am ware erentaitay | Alex-Agassiz= Hibrary of the Wusenm OF COMPARATIVE ZOOLOGY, AT HARVARD COLLEGE, CAMBRIDGE, MASS. -| Founded by private subscription, fn LSE. PDI eer Deposited by ALEX. AGASSIZ. No. Vike ee QUARTERLY JOURNAL OF MICROSCOPICAL. SCIENCE: EDITED BY JOSEPH FRANK PAYNE, M.B. Oxon., B.Sc. Lonp., Fellow of Magdalen College, Oxford ; Assistant Physician to St. Thomas's Hospital ; BE. BAY LANKESTER, M.A., FBS, °8E:8:, Fellow and Lecturer of Exeter College, Oxford; Professor of Zoology and Comparative Anatomy in University College, London ; AND WILLIAM ARCHER, F-.R.S., M.R.1.A., &e. a i eel VOLUME XVI.—NeEw Sentes. With Allustrations on Wood and Stone. -" LONDON: a *& A. CHURCHILL, NEW BURLINGLON STREET. Sz. 1876, = ee a CONTENTS. CONTENTS OF No. LXI, N.S., JANUARY, 1876. MEMOIRS: PAGE On the Structure of Hyaline Cartilage. By G. Tain, M.D. (With Plates I and II) : uf Cn a Polystomatous Condition of the Hydemets of Cider lophora lacustris. By Huen Price, pews of ean College, Oxford 5 23 Further Observations on a yee or Red- Pate ae —Bacterium rubescens. By Professor H. Ray LANKusTER, M.A., F.R.S. (With Plate II) . , ; RT On the Development of Teeth. By Cuarztes 8. Tomus, M.A., Lecturer on Dental Anatomy at the Dental ae of London, (With Plates LV and V) : 40 An Account of Professor HarcKnt’s recent Additions to af Gastrea-Theory. By Professor HE. Ray Lanxuster, M.A., F.R.S. (With Plates VII, VIII, 1X and X) ; 51 Note on Stenogramma eae ate): By Professor E. PrrcevaL Wricut, M.D. : ‘ Ve Preparation of Sections of coral ei Cerebellar Cortex for Microscopic Examination. By W. Bevan Lewis . 69 On the Evolution of Hemoglobin. By H.C. Sorsy, F.R. s., F.LS., F.Z.8., President of the Royal Microscopical Society 76 REVIEWS: The Anatomy of the Lymphatic System. By E. Kiery, M.D., Assistant Professor at the Laboratory.of the Brown Tustitu- tion, London. II. The Lung. Smith, Elder, and Co., 1875 86 The Histology and Histochemistry of Man. By Hxryricu Frey, Professor of Medicine in Zurich. Translated from the fourth German edition by Artuur EH. J. Barker. London, 1874 (pp. 683, 684 woodcuts) : - a |!) Outlines of Practical Histology. By Witiiam RuruErrorp, M.D., Professor of the Institutes of Medicine in the Uni- versity of Edinburgh. London, 1875 - ; . 94 lv CONTENTS, NOTES AND MEMORANDA PROCEEDINGS OF SOCIETIES: Dublin Microscopical Club Medical Microscopical Society . PAGE 95 . 104 . La CONTENTS OF No. LXII, N.S., APRIL, 1876. MEMOIRS: Observations on the Early Development of the Common Trout (Salmo fario). By Dr. E. Kuxry, F.R.S. With Plate VI Recent Researches on the Nuclei of Animal and Vegetable Cells, and especially of Ova. By Joun Prizstxey, Assistant Lecturer on Physiology, The Owens he Manchester. (With Plates XI and XII) Contributions to the History of the Germinal Vesicle, and of the First Embryonic Nucleus. By Epovarp Van Benepen, Professor in the University of Liege. (With Plate XIII) A New Process for Examining the Structure of the Brain, with a Review of some points in the Histology of the Cerebellum. By H. R. Octavius Sankey. (With Plate XIV) . On the Development of the Ova and Structure of the Ovary in Man and other Mammalia. By James Fouuis, M.D. (With Plates XVI, XVII and XVIIT) : On the Genus Astrorhiza of Sandahl, lately described as Haeckelina by Dr. Bessels. By W. B. Carpenter, M.D., C.B., F.R.S. (With Plate XIX) : ; NOTES AND MEMORANDA: Relation between the Limit of the Powers of the Microscope and the Ultimate Molecules of Matter. By H.C. Sorby, F.R.S., F.L.S., F.Z.S., President of the bei caine . 113 . 131 . 153 . 182 . 190 . 221 Society . 225 PROCEEDINGS OF SOCIETIES : Dublin Microscopical Club . 235 Medical Microscopical Society . . 240 CONTENTS. CONTENTS OF No. LXIII, N.S., JULY, 1876. MEMOIRS: PAGE On the Formation of Blood-vessels as observed in the Omen- tum of Young Rabbits. By G. Tu1n, M.D. With Plate XV (figs. 1—5) . : é : , . 241 On the Structure of Muscular Fibre. ere G. nae M.D. With Plate XV (figs. 6—14) : . 251 An Account of the Recent Researches into the vata of the Bacteria, made by, and under the Direction of, Professor Cohn. By F. Jurrrey Brut, Exhibitioner of Magdalen College, Oxford. (With Plate XX). . : . 259 Note on Bacterium rubescens and Clathrocystis roseo- perce By HE. Ray Lanxester, M.A., F.R.S. : . 278 Résumé of Recent Contributions to our Knowledge of § “ Fresh- water Rhizopoda.” Part I. Heliozoa. Compiled by Wit- tiaM ARCHER, F.R.S., M.R.LA. on Plates XXI and XXIT) : . 283 The Process of epiecwiiies in the ieatapleg of ones rotun= difolia. By Francis Darwin, M.B. (With Plate XXIII). 309 Remarks on the Shell-gland of Cyclas and the Planula of Limneus. By E. Ray Lanxzsrer, M.A., F.R.S. (With Plate XXIV) : < . 820 Note on Mihakowics’ New Method of Imbedding, By H.N. Mosetey, M.A. Oxon., Naturalist on H.M.S. Challenger . 327 NOTES AND MEMORANDA . : : P . 330 PROCEEDINGS OF SOCIETIES : Dublin Microscopical Club. : : : . 336 Medical Microscopical Society . ‘ : - . 345 CONTENTS OF No. LXIV, OCTOBER, 1876. MEMOIRS : Résumé of Recent Contributions to our Knowledge of ‘‘ Fresh- water Rhizopoda.” Part II. Heliozoa. Compiled by Wit- LIAM ARCHER, F.R.S., M.R.LA. (With Plates XXI and XXII) d : ; ; . 348 On the Coincidence of the Hoaeke and Anus in Paludina vivipara. By KE. Ray Lanxester, M.A., F.R.S. Bi Plate XXV) : a ONE Note on the Lym kites of — Gikcias. by P. Kiva, B.A. Oxon. (With Plate XXVI). ; . 386 vi CONTENTS. Some Recent Views as tothe Composition of the Fibro-vascular Bundles of Plants. By Sypney H. Vinus, B.A., B.Sc., Fellow of Christ’s College, Cambridge. (With Plate XX VII) The Termination of the Nerves in the Vestibule and Semicircular Canals of Mammals. (Read before the British Association, Glasgow, September, 1876). By Ursan Pritcnarp, M.D., F.R.C.S., Aural Surgeon to King’s College Hospital, Lecturer on Animal Physiology, Kings College, London. (With Plate EX VIER). : : j : ; . On some Foraminifera from the Loo Choo Islands. By Henry B. Brapy, F.R.S. : , ; ‘ REVIEW : Epicrisis Systematis Floridearum. Auctore Jacopo GEoRGIO Acarpu. Lipsie: apud T.O. Weigel, 1876 : NOTES AND MEMORANDA : Dr. Klein’s supposed Mycelial Growth in Sheep-pox PROCEEDINGS OF SOCIETIES: Dublin Microscopical Club Medical Microscopical Society PAGE 388 398 404 407 412 414 418 MEMOIRS. On the Srructurr of HyaLine CArrinaGsE. By G. Tuty, M.D. (With Plates I and II.) HYALINE cartilage is generally believed to consist of a homogeneous ground substance in which are closed cavities harbouring nucleated cells, the opinions. of Bubnoff! and Heitzmann? to the contrary not having hitherto found general acceptance. The author’s views, being founded on results obtained by several methods, some of which are new and others probably not as yet much in use, will be best introduced by a descrip- tion of these methods and the results obtained by them respectively. ‘The application of caustic potash, being the most important of these, will be considered first. Any one who would apply this process to the study of cartilage should first of all apply it to the cornea of a large animal, such as the ox or the sheep, in order to become conversant with the details of the manipulation and to acquire the necessary faith in its results. For it is on the cornea that in the author’s hands it has succeeded with incomparably the greatest frequency. Since first publishing an account of this process® the conditions of its success have been somewhat more closely defined, and with the sheep or ox’ cornea success is now almost invariable. The method by which the author generally operates is as follows :—Fifteen grammes of pure anhydrous caustic potash in stick are ground to powder in a mortar, which if the weather is cold should be previously warmed and well dried ; on the powdered potash are quickly poured fifteen cubic centimeters of distilled water, and solu- tion is favoured by stirring. As soon as the potash is dissolved the solution is poured into a narrow glass vessel or porcelain capsule, and the bulb of a thermometer is suspended in the 1 «Wien. Sitzb.,’ 1868. * ‘Wien. Med. Jahrb.,’ 1872. ; 3 * Proc. Roy. Soc.,’ No. 155, 1874. VOL. XVI.—NEW SER, A 2 DR. G. THIN. upper stratum of the liquid. The heat evolved by the solu- tion of the potash should raise the thermometer above 120° Fahr. When the temperature falls to 107° Fahr. pieces of a cornea are put in one after the other until the temperature falls to 105°. From 107° to 105° is the most favorable temperature. An ox cornea may be cut into four or five portions and allowed to remain in the aqueous humour, which should be saved for the purpose until the solution is ready. Before putting them into the solution the excess of aqueous humour is removed by bringing the portion into momentary contact with the surface of a dry glass slip. After being a few minutes in the solution the portions of cornea are ready for examination. The froth should be removed from the surface of the fluid by a small watch-glass, and a clear drop removed by a glass rod to a glass slip in which a small fragment of one of the pieces is broken up by needles and examined. If the above precautions are carefully observed, it is rare that at least one of the portions is not found to consist of a multitude of the cells described by the author. A solution previously made and heated over a flame has not proved so successful as when the heat pro- duced by the solution of the potash is employed. Fifteen grammes is the smallest quantity that can be relied on as invariably producing sufficient heat. Of course any larger quantity treated with an equal weight of water will be as successful. Ten grammes sometimes, but not invariably, produce sufficient heat. It is necessary that the caustic potash be pure and dry. A cornea successfully treated should show nothing but the cells. If any other substance of any kind whatever, formed or amorphous, is seen, the operation has been only partially successful. Partial success, in which a greater or less num- ber of cells are seen mixed with other substances, is very common. When a perfectly successful cornea has been ob- tained it may be kept some time in the solution in a well- stoppered bottle. The smallest particle from any part of it can then be used for demonstration or study when con- venient. On all other tissues except the cornea the operator must at present be prepared to encounter one failure after another, but by persevering not only will all the results be obtained which have been previously described by the author as demonstrable in connective tissue, nerve, and muscle, and those now to be described in cartilage, but probably many others whose discovery awaits a diligent application of_ the process. ON THE STRUCTURE OF HYALINE CARTILAGE. 8 The cartilage covering the head of a froz’s femur, or that of a small mammal, is well adapted for treatment by the potash solution. The author has generally cut off the head of the bone and placed it in the solution at the temperature indicated. Ina few minutes it falls to the bottom of the vessel, and it can then be removed to a glass slip for examination. The outer pellicle which represents the cartilage can be easily separated by needles and examined. The substance pro- per (matrix) of the cartilage can be always seen, but variously modified in different preparations, and some appearances having relation to its arrangements and structure which have not hitherto attracted much attention can frequently be observed ; but as these can be better studied by methods shortly to be indicated, it is not necessary to describe them here. A successful potash preparation shows flattened polygonal cells adhering to each other exactly like an epithelium. In contour, mutual arrangement, and appearance generally they are absolutely indistinguishable from any ordinary epithelial layer. No histologist who had once observed them could hesitate to classify them as a variety of epithelium, especially when the mode of preparation is taken into account. They are seen differently according to the degree in which the potash has acted on the ground-substance. Sheets of cells and more rarely isolated cells are sometimes seen floating free in the liquid. At other times the cells may be seen lying on the surface of considerable fragments of the ground- substance. These fragments sometimes show depressions corresponding in outline, diameter, and relative position with what have been usually described as the cavities or capsules in which the or- dinary cartilage-cells are situated, and when the flat cells on them are visible it can be seen that the so-called capsules consist of depressions in the ground-substance, and that the depressions and the more even surface in which these are situated are covered with a continuous epithelial layer. Such a preparation at once shows that the idea of a closed cavity in the ground-substance harbouring a cell has been founded on imperfect methods of observation. Between the free sheets of cells and those seen adhering to the ground substance and its depressions there is an appearance some- times seen which is intermediate as regards the solvent effect of the solution. The potash may preserve entire a substance which may be described as an interepithelial framework. It follows the lines and angles between the cells, and can be distinguished as differing both from the cells and the ground- 4 DR. G. THIN, substance properly so called. It is shown imperfectly in Fig. 3. In the sheets of cells found free parts of them are some- times seen to be formed by a double layer of cells. The sheets are often found of considerable size. The cells represented jn Fig. 2 formed about the fourth part of a layer which consisted entirely of similar cells to those drawn, and about a sixth part of which consisted of a double layer. It may be presumed, although it is not capable of direct observation, that some of the layers found free in the fluid belong to a superficial layer forming the surface of the cartilage. But the observation of double layers and of fragments of ground-substance which with their depressions are covered with cells shows that they do not all belong to a superficial layer. The author long searched in vain for the narrow elongated flat cells which he had found in potash preparations of other tissues, but at last succeeded in obtaining a preparation sufficiently distinct to put their existence and arrangement beyond doubt. Fig. 5 represents rather less than the half of a mass of cells, adherent to the ground-substance, isolated from the head of a frog’s femur in May of this year. The cells are in size and arrangement similar to those which he had previously isolated from the mesentery of the frog and from tendon, by the same process. Their extreme minuteness will be estimated when it is