preparation with picric acid and absolute alcohol and subsequent staining, they still exhibited a distinct reticulate structure, and agree fully with the description given by Pringsheim³ of the structure of chlorophyll grains in general.

As to the occurrence of chlorophyll grains in the cell wall, it is scarcely necessary to state that after very careful examination no such case was observed. Were I to attempt to explain Professor Frommann’s mistakes in this direction I might suggest with regard to the occurrence of chlorophyll grains in the cell walls that he was looking down through the thickness of a cell wall upon a chlorophyll grain that had got into a pit; and in the case of the grain in the cuticle, I can only put forward the explanation that he was viewing a chlorophyll grain in the guard cell of a stoma through the cuticle of an epidermal cell.

There is the simpler view, that during swelling, and also by mechanical means, some of the protoplasm was carried on to the cut surface of the cell wall; and fig. 4, Plate I, which represents a subepidermal cell of Dracæna, certainly gives some colour to this idea, although I put it forward with some diffidence. Numerous preparations, treated and stained in various ways, showed no sign of there being either granules or nets, or, finally, any protoplasmic structure whatsoever in the intercellular spaces.

Now, as to the subject of holes, gaps, and crevices in the cell wall. At the outset I cannot but feel that Professor Frommann was somewhat unfortunate in taking for his investigation such small-celled tissue as occurs in the leaves of Dracæna and Rhododendron, and especially so as regards the epidermal cells. In both these leaves, and particularly in Rhododendron, the epidermal and parenchyma cells are very freely pitted, and it is quite evident that what Professor Frommann has taken for open passages between the cells are in reality pits, each of which is closed by its own pit membrane. In Plate II, figs. 4 and 5, he gives a drawing of the so-called holes, but even a cursory examination of carefully prepared and thin sections treated with Chlor. Zinc Iod., or otherwise appropriately stained, will at once convince one that in every case a closing membrane is present, and that the pits are not open. As I have mentioned in the earlier part of this paper, the occurrence of open pits in living cells would be quite opposed to our accepted ideas of cell structure and cell mechanism. In his descriptions, however, Professor Frommann gives passages which suggest that he has noticed, but not identified, the pit-closing membrane. For instance, on page 9 he observes “short threads crossing from one side of the membrane (i.e. the membrane of the cell wall bordering a gap) to the other;” and again, page 17, “a somewhat stouter and more strongly refractive thread not unfrequently unites the portions of membrane bordering the gap in a bridge-like manner, and crosses the threads which pass through it.” On page 111 may quote a passage which shows the want of accuracy in his botanical terminology, for he describes as middle lamella “the layers immediately below the cuticle, which are double or three times as thick as the partition walls between the epidermal cells.”

The last point I have to deal with is the question of the perforation of the cell wall, and the possibility of following protoplasmic structures into its substance. Tangi¹ has shown that it is impossible to see anything of the protoplasmic threads in the cell walls of the endosperm cells of Strychnos, Phœnix, and Areca, by direct observation in such fluids as dilute glycerine, and even by ordinary staining. In each case a special mode of preparation must be employed. I can fully bear out his statements, and, indeed, in some of my most striking examples of the occurrence of protoplasmic filaments in the cell wall, it was quite impossible to see anything of them when examined in the usual manner. I was unable to observe anything of the kind in the epidermal cells of Dracæna and Rhododendron, but I think that some satisfactory explanation can be given of the appearances which Professor Frommann describes. If sections of Dracæna be examined in dilute glycerine, what appears to be a reticulate structure can be distinctly observed, both on the upper, and the side walls of the epidermal cells. The outlines of the walls bounding the cell lumen are not well defined, and, as Professor Frommann says, the protoplasm seems to gradually merge, as it were, into the cell wall, as he endeavours to represent in fig. 25, plate I. Indeed, the whole appearance is most striking. If, however, excessively thin and exactly transverse sections be treated with Chlor. Zinc Iod. the walls swell; the boundary bordering on the cell lumen becomes more distinct; and definite granules can be recognised deposited in the substance of the swollen wall. I made several experiments to test the nature of these granules. When the sections are warmed in dilute potash solution, the granules appear to become somewhat aggregated together, and signs of commencing solution can be recognised. When boiled with a 5 per-cent, solution of potash they are totally dissolved, the substance of the cell wall is left quite clear, and its limits sharply defined. When sections are treated with ether, and afterwards with boiling alcohol, considerable solution takes place, attended with a clearing up of the structure. These reactions appear to indicate that the granules consist of wax, and, as De Baryl has shown, the presence of wax is of frequent occurrence, not only on the surface of the cuticle, but even embedded in the substance of the cuticularised layers of the cell wall. The presence of these granules appears to explain, in a satisfactory manner, both the diffuseness of outline and the appearance of reticulation.

In Rhododendron, in the same way, an appearance of striation approaching to reticulation occurs. When examined in Chlor. Zinc Iod. it becomes apparent that very great cuticularisation of the epidermis has taken place, the cuticularisation extending even to the transverse walls. It is also apparent that the striation is confined to the cuticularised layers, and is separated from the cell lumen by a thin layer of cell wall, which still gives the cellulose reaction. If treated with a solution. of potash, the cuticularised portion still shows striation, while the rest of the cell wall becomes clear and transparent. Examination of thin sections mounted in glycerine supports these observations in every way. I need only refer to Sach’s ‘Text-book’² to show that the occurrence of striation in the cuticularised layers of the cell wall of epidermal cells is a perfectly well known phenomenon. His figure of the epidermal cell of Ilex aqui folium presents a case in point. I used in this investigation one of Zeiss No. 1 microscopes, Oculars 2 and 4, Objectives D and F, and a microscope of Hartnack’s with two very excellent Objectives, No. 10, and an Immersion, known as “Foyer 1 m/m”(No. 13?). Oculars 2 and 4.