con- sidered that the figure represents as accurately as possible their apparent size when examined by a No. 8 objective and No. 3 ocular of Hartnack’s system with the tube full out. The outlines of the nucleus were sharply demarcated, but the cell-substance was represented by aless clearly defined finely granular substance. Until a more certain method of operating on cartilage by the potash solution is discovered, those who would examine these cells for themselves must possess in a high degree the virtues of patience and perseverance. A strong conviction of the existence of a unity of plan in the general structure of the tissues, and observations on the sclerotic of the frog by maceration in aqueous humour, which will afterwards be de- scribed, encouraged the author to continue subjecting car- tilage to the action of potash after a number of failures greater than he can recount, and until he had obtained pre- parations sufficiently distinct to enable him to consider the existence of layers of flat cells, epithelial in their form and ON THE STRUCTURE OF HYALINE CARTILAGE, Or arrangement, in the substance of cartilage as a settled question. This is not intended to discourage other investigators, who may probably have more frequent success, but to suggest the advisability of the attention of histologists being directed to the study of the conditions of success of a process which produces so important results. One of the most successful series of preparations was ob- tained from the head of a frog’s femur, which was operated on after the animal had been left dead twenty hours at the temperature of a room in London in the end of May. A number of vertical incisions were made through the cartilage before the head of the bone was’put into the solution; but as this was amongst the last experiments that were made, it is at present uncertain whether the time that had elapsed after death and the incisions had anything to do with the success. - The results obtained by subjecting cartilage to the action of nitrate of silver are now to be described. : The author believes them to be mostly, if not entirely, new. ‘The success which has been obtained is due not so much to any special merit in the methods employed as to the number of preparations that were made. - The light that was thrown on the structure of cartilage by potash prepara- tions stimulated him to a more persistent application of silver than he would probably have otherwise undertaken. The appearances seen will first be described, and an attempt will afterwards be made to explain them in conjunction with those obtained hy other methods. One of the commonest effects of the action of nitrate of silver on cartilage is a uniform dark-brown staining of the ground-substance. A section so acted on shows a framework of dark substance enclosing in its meshes circular or oval colourless spaces. ‘This appearance is figured by Ranvier in the part that has appeared of his ‘ T'raité technique d’Histo- logie,’ p. 283, and need not, therefore, be repeated here. To the best of my knowledge that is the only effect produced by nitrate of silver that has hitherto been noticed. The cells on the surface of articular cartilage may be demonstrated in the following manner:—The sections are made from the newly disarticulated head of the frog’s femur, one surface of each section consisting of the free surface of the cartilage, and are placed instantly; in a half-per-cent. solution of nitrate of silver. As soon as all the available surface has been so removed, the sections are transferred from the silver solution to a half- 6 DR, G. THIN. per-cent. solution of common salt and allowed to remain in it only a few seconds. They are then either at once placed in a drop of glycerine on a glass slip, or are put for a few seconds in distilled water, and thence transferred to the gly- cerine. ‘The glass slip is placed in sun-light. After a quarter to half an hour it will be seen that the free surface of the cartilage is covered with uncolored, generally rectan- gular, spaces, arranged not unlike the stones that form the pavement of a causeway, and between the spaces are fine lines of a darkly stained substance. The white spaces cor- respond, as will be seen, to cells. It is further seen that the cells are arranged in quadrangular, triangular, or irregular groups, and that the groups are separated from each other by broader lines of dark substance than those which sepa- rate the individual cells. In the best preparations the widest lines of dark substance do not equal in breadth the diameter of a human red blood- corpuscle, whilst the finer lines between the individual cells are only from one half to a fourth of that breadth. A careful examination will show, in some of the cells, a large round nucleus indicated by a fine unstained ring, which refracts light differently from the substance by which it is surrounded. A change of focus brings into view the ordinary ground- substance of the cartilage, stained of a brown colour, and distributed in it the ordinary cartilage-cells, more darkly stained, and irregular in outline. A comparison of these with the nucleated quadrangular cells on the surface shows that they differ in size, form, arrangement, and staining capacity. The surface-cells are seen in Fig. 1. The intercellular substance is exaggerated in the drawing. In the best pre- parations the epithelial nature of these cells is evident enough, even if it had not been demonstrated by the potash solution. The knowledge obtained by the use of the potash solution, that layers of cells epithelial in arrangement exist in the substance of cartilage, and the now-established fact that silver solution can demonstrate epithelial layers in the substance of the cornea, although the cornea had been treated for years by silver without these having been seen, rendered it reason- able to persevere in an attempt to obtain similar silver pre- parations in cartilage, in spite of a long-continued series of negative results. Thin sections made with a very sharp razor transversely through the cartilage covering the condyle of the femur of ON THE STRUCTURE OF HYALINE CARTILAGE, 7 the frog and young rabbits (the animals at my disposal) were transferred directly to the silver solution, and then im- mediately placed in glycerine, and exposed to sun-light. Frogs that have died from disease, and especially frogs and rabbits in which a mechanical or other irritation has been applied for several days to the shaft of the lower third of the bone, are best adapted for its demonstration, but successful preparations can also be obtained from healthy animals. Fig. 6 represents part of an epithelial surface obtained by this method from a transverse section of the condyle of a frog’s femur. In this preparation the extent of surface showing the silver markings (all of it has not been shown) is the largest that was obtained. For a short time after it was prepared the nuclei of some of the cells were distinct. The drawing was made a few days after the preparation was mounted (in May, 1875). Since that time the preparation has considerably deteriorated, but it is still (August 21st) perfectly demonstrative to any one at all familiar with silver preparations. The form and arrangement of the cells will be best understood by an examination of the figure, which represents the preparation with great accuracy. It is well known that nitrate of silver may produce two distinct results when it permeates a tissue in solution. It may diffuse itself through the ground-substance, which is then stained a varying shade of brown, whilst the interstices which are present in the tissues are unstained, and are then seen as colourless canals and spaces, or it may, while still staining the ground-substance a shade of brown, produce in the system of interspaces, by contact with the lymph-fluid which is present in these channels, an abundant black pre- cipitate of albuminate of silver, The spaces and canals which in the former case are indi- cated by their want of colour, are in the latter indicated by the outlines of the black deposit with which they are filled. Both these effects can be produced in hyaline cartilage by nitrate of silver. To begin with that in which there are white interspaces in a dark ground. The author has found three distinct kinds of effects so produced, which are respectively repre- sented by Figs. 10,12, and 14. That represented by Fig. 10 will at once recall the white stellate spaces and communi- cating canals seen in a silver cornea. ‘here is a difference to be noted, due, probably, to the unequal permeability of the tissues, rather than to a difference in their relative struc- ture. In a silver cornea the stellate spaces and lines are found at all depths of the substance acted on. In cartilage 8 DR. G. THIN. the author has only found them on the surface of sections cut fresh, and then submitted to the action of the silver. By depressing the focus the uniformly brown ground- substance, with the more darkly coloured cells interspersed, was in such sections invariably brought into view. There is a certain similarity in the figure to the drawings which have been published by Bubnoff and Heitzmann. Although in strict detail it corresponds with neither, the author considers such preparations as confirmatory of the general principle laid down by these histologists, that there are no closed capsules in cartilage. Heitzmann interprets his preparations as demonstrating that the cartilage-cells com- municate by elongated protoplasmic processes. Without at present entering on the question of the existence of such processes, it may be stated that in the author’s opinion Heitzmann’s drawings show that what he had under his observation was a system of communicating spaces formed by the apposition of two layers being at certain points incomplete. Another effect of the impregnation of the ground-substance is that shown in Fig, 12. Such preparations are obtained by removing to an object- glass slip any flat cartilage which is sufficiently thin to be examined entire. A piece of solid nitrate of silver moistened with distilled water is rubbed efficiently over both surfaces. ‘The cartilage is exposed to the light in glycerine, and when it has become quite dark is freed from the perichondrium under a dissect- ing lens. (In the frog several distinct layers of large flat polygonal cells, joined like any ordinary epithelium, are found in the perichondrium, and on its surface a thick net- work of lymphatic capillaries is indicated by a black silver deposit.) When sufficiently freed from its investing mem- brane the dark ground-work is seen intersected by a network of white lines which branch from a system of wider and nearly straight spaces, such as that represented in the figure. Hither of the sternal cartilages or the thin cartilages which form the walls of the larynx of the frog are well suited for treatment by this method. By changing the focus it is seen that the white lines are present at all depths of the cartilage. The third effect of impregnation of the ground-substance which I have to describe is of a different kind.- It is repre- sentedin Fig. 14. The condyle of the femur ofa kitten a day old was firmly and persistently rubbed over by solid nitrate of silver, which was several times moistened during the opera- ON THE STRUCTURE OF HYALINE CARTILAGE, 9 tion, The cartilage was then cut in thin sections more or less perpendicularly to the long axis of the bone, the sections being placed as they were cut in glycerine. They were then exposed to sunlight, and after a few hours examined. In some of the sections the ground-substance was found to be impregnated a dark brown in the form of uniform parallel straight bands or ribbons about the breadth of the diameter of a human red blood-corpuscle, which were separated by white (unstained) lines of a quarter to half the breadth. The alternation of the broader brown and narrower white lines is of the most regular kind. At the spaces which indicate the position of the ordinary cartilage-cells these bands are interrupted. The part of the cartilage next the bone shows them most frequently. ‘They are found, however, in all parts of the cartilage except the most superficial layers, but this may be accounted for by proximity to the directly cauterized surface. From sections of the condyles of three kittens so treated and examined about a score of available preparations were obtained, and as they are still (after about four months) good it is hoped they may be permanent. Opportunity was wanting to extend this series of observa- tions to other mammals, but two similar preparations were obtained and preserved from the cartilage of the head of the femur of the frog. As they differ in no respect from those seen in the kitten’s cartilage, except in so much as the white lines were relatively broader, no drawing of them has been given. No attempt has been made in the figure to imitate the colour of the brown staining, and the lines are more wavy than they are generally seen. Otherwise the repre- sentation is accurate. Only a fraction of a preparation so marked has been drawn, the extent of surface showing this peculiar impregnation being in many of the preparations very much larger than that shown in the figure. The interpreta- tion of this appearance will find its place further on. In some silver preparations there is a more or less abund- ant deposit in ‘the substance of the cartilage, and in every instance the form in which the deposited substance is seen may be placed under one of two types. One of these types is shown in Figs. 7 and 8, and the other in Fig. 9. Pre- parations like that copied in Fig. 8 are obtained either by rubbing over the surface of the cartilage with the solid nitrate or by placing sections in the silver solution. What the con- ditions are that produce this appearance, and not the others previously described as being sometimes obtained by a similar procedure, have not been determined, ; 10 : DR. G. THIN, It will be seen from the drawing that the silver is depo- sited in a connected series of black curves and circles, and when the deposit is not too abundant it can be observed that the ordinary cartilage-cells are mostly found in the area of the deposit. This is still better seen in preparations like that represented in Fig. 7. This drawing represents a section through the cartilage of the knee ofa frog that died from disease. The section was placed in a silver solution, and as is shown in the drawing a large part of it was covered with a network of silver deposit. Where the network was com- plete it was found that the encircled ground-substance was stained a faint brown and that no cells were visible. That the cells were in line with and covered by the deposit was shown by an examination of the edge of the area of deposit where it was incomplete. At this part the position of the cells was indicated some- times by a white unstained circle, and sometimes bya similar circle partly filled by the silver precipitate. It is evident, then, that the precipitate takes place most easily in the circular spaces, and afterwards in curved tracts of tolerably uniform breadth which connect the spaces. A general or unsystematic precipitate of silver in such pre- parations was never observed, nor was there ever a departure from the usual type either in the size of the meshes of the net- work or in the breadth of the tracts of which it is composed. The similarity between this network and that of the lymphatic capillaries which are indicated by silver deposit in the perichondrium is evident. Metallic deposit in silver preparations is sometimes found in a different relative position. In that part of the articular cartilage which is near the bone the ground-substance has, as is well known, a peculiar arrangement. ‘The cavities or depressions known as capsules appear much larger, and the intermediate substance assumes somewhat the character of a framework, arranged in parallel beams, which are connected by transversely disposed arches. The metallic precipitate at this part of the cartilage is frequently found deposited in the centre of the beams and arches, whilst the contained cellular spaces may be free from it. In preparations of this kind, in which a small portion of bony substance has been removed in continuity with the cartilage, I have been able to trace the continuity of the tracts of deposit with similar tracts in the bone, a fact of which the significance in relation to the nutrition of cartilage is sufficiently evident. This form of metallic deposit is shown in Fig. 9. ON THE STRUCTURE OF NYALINE CARTILAGE, 1l In a paper presented to the Royal Society, an abstract of which is published in the ‘ Proceedings,’ No. 158, 1875, the author has described a number of appearances seen by the application of a method which he thus summarised :—“ Trans- parent animal tissues, when sealed up fresh in aqueous humour or blood-serum by running Brunswick black round the edge of the cover-glass, undergo a series of slow changes, by which, generally within a period of two to five days, anatomical elements, mostly otherwise invisible, become distinct.” It was in studying cartilage in the sclerotic of the frog that in the spring of 1874 the author observed for the first time that by this method flat cells could be seen in layers in cartilage. The successful preparations sealed up as de- scribed in aqueous humour, andallowed to remain for 24—48 hours (at the temperature of London in June), showed, in addition to a large polygonal epithelium, rows of narrow cells joined to each other end to end, and in contact with other rows on each side. ‘These cells are represented in Fig. 17. The cells were perfectly hyaline in appearance, with the nucleus well marked. Attempts made this year to obtain. similar preparations in the sclerotic of the frog have failed. A series of observations made on sections of the articular cartilage of the carpus of the sheep treated in this way gave the following results. Very thin transverse sections embracing the free surface of the cartilage, show after macera- tion a distinct layer of rounded cells with a central nucleus. Smaller layers of similar cells may be sometimes seen in very thin transverse sections which do not include the surface of the cartilage, but this appearance is rarely found. In both cases the cells are distinguishable only by the very fine borders which indicate the cell and nucleus, their refractive power differing so little from that of the surrounding medium that it is only when the section is very thin, and with good’ light and a good lens, that they need be sought for. There is no granular protoplasm in the cell. The ordinary carti- lage-cells, much fewer in number and more or less widely separated from each other, can be always observed in such preparations ; and as they are in these circumstances seen as well-marked, granular, irregular protoplasmic bodies in the centre of the cellular spaces, it is not possible to confound the two kinds of cells. The narrow elongated cells’ seen in the sclerotic and found in the cartilage of the frog’s femur, by solution of potash, were not seen in these preparations. In many of the sheep-cartilage preparations isolated flat cells were found free in the fluid. These were found when 12 DR. G. THIN. every precaution had been taken to ensure that the sections did not include the free surface of the cartilage. Individual cells so seen are represented in Fig, 16. In regard to the cells usually seen in cartilage the fol- lowing is to be added to the methods explained in the text-books of histology:—An ordinary, not necessarily very thin, transverse section is placed on a glass slip, in aqueous humour or blood-serum, and cut in pieces by a sharp knife, an oblique direction being given to the incisions. ‘The pieces are then sealed up in the ordinary way. ‘To increase the chances of success it is advisable to seal up a number of. preparations at one time. On the second day or afterwards the obliquely cut edges should be carefully examined. Supposing a preparation to have succeeded, in the oblique edge of the incised surface the same protoplasmic clumps that indicate the cartilage-cells in the rest of the section will be found, but also there may be detected at intervals pear- shaped protoplasmic masses lying free in such of the cellular cavities as have been laid open by the incision. The pro- jecting end of the protoplasm is irregularly round, but the eud which is towards the section tapers toa fine prolongation, which is lost in an exceedingly delicate, somewhat glistening fibre, which enters and is lost in the substance of the cartilage. It is easy to identify this branched mass of protoplasm as being of the same nature as the cellular masses which are covered by the cartilage-substance, and to satisfy one’s-self that the process has been rendered visible, because the cayity in which it is contained has been laid bare. This appearance is represented in Fig. 20, the depth of shading in the drawing being, it is to be understood, intended to make more prominent the contours of the different ele- ments rather than to indicate the relative degree of difference of their refractive properties. In sections so sealed up the substance of the cartilage can sometimes be seen to be composed of parallel bands, having approximatively the diameter of a human red blood-corpuscle. The contour of these bands is well defined, resembling in breadth and arrangement the similar bands that have been described as being sometimes seen in silver preparations. The size and arrangement of these bands are rendered in Fig. 15, which may be compared with Fig. 14. In Fig. 15 the depth of shading is intended to enable the reader to realise more easily the described arrangement. In the pre- paration the contours are rendered visible only by slight differences of refraction. In such preparations these bands may be sometimes seen ON THE STRUCTURE OF HYALINE CARTILAGE. 13 to be arranged in groups, the divisions between the groups being wider than those between the individual bands. It has not been considered necessary to give a drawing of these groups, which morphologically find their analogues in other forms of connective tissue. In sealed preparations of cartilage there are sometimes to be seen lying free on the surface of the section, or more frequently free in the fluid near its edge, isolated flattened cylindrical bands, of a like breadth to those described as being arranged in parallel layers in the cartilage-substance, but differing from them when in situ in the following par- ticulars:—They are puckered transversely, the puckerings being formed by parallel transverse ridge-like discs separated from each other by ahyaline substance. Frequently the end of such an isolated band bulges over the end of the cylinder as a flocculent coherent mass. Jig. 19 is a copy of a drawing made by Dr. Ewart from a preparation of the head of a frog’s.femur an hour after it had been sealed in blood-serum, which was obtained by beheading the animal. Such isolated bands can be seen in preparations of other _ forms of connective tissue similarly treated, and more abundantly if the tissue has been inflamed. When seen isolated it is impossible to distinguish the form of tissue from which they have been separated. The appearance of such an isolated cylindrical body, whether from hyaline cartilage, neurilemma, or an inflamed cornea, is identical. As in some tissues this cylindrical band is farther resolvable into a num- ber of exceedingly fine fibrillee, the author in his previous histological papers, taking the fibrilla as the ultimate unit, has applied the term primary bundle to such a cylinder or rod. Now follows the history of a remarkable preparation. In December, 1874, a number of aqueous-humour prepara- tions of transverse sections of the cartilage of the metacarpus of the sheep were sealed. One of these, about which nothing was noted, although it was presumably frequently examined during the first fortnight, was laid aside in order to observe any further changes that might follow. It was not looked at again until July of this year, and when then examined it presented the following microscopic characters : —The substance of the cartilage, to judge from its uneven appearance, had apparently undergone some disintegration ; the granular protoplasmic cells had become very small, and lying free in the surrounding fluid were swarms of large, irregularly and variously shaped, finely granular bodies, beset with yacuoles which corresponded in shape and size with 14 DR. G. THIN. nuclei. It was impossible not to observe the identity of ap-~ pearance with that of the bodies described as giant cells or myelopliques. By a careful examination the presence of similar bodies on both surfaces, and in the thickness of the sealed cartilage, could be detected. A drawing of one of the free “ giant cells” seen lying close to the edge of the pre- paration is given in Fig. 18. The curious part of the history is what follows :—The pre- paration, with others, was taken from the author’s house to Dr. Ewart’s room in the museum of University College, and must have remained essentially unchanged there for more than twenty-four hours, as the day after he had received the preparations Dr. Ewart was able to demonstrate the struc- tures in question to Professor Ray Lankester. During the following week the preparation remained in the same room, but was not further examined. It was then brought back to the author, and again examined by Dr. Ewart and himself. To their astonishment every trace of the giant cells had disappeared. The fluid contained threads of streaky looking protoplasmic substance, and swarmed with bacteria, in active motion, in the forms known as coccos, rods, and leptothrix. The development of bacteria in the preparation took place . whilst it was in University College. This, be it remembered, was in the end of July, the preparation having been sealed in the middle of December. It is well to add that the section of cartilage was from a full-grown sheep, and quite clear of the bone. . One other preparation remains to be described. A pin was pushed through the knee-joint of a frog from side to side between the femur and tibia, the cartilage not being injured further than would result from the presence of the smooth foreign body in the joint. After seven days the frog was killed, and the condyle of the femur placed for three days in a quarter-per-cent. solution of osmic acid. ‘Transverse sections were then made and mounted in glycerine. Thin sections only were sufficiently transparent. The colouring was found to be unequally distributed. Broad winding tracts, darkly stained, contrasted with comparatively faintly stained intermediate portions, and what was especially note- worthy, the coloured tracts contained the ordinary carti- lage-cells. The intermediate faintly stained, homogeneous- looking substance contained no visible cells, and indicated by an appearance of splitting the described division of car- tilage into polygonal areas. The deeply stained cellular areas and lightly stained in- ON THE STRUCTURE OF HYALINE CARTILAGE. 15 termediate portions are represented in Fig. 13, but the transition between the two is more abrupt in the plate than it is in the preparations. An attempt will now be made to construct a theory of the structure of hyaline cartilage by which the above data will receive a sufficient explanation. In regard to the appearances described, as seen by treat- ment with potash and by maceration in sealed aqueous humour or serum, the facts being once admitted, there can be little difference of opinion as to their interpretation. With regard to the nitrate of silver appearances, there may not be so much unanimity. It may, however, be taken for granted, without further discussion, that the dark lines represented i in Fig. 6 indicate the dark lines of epithelial (endothelial) cells. In regard to the uncoloured spaces represented in Figs. 10, 12, and 14, there is room for discussion. Fortunately the same appearances are found in silver preparations of tissues, in regard to whose structure more definite informa- tion is obtainable. For example, let the eye of a frog be placed entire in a half-per-cent. solution of nitrate of silver, after a minute or two let the anterior corneal epithelium be romoved, and the eye replaced in the solution for some minutes longer, and then finally removed. ‘The cornea is then to be excised, placed for a second in half-per-cent. salt solution, and then exposed to the light in glycerine. After it has become brown let the upper layer of the cornea be removed at some parts under a dissecting-lens. The following fact is then frequently to be-made out :—the silver has diffused itself equally through the whole thickness of the more super- ficial layer, but has not penetrated to the deeper layer. There has been evidently an obstacle to the penetration of the solution from the upper to the subjacent layer. The nature of the part or whole of the structure which constitutes this boundary or obstacle may be learned by a rare chance in the frog’s cornea itself, but may be more easily shown in the cornea of the mouse. The mouse’s cornea is peculiarly well adapted for the demonstration of many points in regard to the histology of that structure. Ifa mouse’s cornea be ex- cised by a single stroke of the scissors and placed in silver solution, and be thence transferred to the light in glycerine, the epithelium of Schweigger-Seidel, of the existence of which it has taken so long to convince some histologists, may be demonstrated with comparative ease. It is not meant that success in its demonstration is inva- riable in the cornea of the mouse, but it succeeds in that animal as frequently as many of the ordinary histological processes 16 DR, G. THIN, which are not supposed to be attended with any special difficulty. The layer of larger flat cells indicated by the silver lines will be found on disintegrating the tissue by needles to correspond to the surface which limits the penetration of the silver in the instance above indicated. It may be taken, then, as a fact that a silver solution may permeate through a tissue, but be arrested by an epithelial layer in the depth or thickness of the tissue. ‘To associate the narrow and anastomosing colourless linear spaces that permeate the substance of a silver cornea with the cells of a similar nature seems justified when it is considered that by potash solution there can be isolated from the cornea multitudes of elongated, narrow cells, and that in gold preparations of the mouse’s cornea, and more rarely in the cornea of the larger mammals, rows of similar elongated cells can be-traced, taking their departure from layers of polygonal flat cells, and penetrating the interstices of the tissue. In the mouse’s cornea treated by gold the layers of flat cells capable of demonstration are, in the author’s expe- rience, composed of smaller cells than the layers of larger cells which are seen in silver preparations. In potash preparations cells corresponding to those of both layers areisolable. The smaller cells can be seen occasionally ina healthy mouse’s cornea treated by gold, but more readily when the structure has been inflamed for a few hours before the animal has been killed. In the cornea of a frog sealed up in aqueous humour the author, in conjunction with Dr. Ewart, saw the elongated narrow cells in situ covering the primary bundles, applied, therefore, to structures having a signification similar to the darkly-stained bands in the silver-treated cartilage (see Figs. 14 and 15). The unstained lines in the cartilage pre- parations correspond to the rows of cells seen by them in the cornea. When it is considered that elongated narrow cells, similar to but shorter than most of those which are seen in the cornea, exist in cartilage, the presumption becomes very strong that in the cartilage, as in the cornea, they invest the narrow bands of tissue on which they lie, and that the un- stained lines seen in section correspond to the cellular invest- ment of the darkly stained cylinders of ground-substance into which the silver solution has permeated. Recklinghausen’s doctrine of Saft-kandlchen rests mainly on the fact that in silver preparations communicating colour- less spaces are seen in a dark ground, and the unstained stellate spaces in the cornea have been regarded as peculiarly well adapted for its demonstration. Recklinghausen, believing - ON THE STRUCTURE OF HYALINE CARTILAGE, Vy that the fibrillary tissue is knit together by a cement-sub- stance, has taught that this cement-substance of the cornea is tunneled by a system of communicating canals, and that it is this system of canals which is not stained by silver. The author dissents from this opinion, believing that the demonstration of layers of flat cells covering free spaces in the cornea, and of the stellate cells as being situated in spaces formed by the separation of these layers from each other at given points warrants the conclusion that Recklinghausen’s Saft-kandlchen are not closed canals, but are spaces left by apposed membranes receding from each other. In narrower spaces thus left the branches of the stellate cells ramify. Recklinghausen confounded these spaces between the layers (Saft-Landlchen) with Bowman’s corneal tubes, which the author subsequently confounded! with the lymphatic chan- nels in which the nerves lhe. The corneal tubes described by Bowman are different both from the so-called Saft-kandlchen of Recklinghausen and from the lymph-channels of the nerves. They are spaces between the primary bundles of the cornea, and have never been described or figured as white spaces in a silvered cornea. In Schweigger-Seidel’s plates the injected mass is seen penetrating these straight spaces or corneal tubes, and the author has seen them indicated by rows of fine black gran- ules in silver preparations. Recklinghausen’s Saft-kandlchen and Bowman’s corneal tubes are neither of them closed channels. The Saft-kan- dlchen are spaces between the lamine or larger bundles, and the corneal tubes are the spaces between the primary bundles of fibrillary tissue of which the larger bundles are composed. It is evident that the size of the spaces may vary in accord- ance with the quantity of lymph fluid that passes along the channels, an increase of the calibre of these latter involving no other change than an increase in the extent to which the layers and bundles are separated from each other. This doctrine, in its full integrity, is now—based on the facts detailed above—sought to be applied to hyaline car- tilage, in which the author’s preparations show that both the stellate and the parallel systems of lymph-channels exist. The parallel system—corresponding to Bowman’s tubes in the cornea—has been shown by an application of the silver method which has not yet succeeded for the cornea, the pro- duction, namely, of straight colourless spaces between darkly stained primary bundles. In interpreting the significance of the epithelial layers 1 ‘Lancet,’ Feb. 14, 1874. VOL, XVI.~=NEW 8ER. B 18 DR. G. THIN. found free in potash and seen in sections the following facts are to be borne in mind:—In silver preparations of the surface of articular cartilage a layer of cells can be demon- strated in which the intermediate dark substance is so scant as not only to suggest but to justify their classification as epithelial. In potash preparations layers of cells are found free which, making due allowance for the difference in the mode of preparation, can be identified with a probability which amounts almost to certainty as being similar cells to those which are seen in the silver preparations. The differ- ence between these cells, when demonstrated by silver, and ordinary epithelium similarly treated, the author attributes, not to a difference in the cells themselves, but to their rela- tion to the cartilage-substance, on the surface of which they may be considered to be firmly imbedded. Set free by potash, no difference can be observed between them and ordinary epithelium. In regard to the epithelial layers seen by silver in sections it is to be considered that in potash preparations layers are seen presenting two kinds of appearance. They are either free in perfectly flat sheets, or they are seen applied to the convex prominences and concave depressions of fragments of the cartilage-substance. It is impossible to be certain that the free sheets in the one case are not the same cells seen still undetached in the other. The analogy of the cornea would seem to suggest that they are not the same. In the cornea the layers of polygonal flat cells are of two kinds. One kind is most easily demonstrated by silver, and consists of flat layers composed of large cells; another kind is most easily seen by gold, and, although in fortunate cir- cumstances demonstrable as continuous broad layers, is most often seen partially as forming a broad network of small poly- gonal cells following the channels formed by the separation of the lamelle in the position of the stellate cells. The former suggests the idea of a thin fascia investing the larger portions of the cornea-substance, the latter of layers of cells investing its larger bundles. Similarly it is suggested that the layers of flat cartilage-cells found free in the potash solu- tion, and those whose outlines and nuclei are represented in Fig. 6, as they were seen in a silver preparation, belong to a thin layer of substance of the nature of a fascia, and that the cells seen adhering to the cartilage-substance in potash pre- parations invest more closely the cartilage-substance proper. The explanation is hypothetical, but it is a hypothesis sug- gested by facts. In regard to the elongated narrow cells nothing can be ON THE STRUCTURE OF HYALINE CARTILAGE. 19 considered as proved except their existence in cartilage, but analogy with other tissues suggests that they are applied to the surface of the cylindrical bodies seen in silver and serum preparations (primary bundles of the author as previously described in regard to other connective tissues). In reference to the appearance of a coherent circular mass bulging from the end of a separated portion of such a band or cylinder the following explanation is suggested :—It is highly probable, for reasons which need not now be entered on, that in all the forms of connective tissue these bands or primary bundles are invested by a resistant delicate mem- brane on which the narrow elongated cells lie. In all tissues in which the author has seen them these bulgings have been occasionally observed, and have been invariably snared at the point of their escape from the cylinder by a sharply defined ring continuous with the contour of the cylinder. These appearances may be well studied in the cornea of a frog which has been broken down by acute inflammation, however produced, which has lasted about a week, the cornea being then stained in gold. The production of giant cells from healthy cartilage by simple prolonged maceration in aqueous humour in a sealed preparation must be considered, in the present state of patho- logical inquiry, as a fact of great importance. During the author’s experiments with caustic potash, especially in cartilage, he had frequently seen irregular masses occurring in imper- fectly successful experiments which resembled in contour, size, and the arrangement of nuclear vacuoles the bodies known as giant cells, whilst at the same time they could be recognised as partially disintegrated single and double layers of epithelial cells and intermediate substance. So accurate was this re- semblance that the hypothesis which the author believes to have received complete confirmation from the aqueous- humour preparation had already presented itself to his mind. If giant cells can be isolated from healthy adult cartilage by simple maceration, it is unnecessary to assign any other reason for their presence in the neighbourhood of cartilage and bone which are undergoing absorption during the process of ossification than that, during the disintegration of the absorbed structure, sheets and fragments of sheets of flat (epithelial) cells are set free, but before they are loosened from their connection they have become so much disintegrated that the lines of junction of the individual cells have become obliterated, and nothing remains but a shapeless protoplasmic mass, containing either nuclei still entire or vacuoles cor- responding to nuclei that haye undergone destruction. ‘The 20 DR. G, THIN. physiological connection of the structure with the living organism has ceased to exist. The strongest objection that could be raised against this interpretation of “‘ giant cells ” —namely, want of proof that layers of flat cells exist—is answered completely as far as cartilage is concerned by the observations recorded in this paper. Kolliker does not bring forward a single argument in support of his hypothesis that giant cells or osteoclasts are actively concerned in the absorption that takes place in growing bone, except that they are found on surfaces in which absorption is taking place, a fact which finds a much simpler interpretation on the hypothesis that they are a product of the absorption. ‘That they are found in ossification that is taking place where there is no cartilage to absorb does not materially affect the argu- ment when it is admitted that they exist in cartilage. Hitherto layers of flat cells have been as little suspected to exist in cartilage as in bone. But even in bone the author relies on more than mere analogy in believing them to exist. He succeeded on one occasion in isolating by potash from the shaft of a frog’s femur great numbers of oval cells, and on several occasions he has seen on fragments of bone treated by the solution numbers of round nuclei, the distribution of which corresponded with the supposition that they belonged to cells whose outlines were imperfectly visible, but which in so far as they could be seen suggested an epithelial arrangement. In regard to the “ giant cells” found in pathological pro- cesses it is highly probable, in the light of the above observa- tions, that they are simply fragments of altered and disintegrated membranes, covered with flat cells such as are present in all connective tissues. Ifthe author may be allowed with diffidence to express an opinion in regard to an appearance that has been studied by so many eminent observers, he would suggest that many of the giant cells (osteoclasts) found in the marrow of young bones present when examined under the microscope (no regard being had to any theory) the characters of effete and disintegrating tissue. It remains to consider what relation the structures above described have to the spaces known as cartilage ‘‘ capsules” and their contained cells. As has been already stated, fragments of cartilage can be isolated from a mass treated by the potash solution in which a series of concave depres- sions are distributed in perfect analogy with the distribution of the “‘ capsules.” A so-called capsule consists of two such concavities being applied to each other in such a way that ON THE STRUCTURE OF HYALINE CARTILAGE, 21 a wide space, unoccupied by cartilage-substance, is enclosed by them. ‘The edges of this space are formed by the appo- sition of the edges of the concavities, but although in close apposition the two layers remain anatomically distinct. In sec- tions treated by silver white tracts connecting the spaces, such as are represented in Figs. 10 and 11, or as have been shown by Heitzmann,can be obtained, and areindicative ofthe continuity of the cell-covered membrane that forms the free surface of the concavities and the intervening flat surface. As can be easily shown for the cornea, so is it equally true but more difficult to show for cartilage, that the cell-spaces are nothing . more than the separation from each other of distinct lamine. Positive evidence of this can be found in potash preparations, but itis also supported by silver preparations, and by the result of such a treatment by osmic acid as is represented in Fig. 13. A white space in a brown ground, as seen in silver preparations of connective tissue, depends upon the fact that the thickness of a space from one epithelium (endothelium) to the other of the two membranes whose separation produces it is sufficient to prevent the dark ground-substance coming into focus. Where the cell-covered membranes are in closer apposition it is impossible to obtain a focus that does not include the dark ground-substance that is above or below the layer of cells. Consequently the unstained thin layer is optically lost in the stained tissue. No tissue is better suited to work this out practically than a well-silvered cornea studied under a good immersion-lens. The ordinary granular protoplasmic cells of hyaline carti- lage are analogous, according to the views of the author, to the stellate cells of the cornea and connective tissue gener- ally. In osmic-acid preparations, and in sections soaked in solution of logwood and alum and subsequently treated by acetic acid, when the protoplasma of the cell had shrunk round the nucleus in the centre of the space, he has seen fine glistening fibres enter the cartilage-substance, into which, however, he has not been able to follow them. Such prepa- rations have not been drawn, because it is believed that pre- parations of a more demonstrative nature may yet be obtained, and because none of them have been so conclusive as the results of maceration in blood-serum shown in Fig. 20. That only one process has been traced for each cell in such a preparation is due to a law that seems to hold good for stellate cells in other tissues, and especially in those of bone, that although there are many fibres given off the fibres which are continuous with the long axis of the cell are not unfre- quently, more developed than those which spring from its 22 DR. G. THIN. sides. The terminal fibres at opposite poles are accordingly often visible when the others are not seen. In the present case one of the terminal fibres is necessarily cut off when the section ismade. It is worthy of remark that although when covered by the cartilage the cells in question show no special disposition to assume an elongated form, they invariably do so when the cavity has been laid open. The drawings of Bubnoff and Heitzmann theauthor believes to represent spaces that exist between the layers of cartilage. He is thus in accord with Bubnoff more than with Heitzmann. Heitzmann believes that the appearances which he has re- produced are those of a cell and its protoplasmic processes. The author interprets the appearances shown in Heitzmann’s own drawings as representing stellate spaces, and sees nothing in them that he can interpret as cell-processes; being thus at one with him as regards the fact observed, but differing from him in regard to its interpretation. In that part of cartilage which adjoins the demarcating line characterised as the zone of absorption when bone is being formed there is a preliminary absorption which ex- tends further into the cartilage than is generally supposed, although, not being permeable to blood-vessels, it is not so evident. For some distance into the cartilage the separation of the layers which compose the walls of the spaces increases in extent until the intervening narrow communication between two spaces is swamped, and what was originally two spaces with their intervening communication becomes one large space. A section through cartilage at this point, if stained in carmine or picrocarminate, shows the nucleus of each of the cells which lay in the original spaces lying at opposite ends of the newly formed large space, an appear- ance which without more ado has been deemed conclusive proof of the division of one cell into two. A further separa- tion of the lamine permits the entrance of new blood-vessels into the space, and in the language usually employed the “capsule” or “‘ cell’’ is said to be ‘‘ opened.” In this series of changes the relation of cause and effect between the two processes of increased entrance of lymph- fluid and thinning of the walls of the laminz has not been determined by direct observation. The author acknowledges his obligation to Dr. Ewart for executing the following drawings, and other valuable assist- ance rendered him whilst engaged in the studies which have served as a basis for this paper. August, 1875. POLYSTOMATOUS CONDITION OF CORDYLOPHORA, 23 On a Poxtystomatovus Conpition of the Hyprantus of CoRDYLOPHORA LACUSTRIS. By Hucu Pricr, Demy of Magdalen College, Oxford. Specimens of the beautiful hydroid polyp Cordylophora were brought by me last summer from the Victoria Docks for the use of the class of practical zoology at University College, London. Professor Ray Lankester drew my attention to certain abnormal-looking hydranths amongst these specimens which appeared to possess a number of supernumerary oral cones. I isolated some of these abnormal specimens and made them the subject of further study. It should be stated that these observations were made at the end of June, 1875, on specimens which had apparently already produced their crop of gonophores, and consisted almost entirely of nutrient hydranths which were develop- ing with some vigour, as evidenced by the clean and pale flesh-coloured appearance of their supporting stalks. I presume that a crop of gonophores had been already pro- duced by these specimens, since others obtained from the same locality were in full sexual maturity. An outline sketch of two of the polystomatous hydranths is given in Figs. 1 and 2, where o ¢ indicate the oral cones. Fig. 1 is a pentastomate, fig. 2 a tristomate hydranth, each cone having an oral aperture at its apex. The oral cones are larger in the latter than in the former, and are seen to present scattered tentacles on their surfaces, in addition to those which are present on the common body uniting the three cones. The questions which presented themselves for solution in connection with this hitherto unrecorded mode of growth in Cordylophora were—1. Is such a production of super- numerary oral cones a normal phenomenon in this genus? 2. Do the cones proceed to develop into complete hydranths and become divaricated by the production of interposed hydrocaulin? or do they become detached as fissiparous buds? or does the monstrous condition maintain itself per- manently? 3. Have the supernumerary cones developed in consequence of an injury to the parent hydranth? or were they indicated already in the earliest condition of the hydranth which bears them ? These questions 1 have not been able satisfactorily to resolve, but think it desirable nevertheless to place the facts observed on record as well as some observations on arti- ficially produced polystomatous hydranths of Cordylophora, 24 HUGH PRICE. which may tend to explain the facts observed in the naturally produced specimens of the same kind. As to the first question, it would appear from the fact that Cordylophora has been attentively studied by two ex- cellent observers, Prof. Allman and Prof. Eilhard Schultze, Fie. 1. who have not recorded the existence of polystomatous hy- dranths, that their occurrence must be exceptional. On the other hand, they were not uncommon on a particular mass of the Cordylophora colony, though they were not found on all colonies gathered with this mass—and have not been previously seen by Prof. Lankester in specimens taken at the same season from the same locality. As to the second question, I endeavoured to obtain a definite answer. I isolated some fragments of the colony bearing each a single polystomatous hydranth. Unfortunately I was not able to follow these specimens with sufficient care, but it appeared after a few days that the polystoma- tous hydranths had disappeared, not only in the isolated specimens, but in those still living in the general aquarium containing the mass of the specimens. From this it may be concluded that the polystomatous hydranths had proceeded to develop in some way or other. The opinion that the supernumerary oral cones are but a transitory stage in an exceptional mode of development is borne out by a com- parison of Figs. 1 and 2, in the latter of which the cones are relatively larger than in Fig. 1, and have, further, a POLYSTOMATOUS CONDITION OF CORDYLOPHORA. 25 number of tentacles upon their sides, which is not the case with the specimens drawn in Fig. 1. Question three is suggested by some observations which I made by snipping with scissors the oral cone of normal hydranths. These observations demonstrate that there is a very remarkable power of repair and what may be called “polarity ” in Cordylophora. When a cut is made along the long axis of the hydranth the sides of the separated parts almost immediately come together, and after twenty-four hours both halves of the slit hydranth appear as complete hydranths with oral cone and tentacles (Figs. 3 and 4). If the cut is made so long as to extend to the proximal end of the hydranth experimented on, the two portions are in a few days found to have developed each a distinct segment of hydrocaulus surmounted by a complete hydranth. These facts lend support to the view that the supernumer- ary oral cones observed in the fresh specimens from Victoria Docks may be due to the injury of the parent hydranth by the attack of some crustacean or to other mechanical injury. Other experiments which I made do not bear directly on the production of polystomatous hydranths, but serve to illustrate the reproductive power of the tissues of Cordy- lophora. Some of the hydranths, with a bit of hydrocaulus, were snipped off from a healthy colony and placed in a vessel 26 HUGH PRICE, which was kept undisturbed for some days. The cut-off hydranths were found to attach themselves by the cut end of the hydrocaulus to the sides and bottom of the vessel and to give rise to new colonies. Thin cover-glasses were placed on the bottom of the vessel, and to some of these the detached hydranths affixed themselves, thus giving a means of study- ing the mode of attachment. The cut end by which the animal appears to attach itself is seen under the miscroscope to have given rise to a knob-like swelling, consisting of endoderm and ectoderm. But it is not by means of this knob directly that the hydranth becomes affixed. Delicate filaments, continuous with the horny perisare are seen ex- tending from the neighbourhood of the knob, and these fix the organism to the glass. The knob, on the other hand, proceeds to point in a direction away from the surface of attachment, and gradually develops a bud, which elongates into a complete hydranth, with a rapidly growing stalk or hydrocaulus. Thus two main stems are united at the point of attachment, viz. that of the original hydranth cut off for experiment and that of the new hydranth which has grown out from the cicatrix or knob. Further (at this time of the year and in these particular specimens) there is a great tendency on the part of the proximal segments of hydrocaulus still attached to the colonies from which hydranths have been cut to produce new hydranths. I have even noticed this on old pieces of hydrocaulus overgrown with Vorticelle and Alge. I had occasion to cut a considerable piece off from a main stock, and some of the oldest part of the colony was removed with it. After five days there was developed from the cut end of that portion of the old stalk still attached to the main colony a new hydranth with about half an inch of new stalk attached to it. These observations on reproduction from the hydrocaulus correspond in the main facts with those of Dalyell and Ailman on Tubularia, but the production of a new hydranth- bud from the cicatrix-knob of the radical end of a divided hydrocaulus is not recorded in the case of Tubularia. The facts above noted show that Cordylophora is well adapted for the study of some of the phenomena of artificial repro- duction.! 1 T may add to these notes that Cordylophora is by no means difficult to keep alive if provided with a very large quantity of water and kept in the dark. Specimens taken in June are still alive (October). These specimens consist of fragments of a colony numbering only some half dozen hydranths. Each fragment has been kept in a covered half-pint jar in a dark cup- board.— HK. Ray LANKESTER. ON A PEACH-COLOURED BACTERIUM, 27 FURTHER OBSERVATIONS on a@ PEACH- or RED-COLOURED BacrErium—Bacterium rubescens. By KE. Ray Lan- KESTER, M.A., F.R.S. (With Plate IIL.) Tux growth varying in colour from peach-blue to a ruby- red which I described in this Journal in 1873 (vol. xiii, page 408, Plates XXII, X XIII) has been under my observation from time to time during the last two years, and I have ascertained a variety of new facts with regard to it which add very much to its interest. Frequency of Occurrence.—1 have had no difficulty in ob- taining any quantity of the red growth formed by B. rudes- cens ; it is probably familiar to most microscopists who occupy themselves with the lower organisms of fresh waters. Almost any mixture of Alge and Infusoria which is allowed slowly to putrefy becomes the seat of its development— thick, red-coloured crusts forming on the sides and bottom of the jar in which the putrefaction is proceeding, and resembling the coloured films which form on the sides of a bottle of Burgundy wine. I have received a particular form of this growth (of which more below) from my friend, Mr. Charles Stewart, F.L.S., of St. Thomas’ Hospital, who finds it in large quantities in the macerating tubs which he uses for the preparation of skeletons. Further, from my friend, Mr. Archer, F.R.S., of Dublin, I have received other samples taken ina pond fifty miles distant from Dublin, which I do not feel any doubt are to be considered as gr owths of Bacterium rubescens. To these also I shall allude further below. Lastly, I am very much disposed to believe that the pink- coloured Spirillum, described by Dr. Klein in the October number of this Journal for the year 1875, is a spirillar phase of Bacterium rubescens. Dr. Klein was good enough to show me his specimens, and I base my opinion on a micro- scopical examination of the growth and on Mr. Page’s state- ment, given by Dr. Klein, as to the absorption-spectrum, which so far as it was observed (the colour not being very intense) corresponds with that of Bacterio-purpurin—the colouring matter of B. rudescens. ¢ Variation in Colour.—The main fact on which I rely for the identification of the various forms and aggregations of plastids, assigned to Bacterium rubescens as members of a series or physiological species, is their agreement in colour. 28 PROFESSOR LANKESTER, I assume it to be highly improbable that two or more speci- fically distinct organisms would develope in a jar simulta- neously, each tinted by the peculiar substance Bacterio-pur- purin. At the same time the hypothesis that the various forms observed do form a series belonging to a protean species is confirmed by the observation of all conceivable intermediate annectent forms. In my former paper I pointed out that the colour of the films was liable to a certain variation, tending at one time to blue and at another time to red. ‘This variation in tint, which I have now constantly observed, occurs in response to definite variations in the conditions of growth, and is, I believe, satisfactorily explained by the predominance of either a blue or a red element of the complex colouring matter Bacterio-purpurin. A certain brownish tinge is also obsery- able under some conditions, and is, I conceive, to be ex- plained similarly. The bluer peach tint occurs when the growth is young, and has been especially noticed by me when it occurs in the form of a dense film (the plastids being arranged like a mosaic or “ tesselate,” as in the mycoderma phase of Bacterium termo, or in ‘ catenular” series as seen in Plate XXII, fig. 3, of my former paper), encrusting vegetable matter such as dead twigs of trees. ‘The redder colour is assumed when the growth is more luxuriant, and is espe- cially brilliant when the growth takes on the form of large homogeneous discs (to be described in the present commu- nication). In small specimens of these discs and also in small biscuit-shaped plastids from Mr. Stewart’s macerating pan I have seen the colour of the greatest intensity, only to be compared to the deepest magenta dye. Also in the same conditions which furnish these most brilliantly coloured examples are to be noticed others of a decidedly brownish tint (Plate ITI, fig. 4, fig. 20). As will be mentioned below, the conditions under which the homogeneous discs are developed have not been deter- mined, but their occurrence seems to be due either to the exhaustion of nutriment in the jar containing the growth, or to the dying down of the green unicellular alge with which Bacterium rubescens is usually most intimately asso- ciated and upon which it may be dependent for oxygen, and such services as the gonidia of the Lichen render to the hyphal fungus. Variation in the size of the Plastids——As was pointed out in my previous paper, the form of the plastid of Bacterium rubescens may vary greatly. It may be spherical, biscuit- ON A PEACH-COLOURED BACTERIUM, 29 shaped, filamentous, or irregular. We shall see now that it may also be discoidal. I pointed out previously that there was considerable variation in the size of the plastids, and gave figures illustrating this. The discoid plastids now to be described, whilst retaining a perfectly homogeneous structure, may attain each to the diameter of ;1;th of an inch, whilst the largest biscuit-shaped plastids described in my former paper scarcely exceed the ,-4;th of an inch in length. Under certain conditions it seems that very small biscuit-shaped plastids (very similar in shape and size to the particular form-species distinguished by Cohn as Bacterium termo) are uniformly produced by Bacterium rubescens. A group of these is seen in Plate III, fig. 4. The plastids do not exceed the z,4,,th inch in length. In this case I am able to point to a special condition of life as connected with this uniform shape and small size. These are the only form of Bacterium rubescens occurring in the macerating pans of St. Thomas’ Hospital.* They occur in immense quantity, covering the bones with a red film and forming crusts on the side of the vessel. It appears to me that the small size of the plastids and their uniform character may be due to the unconscious process of cultivation and domestication to which the museum curator subjects them in his macerat- ing tub. A uniform temperature is maintained, an immense excess of nutritive matter is continually present of a uniform character (namely, decomposing bones), and the macerating tub once established is kept in operation for many years. Under these circumstances it is not unlikely that a par- ticular “ breed” or “race” of the protean Bact. rubescens has become established, just as very possibly in other par- allel cases (for instance, that of Saccharomyces cerevisiae) races or even species of ferment-organisms have been estab- lished by the unconscious operation of mankind. I made some experiments with the view of ascertaining whether by changing the conditions of life of the maceration-breed of Bact. rubescens its form or colour could be affected in any way. Some of the red scum from the macerating pan was isolated and placed with distilled water in a test tube. It was thus cut off from further accession of nutriment. It was left undisturbed for three months; at the end of this time no change visible to the naked eye had occurred in the 1 The colouring matter, which is very rich, gave the characteristic bands of absorption of Bacterio-purpurin. 2 It should be very explicitly stated that a large proportion of green unicellular alge is present with the red Bacterium in the macerating pans, intermixed with it. 30 PROFESSOR LANKESTER. red sediment formed by the scum, excepting that its colour was a little more intense. On examination with the micro- scope, however, the plastids were found to have altogether changed the character of their growth. Instead of keeping down to a small size by repeated transverse fission they had all increased individually in size. Some were a little more than double the length of the specimens taken fresh from the macerating tub, and presented two, three, or more brightly red-coloured, highly refracting granules in their substance, similar to those seen in the plastids drawn in fig. 3, Plate III, which do not, however, represent these particular specimens. This change of size and character in the plastids in attendance upon change of environment (removal of excessive nutrition) warrants the inference which I had already drawn from the large range of variation in the forms and mode of aggregation of the plastids of Bac- terium rubescens—that we have in it (and probably also in the true physiological species of colourless Bacteria, dis- tributed under such form-genera as Micrococcus, Bacterium, Vibrio, &c.) an example of an organism which is highly sus- ceptible of change of form in response to minute changes of life-conditions, and in which such changes of form have full license because form has very little importance in relation to the essential chemical phenomena which characterise the life of this class of organisms and give them specific limits. Movements of B. rubescens.—In my former account of this organism I stated that I had not observed active “ vital ” movement exhibited by the plastids except by the “ acicular ” form. I am now able to modify this statement very essen- tially. I have frequently observed active vital movement exactly corresponding to that of Bacterium termo and B. lineola in the biscuit-shaped or bacterioid form of plastid of B. rubescens. The plastids drawn in Fig. 2 formed part of a growth in which nearly all the plastids were of this homo- geneous character and exhibited the active darting move- ment of living Bacteria. On other occasions I have found growths of plastids of the character of those given in ' Fig. 3, which also exhibited the unmistakable vital move- ments known and described in other Bacteria. Further, I have on more than one occasion observed slow but continuous undulating movement in the Leptothrix forms of B. rubescens, such as are figured in my former paper (Plate XXIII, figs. 24, 25). The maceration variety from St. Thomas’s Hospital (fig. 4) also exhibits active vital movements. From recent study of B. rubescens and other forms of Bac- ON A PEACH-COLOURED BACTERIUM. él teria I am inclined to think that the former is not, on the whole, a more sluggish form than are the colourless species. In the gleeogenous condition all Bacteria are motionless, and it is only the biscuit-shaped form of plastid or still more elongated forms which exhibit “ vital’? movement. The question as to the mechanism of locomotion in the Bacteria is one which I have not been able to assist in solving by examination of active B. rubescens. A priori, 1 suppose all biologists are inclined to adopt the view that the motile plastid of Schizomycetes is provided with one or two protoplasmic flagella. ‘The evidence in favour of the exist- ence of such flagella is very nearly as convincing as any that could be obtained by the actual inspection of a filament attached to the plastid. I confess that I have not been able to see the flagella in any form of Schizomycetes which I have studied for the purpose, but I think there is every reason for admitting the truth of the recent observations of Messrs. Dallinger and Drysdale on this point. New Transition-forms of Plastids and Aggregates.—lIt will not be necessary for me to repeat, on the present occasion, the enumeration of the various kinds of plastids presented by B. rubescens, and the various modes in which these plastids unite to form aggregates, linear, globose, retiform, and tes- selate. I shall, without any preface, draw attention to two interesting forms which I have observed since 1873, refer- ring to my previous paper for a general account of the forms assumed by the species. Fig. 3, Plate III, represents a series of plastids from a growth in which such forms constituted the greater part of the sample. They may be described in the terminology which I have adopted, as multilocular bacterioid plastids— which occur in this case isolated—and free from a gelatinous matrix. ‘Those to the mght hand side are interesting on account of their elongation, furnishing, as they do, a transi- tion-form to the filamentous plastids (leptothrix-form) figured in vol. xii of this Journal, Plate XXIII, figs. 24, 25. Fig. 5in Plate III is a small specimen ofa form of B. rubes- cens which is rare,and of very great beauty and interest. I had only met with it on two occasions (one specimen being that here figured), when I received, in September last, from my friend Mr. Archer, of Dublin, a gathering which contained a great quantity of it, aud very much larger aggregates of it than that here figured. ‘This form, which consists of strongly marked unilocular bacterioid (biscuit-shaped) plastids (almost like an hour-glass in shape), united with the greatest regu- 32 PROFESSOR LANKESTER, larity in rectilinear series, may be described as the rectilinear tesselate form of aggregation. Sarcina, and more especially Merismopedia, furnish very similar aggregates of their con- stituent plastids. ‘The gathering which I received from Mr. Archer contained sheets of plastids in which the plastids were of this hour-glass shape, and arranged in this same rec- tilinear manner; but the-sheets were of larger size, consist- ing of as many as twelve or twenty rows. The aggregates of this kind very generally are nearly square plates, and have so symmetrical an aspect as to suggest rather a work of art than a natural growth. Frequently, at the sides of the square plate, a group of four, six, or eight plastids has become de- tached and fallen away, leaving a square hole or blank in the series. I hesitated for some time about regarding these regularly arranged plates as forms of B. rubescens, until the study of Mr. Archer’s specimens enabled me to satisfy myself that the plastids are really coloured centrally with the cha- racteristic purple-red pigment of that species, and also fur- nished additional proof of the connection of this form with the other varieties of B. rubescens, in the fact that they oc- curred in his gathering (as they had in those I first observed) in connection with a great abundance of other varieties assign- able to that species, such, for instance, as fig. 16 and fig. 21 of Plate XXIII of my former memoir. The form of the individual plastids, though very strongly marked, was already familiar to me. Some of this form are drawn in figs. 13 and 26 of my former memoir. In fig. 6, Plate III, I have represented a plastid of the same form, which is interesting as giving a transition between this very regular unilocular form and the multilocular forms. In ad- dition to the central hour-glass-shaped cavity filled with coloured material, a small eccentrically placed granule is seen, eating its way, as it were, into the thick colourless wall of the plastid. The plastids of the regular tesselate form of aggregation cohere laterally by means, no doubt, of the gelatinous sub- stance which forms a sheath to each of them. One would suppose, from the analogy of tissues and cell-development generally, that such a regular aggregate had been formed by a regular process of cell division im sttu. I am inclined, however, to believe that this is not the case, and that the tesselate aggregations of all kinds of Bacteria, as well as the retiform (such as that drawn in fig. 19 of my former paper), take their origin by the spontaneous apposition of indepen- dently developed plastids, just as the network of Hydrodictyon is developed. ON A PEACH-COLOURED BACTERIUM. 83 Macroplasts or Reproductive Discs.—The form of plastid which I have now to describe is one of altogether special interest, since it seems possible that we have in it a definite reproductive form, whilst the phenomena of division pre- sented by it, resulting in the formation of new colonies of plastids, are so exceptional as to be in themselves a matter of interest from the point of view of the histologist. At the same time, should it be found that the colourless Bacteria develop reproductive discs, or macroplasts of the same charac- ter as those of B. rubescens, we shall have ascertained a pos- sible source of origin for those excessively minute germs which are so widely distributed, and which so readily develop in putrescible liquids. In the jar of fresh water in which my Bacterium rubescens originally developed the growth was continually kept up by the addition on my part of dead animal matter, such, for instance, as an earthworm or a moth, or a limb of a crayfish. After luxuriating for a time, the red growth would always languish and become reduced to a small area, varying in colour from peach colour to brick red, and this film would lie at the bottom of the vessel beneath the black deposit of carbonised débris. When it had assumed this state I found that I could always call forth an abundant crop by the addi- tion of fresh animal matter. In September of 1873 the growth had been allowed to dwindle to its very smallest limits, and I then examined some of the very deeply coloured film which lay at the bottom of the glass jar, and was visible by inspection from below. I found it to consist of a felted mass of green unicellular alge, desmids, various organic filaments, and a large quantity of gleogenous plas- tids of Bacterium rubescens, some in the condition of sheets free from any excess of gelatinous matrix, and forming very highly coloured expanses. Such mycoderma-like frag- ments (but of much larger extent) as those drawn in Plate III, fig. 10 and fig. 22, were abundant, agreeing in the cavernous shape of the aggregate or frond with the Clathrocystis of Henfrey.! The most remarkable appearances, however, were a large number of disc-like bodies, of very various size, deeply impregnated with the red colour, distinguished as Bacterio-purpurin. These bodies varied in size from the dimensions of an ordinary biscuit-shaped plastid of B. ru- bescens to that of a circle with ;4,th inch diameter. The ‘ I am informed by Professor Ferdinand Cohn that he has referred this form of B. rubescens to Henfrey’s genus as Clathrocystis rubeo-persicinus. He also considers some of the forms of my B. rudescens to be the Monas Okeni of Ehrenberg. VOL, XVI.—NEW SER. Cc 34 PROFESSOR LANKESTER. average diameter of these discs was about the =1,th of an inch and less. A series of them is represented in the plate (Plate III), which gives a representation ofa possible group of these bodies (figs. 7 to 28), as obtained by teazing such a film as that described. With a power of 600 diameters the disc-like bodies are seen to vary both in colour and struc- ture. Some are of: the very deepest ruby red (figs. 14, 23); others are paler, but still of a rich colour (fig. 16); whilst others, again (figs. 19, 20), have a decidedly brownish tint. With the power of 600 diameters (Hartnack’s No. 8 objec- . tive and No. 4 eye-piece) many of the discs appear homo- geneous, devoid of all granulation, and of gelatinous border or capsule. This is the case equally with figs. 16, 17, 18, 19, 20, and 21. On the other hand, with the power named, figs. 12 and 13 appear granular, and fig. 9 is seen to consist of a closely adherent mass of plastids with a gelatinous border. Fig. 15, again, is seen to have a relatively larger amount of gelatinous matter separating its highly coloured loculi (each of which corresponds to a plastid), whilst in fig. 7 we have a form in which the primitive gleogenous sphere is breaking up into secondary spheres, each secondary sphere containing numerous highly coloured loculi. When a higher power of the microscope is applied to these discoid objects—namely, the No. 10 immersion of Hartnack, with No. 4 eyepiece, giving an amplification of 1100 dia- meters with the best definition and penetration which I know of at that point of magnifying power—some of the apparently homogeneous discs are resolved, and are found to present a minutely punctate structure. Others resist the test and still appear—as I conclude they are (within the physical limits of the word)—homogeneous. Such homogeneous discoid plastids are represented in fig. 14, fig. 16, fig. 20, and in the series of smaller bodies to the left of fig. 19. The most minutely punctate forms are seen in figs. 18, 17, and 21. When teased or broken by pressure of the covering glass these discoid bodies give evidence of being composed of a somewhat tough coherent substance, and do not diffuse or break up into granules. The following is the explanation which I have to offer of these appearances. Under certain conditions of growth, which I have not been able to determine specifically, but which are possibly amongst others those of diminished nu- trition—the plastids of B. rubescens cease to multiply by transverse division or to form filamentous or other irregularly extended growths. They cease also to form gelatinous envelopes and take on a physiologically homogeneous cha- ON A PEACH-COLOURED BACTERIUM, 35 racter (indicated by the uniform distribution of the colouring matter) and grow equally at the periphery so as to form small discs. ‘These are represented in fig. 11 when of small size. This uniform homogeneous growth may proceed until single plastids attain the very large diameter of ~1,th of an inch or more. This is accompanied frequently by great in- tensification of the colour, indicating a special physiological activity in the development of Bacterio-purpurin. Not unfrequently, however, the process which leads to the ela- boration of Bacterio-purpurin is so modified as to give rise to the brown variety of that pigment. Such discs, on account of their large size, I would call ‘‘ macroplasts.”’ These macro- plasts are certainly of a somewhat permanent character. I have kept one under observation for fourteen days without observing any change in its size or structure. The conditions which are necessary for this change are not known, but clearly enough they exist, and at one time or another the macroplast falls under their influence and takes on a new development. This may occur either when it is of comparatively small size or when it is of larger growth. The addition of fresh nutrient material to the jar containing the macroplasts led to their ultimate disappearance and their replacement by colonies of plastids of types similar to those previously described. Hence I am inclined to infer that under favorable circumstances as regards nutrition the macroplasts break up into immense numbers of small plastids. The first step in this process is seen in the excessively deli- cate granulation of such specimens as figs. 17, 18, and 21. In fig. 12 we have a small specimen in which the newly forming plastids have attained larger size and so on through the series—a gelatinous exudation being produced as the process of segregation advances (figs. 13, 9, and 15). If this be a correct interpretation of the appearances de- picted in the plate (Plate III), we have an instance of multiplication of “ centres of organization ” which does not conform to the type observed in ordinary cell-division, and which is comparable to free cell-formation in a blastema. Each plastid in any growth or aggregation of Schizomy- cetes may be regarded as a unit or centre of organization— just as much so as a true cell, though the Schizomycetous plastid differs from a cell in not possessing a central mass corresponding to the nucleus. The transverse division of an elongated or filamentous plastid of one of the Schizomycetes is equivalent to an act of cell-division and so far the proto- plasm of these organisms conforms to the general mode of ¢ ¥ 4 36 PROFESSOR LANKESTER. multiplication of units or centres of organization which is observed in other masses of protoplasm. Just as we find exceptional cases in animal and vegetable cells in which a mass of protoplasm gives rise simultaneously to numerous nuclei, each of which becomes surrounded by a segregated mass of protoplasm and produces a numerous cell-progeny by multicentral segregation, so it appears that in the large discoid macroplasts of Bacterium rubescens a formation of innumerable new plastids occur—not by a pro- cess of progressive division into two, four, eight, &c¢.— but by a simultaneous multicentral segregation. Such appears to me to be the explanation of the minutely granular structure detected only with the highest powers in such specimens as figs. 17, 18, and 21. The granules indicate so many new units or centres of organization, and each of them takes on independent growth, enlarges and finally becomes separated from its fellows by a gelatinous envelope. It would be necessary in order to establish this view to watch the steps of the process in one and the same macro- plast; and it is clear that during the period of increase in the size of the new units, the whole mass must greatly en- large. I have not succeeded in actually watching the process of development which I suppose may take place, but I draw attention to the fact that there is on the whole evidence of a proportionately larger size in those macroplasts which are coarsely as compared with those which are finely granular. This fact does not, however, count for very much, since one observes granular macroplasts of very various sizes from the so'ooth of an inch upwards. A second development of the macroplasts took place in the same jar after an interval of six months, during which time the “growth” had been carefully fed up, and it had become difficult to find any specimens of them still remain- ing. The second development of these bodies occurred after the supply of nutrient matter to the jar had again been dis- continued for some weeks. Specimens of the macroplasts in various stages of segrega- tion were mounted in glycerine, where they preserved their colour for a month or so, but subsequently assumed a pale, dirty green tint. Such specimens were sent by me to various naturalists interested in the study of the Bacteria, whilst my friend, Professor Thiselton Dyer, examined the growth in its living condition in my laboratory at Exeter College. Supposing the most finely granular macroplasts to be mechanically broken up into their minute constituent gran- ules, we should have a very prolific source of those exccs- ON A PEACH-COLOURED BACTERIUM, 37 sively small “ germs” from which Bacteria are supposed to take their origin in experimental infusions. Reasons for assigning Bacterium rubescens to the Schizo- mycetes and the Genus Bacterium.—lIt has been suggested to me by some observers who have examined samples of the growths which I have called Bacterium rubescens, that the organism in question is not to be regarded as a colour-bearing species of the group of Schizomycetes, but is rather to be classed with some of the glaogenous forms of the so- called unicellular Algz, such as Gloeocapsa. This view appears to rest upon two facts—firstly, upon the assumed absence of a motile phase of the plastids of our organism ; secondly, upon the presence of a strongly developed colouring- matter. The really important agreements between Bacterium rubescens and the Schizomycetes appear to me to lie in the following facts: that B. rubescens is one of the chlorophyll- Free Protophyta (consequently physiologically similar to the Schizomycetes and Fungi), and that it exhibits the charac- - teristic vegetation forms of the Schizomycetes, namely, spherical, biscuit-shaped, elongated or filamentous plastids, devoid of nucleus, and aggregated either in linear series or in sheet-like and massive fronds by means of a jelly-like matrix. The assumed absence of a motile phase is anerror. Pre- cisely under the same conditions and with the same constancy as is observed in thecolourless Schizomycetes, active darting movements of the plastids of Bacterium rubescens are observed. It is a mistake to allow the commonly observed active condition of the Schizomycetes, such as Bacterium lineola and B. termo, to dominate altogether in our conception of the essential features of the Schizomycetes. ‘These species exhibit, quite as commonly and abundantly as B. rubescens, quiescent phases and gloogenous aggregations distinguished by Cohn as “ zooglea.” There is very good ground for supposing that the common micrococcus which appears before and with the biscuit-shaped Bacterium termo in putrescent infusions, and which forms *‘ yosary-chains ” and other growths is a phase of the species, whose active condition is distinguished as Bacterium termo ; and we have similarly in B. rubescens—a micrococcus-phase (the spherical plastids described in my previous communica- tion), and catenular rosary-chain aggregations belonging to a species which in a certain phase of growth produces biscuit- shaped plastids, such as those drawn in Pl. III, figs. 2 and 4, These are undeniably referable to the form-genus Bac- 88 PROFESSOR LANKESTER, terium, as limited by Cohn, and often exhibit active, darting movements. The objection which is founded on the presence of colouring matter in B. rubescens is easily met. The pigment of B. rubescens contains neither phycocyan nor chlorophyll, and one or both of these colouring-matters is present in all Pro- tophyta and Thallophyta, which are not referable either to the Schizomycetes, Saccharomycetes, or to the so-called Fungi. On the other hand, pigmentary matter similar to Bacterio- purpurin has been observed in other forms recognised as Schizomycetes, and is not uncommon in various groups of Fungi. It seems to me that the presence of a red colouring matter in B. rubescens is a fact of no significance whatever, as far as its relationship to other forms is concerned. In considering the question of those relationships we should mentally bleach B. rubescens. Further as to the choice of the generic term Bacterium for the designation of our organism, I have this to say. From the known facts as to the mode of occurrence of those forms of Schizomycetes which Cohn has classed as Sphzro- bacteria, Microbacteria, Desmobacteria, and Spirobacteria, and from what I have been led to infer as to the connection of the various forms of plastids in my red growth, I consider that we must definitely accept what Professor Cohn himself, I believe, would approve—namely, that his groups and their genera are simply assemblages of forms and may include but a few real species, which are represented by phases of growth in each of his form-genera and form-families. This being the case, it becomes necessary to select generic terms for the designation of the true physiological species. From the widely accepted use of the term Bacterium, I am led to adopt it for the generic name of all those Schizomycetes which under certain conditions of growth present biscuit- shaped plastids capable of active locomotion. Accordingly I should place the red organism under con- sideration in the genus Bacterium as B. rubescens, together with B. lineola and B. termo. I am not in a position to discuss the whole question, but I think it probable that Bacillus is a really distinct genus and that the Spirobacteria are only form-phases of Bacterium and Bacillus. It appears to me to be an open question whether B. termo may not be a diminutive breed or specialised race of B. lineola, standing in the same relation to it as does the St. Thomas’ maceration-breed of B. rubescens (Pl. III, fig. 4) to its larger biscuit-shaped phase (PI. III, fig. 2). The con- ON A PEACH-COLOURED BACTERIUM. 39 ditions under which the larger and the smaller form respec- tively occur in the two cases are similar. The smaller in both cases occurs where nutrition is abundant and the sur- roundings rendered special by human agency; the larger occurs in natural waters (ponds and streams), where putre- scence is going on in the midst of conditions favorable to the maintenance of other forms of life. The Causes of the Aggregation of Schizomycetous Plastids to form Compound Structures of regular pattern.—I devote a separate paragraph to this subject, not because | have much to say with regard to it, but in order to draw attention to the great peculiarity (already noticed above) in the mode of for- mation of many of the aggregates of Schizomycetes. There is no doubt that the zoogicea or gloeogenous masses of spherical or cavernous (Clathrocystis-like) form increase in bulk by the transverse division of their constituent plastids. But there is reason to doubt whether division along the longer axis of these plastids ever takes place. In the case of the delicate films of Schizomycetous plastids which have been spoken of as the “‘mycoderma-phase”’ of growth, it results from observations of the arrangement and connections of the plastids that they must take up their positions to a large extent as the result of a spontaneous movement of apposition —a kind of mutual attraction, similar to that exhibited by mammalian blood-corpuscles when uniting to form “ rou- leaux.” The operation of this process of adhesion is modified no doubt by the continued growth of the plastids and their self- division at right angles to their long axes. The definiteness and beauty of the patterns resulting is very striking. Some of the most curious, exhibiting a very elagant combination of curved lines, are due to colourless species of Schizomycetes, and have never, as far as I know, been figured. The most striking of these patterns due to the ** fortuitous concourse ” of the plastids of B. rubescens are those seen in the “ regular tesselate ’ form, Plate ITI, fig. 5, and Plate XXIII of my former paper, figs. 19 and 21. Addendum.—I have omitted to describe two additional frond- forms, or aggregates exhibited by Bacterium rubescens. The first is not uncommon in very abundant growths of small biscuit-shaped plastids. The plastids are arranged in star-like groups consisting of 10 to 20 plastids, meeting one another at acentralpoint. This form of aggregate I mentioned in my former paper as observed in the case of the acicular form of plastid (see Plates XXII, XXIII, of former memoir, 40 CHARLES S. TOMES, figs. 2and 28). It*now appears that this stellate condition is assumed also by the more common biscuit-shaped plastids. The second additional form of aggregation was observed very abundantly in Mr. Archer’s gathering of B. rubescens. It may be described as cylindrical. Suppose a number of stellate aggregates superimposed and you have a cylinder. These cylinders are often six or seven times as long as they are broad. On the DEvELOPMENT of TEETH. By Cuaruzs S. Toms, M.A., Lecturer on Dental Anatomy at the Dental Hos- pital of London. With Plates IV and V. For many years past it had been thought that, in its broad outlines at least, our knowledge of the development of teeth was accurate; few persons have hence devoted their time to the verification of the current descriptions, and even now, though several years have passed since Kolliker and others first published their results, and proved that inaccuracies of moment existed in the received accounts, the old theories still hold sway in most of our text-books. This being the case, I propose to ggive a short outline of the leading facts in tooth development as they are at present known, not confining myself to my own researches (which relate chiefly to the teeth of reptiles, amphibia, and fishes), which are more fully detailed in papers published in the ‘ Philosophical Transactions.’ In the papers referred to, and in a manual of dental anatomy now in the press, I have given full references to the authors who have of late years contributed to onr knowledge of the subject ; and I will, therefore, not attempt to do more in the present communication than allude to a very few of their more important papers. Until within the last few years the researches of Goodsir were taken as the foundation of our knowledge of the develop- ment of human and other mammalian teeth, and the generali- sations of Professor Owen as authority for the modifications to be met with among reptiles and fish. The starting-point of Professor Owen’s generalisations hap- pened, however, to be just that portion of Goodsir’s descrip- tion which is not quite in accord with the facts since observed, so that subsequent investigations controvert very much that is ON THE DEVELOPMENT OF TEETH. 41 laid down in his ‘Odontography’ and in his more recently published works. According to the Goodsir theory, the first step was the formation of an open groove (primitive dental groove) ; from the bottom of this groove free papille were supposed to rise up (dental papille) ; while their subsequent enclosure or en- capsulation was believed to be brought about by an upgrowth of the walls of the groove, which arched over and enclosed the papilla, the development of an enamel organ being a secondary process. More recent investigations have compelled us to adopt several modifications in thisaccount. In the first place there is never an open groove at any period, and, consequently, there are never any uncovered free papille; and, further, there is no process of encapsulation, as there described. What really takes place is this: from the deep layer of the epithelium (the rete Malpighi) an ingrowth, consisting of a double layer of cells, takes place, burrowing down into the submucous tissue, and looking, in sections transverse to the jaw, like a tubular gland (c in figs.5 and 6). This inflection of epithelium is said to take place uniformly, all round the circumference of the jaw, and thus far there is no indication of the position of the individual teeth (Kolker, Waldeyer, Thiersch, Frey). The next stage consists of an active growth of cells which takes place in the deepest end of this inflection of epithe- lium ; while simultaneously, or closely following upon this epithelial development, the tissue immediately subjacent to it becomes elevated at corresponding points, bringing about the condition of things represented in fig. 2; these changes taking place only at those spots where teeth are to be de- veloped. The subjacent tissue forms a conical eminence (dentine papilla, dentine pulp), which is invested above by a bell-like cap of epithelium (enamel organ)—(Kolliker, Waldeyer). It is to be noted that— 1. There is no breach of surface, but that the whole process takes place in the midst of solid tissue. 2. The enamel organ is in no sort of way a secondary formation, but that its appearance is coincident with, if not antecedent to, that of the dentine papilla. 3. There is not, and cannot be, any process of encap- sulation such as was formerly described. The significance of these results becomes more apparent when the phenomena observed in the jaws of fish and reptiles come to be described; but it may be worth while to point 42 CHARLES 8. TOMES, out how Goodsir may be supposed to have fallen into the error involved in his description. Ifa foetus be kept in spirit the epithelium is very apt to peel off, or to become but loosely adherent. A very slight amount of manipulation would tear open the line of inflected epithelium, thus exposing at its bottom the eminences form- ing the dentine pulps. ‘These would thus be described as free, uncovered papille at the bottom of a deep groove; the error of description is thus very intelligible, and was hardly to be avoided at that date, when the methods of microscopic research were in their infancy. At this stage we have distinguishable the enamel organ and the dentine pulp, an indication of the future dental sac being visible in the form of prolongations from the base of the dentine pulp, which pass up outside the enamel organ (see A in fig. 1). The latter, formed as it was from an ingrowth of the cells of the rete Malpighi, retains for some time its connection with the oral epithelium through a narrow band of cells called the ‘ neck of the enamel organ” (ce in figs. 1, 2, &c.). After awhile the neck of the enamel organ becomes broken, and its connection with the oral epithelium lost, while the tooth-sac is completed by the upgrowths from the base of the dentine papilla before alluded to (A in fig. 1), arch- ing over the top of the enamel organ and meeting above it. In the meantime the enamel organ itself has become modi- fied, so that it now consists of a continuous sheet of large cells, extending round its periphery, while its interior has become transformed into a “ reticulum”? of stellate cells (fin fig. 1). The peripheral epithelial cells, where in contact with the dentine papilla, form a regular pavement of columnar or prismatic cells (enamel cells, e in the figures), and take the name of “internal epithelium of the enamel organ,’ while those forming the outer wall of the enamel organ are more rounded, and go by the name of the “ external epithelium of the enamel organ.” The enamel is formed by the direct calcification of the cells of the internal epithelium of the enamel organ, the share taken by the other portions of the enamel organ being com- paratively insignificant. Thus far the origin of the tooth-sacs of the temporary teeth only has been alluded to; the permanent tooth-sacs, originating at a much earlier period than has generally been supposed, are developed in a precisely analogous manner, save that the epithelial processes or enamel-germs whence are formed their enamel organs do not spring directly from ON THE DEVELOPMENT OF TEETH, 43 the oral epitheliums, but bud out from the necks of the enamel organs of the corresponding temporary tooth-sacs (see fig. 1). ‘Thus the only connection which the permanent tooth-germ as with that of the temporary tooth is through the medium of its enamel germ ; its dentine papilla and sac originate perfectly independently, at a spot which appears to be determined by the position attained to by the enamel germ. ° In this manner the enamel organ of each successional tooth- sac is derived from a part of that of its deciduous prede- cessor; while those teeth which have no such predecessors— z, e. the true molars—originate in the following manner :— The enamel organ of the first true molar is derived from the back end of that same epithelial inflection whence sprang all the temporary tooth-germs ; in its origin it, therefore, ranks to some extent with the deciduous teeth, being, like them, derived from a primary ingrowth of epithelium. The enamel organ for the second permanent molar buds out from the neck of that of the first; that for the third or wisdom tooth from the neck of that of the second (Legros and Magitot). The development of mammalian teeth had thus been fairly well worked out, and the subject was placed on a satisfac- tory basis of observation, but little had been done towards the elucidation of the process as exemplified in reptiles and fish. Professor Huxley, as early as 1853 (‘ Micros. Journal’), had arrived at some conclusions very far in advance of the knowledge of the time, and uttered a protest against the universality of the papilla theory; Dr. Lionel Beale (‘ Ar- chives of Dentistry,’ 1863) had arrived at a similar conclusion with regard to the teeth of the newt, and, although I cannot wholly agree with his views, had described several points of great importance with entire accuracy. With the excep- tion of these, and a more recent paper by Santi Sirena, the writings of Professor Owen practically held sole possession of the field. The following may, I think, be taken as a fair summary of Prof. Owen’s views (his own words I have quoted in my papers before referred to). The whole process of development of the teeth in certain fish might be taken to represent an early stage only of the development of mammalian teeth; thus the teeth of sharks are said to be developed from germs which never become encapsulated: in fact, never pass beyond the papillary stage, 4A CHARLES S. TOMES. Of Reptiles and Batrachia, on the other hand, he says, that in all cases the papilla becomes enclosed in a capsule; that it never stops short at the papillary stage ; and as a deduction from this, that the teeth of all reptiles consist at least of dentine and cementum, enamel being less constant in its oc- currence. In his various writings abundant reference is made to the open, grooves in which the papille destined for the teeth of reptiles originate. We are thus led to believe that in fish, batrachia, and reptiles there is displayed some sort of progression towards the more complete and more complex process characterising the development of the organs in mammalia; in other words, that the transitional stages of the latter may be taken as representing all that occurs in the former. But notwithstanding the @ priori probability of some such relation, I shall have to show that no such generalisations can be drawn—at all events, not at all in the form originally set forth. At this point I took up the investigation, and by a curious concidence published my results at almost exactly the same time as a German observer, Hertwig,! whose observations cover a good deal of the same ground, and whose figures correspond very closely with my own; but although the dates of publication very nearly correspond, my paper, I believe, takes priority. 1 Hertwig, ‘Ueber Bau und Entwickelung ‘der Placoidschuppen und der Zahne der Selachier. Jenaische Zeitschrift’ (published September, 1874). Hertwig, ‘‘ Ueber das Zahnsystem der Ampbibien und seine Bedeu- tung,” &e. é AvGhir f. Mikr. Anat. Supplementheft’ (published December, 1874). Chas. S. Tomes, ‘‘ On the Development of the Teeth of the Newt, Frog,” fe ‘ Philos. Trans.,’ 1875 (paper received July, 1874, read November, 1874. Chas. S. Tomes, ‘‘On the Development of the Teeth of Ophidia” (¢ Phil. Trans.,’ 1875. Received October, 1874, read November, 1874.) Chas. S. Tomes, “On the Development of the Teeth of Fish’’ (received March, read April, 1875). I have only very lately become acquainted with Hertwig’s papers; it will be seen that my papers on the teeth of amphibia, &c., were in the hands of the Royal Society, and the abstract published before the publication of his paper ; whilst his paper on the development of the teeth of Selachia, was published before my paper, which, however, included also the osseous fishes. Whilst I am desirous of stating that my results were arrived at entirely independently, and some of them published before his, I must add that he has gone into some questions with much more minuteness than I had; and that although there area good many points in his paper with which I do not agree, it contains a very large amount of valuable and interesting research, ON THE DEVELOPMENT OF TEFTH. 45 An observation of my own upon the tooth-germs of the armadillo, and one of Mr. Turner’s upon that of a fetal narwal, had served to indicate the probability that within the limits of the mammalia class the presence of an enamel organ would be found universal, as in neither of these animals is there any enamel upon the completed tooth. My own further researches upon the tooth development of fish, batrachia, and reptiles has shown that the presence of an enamel organ is quite universal, and that the ingrowth of epithelial cells (enamel germ of Kolliker) is the earliest recognisable step towards the formation of any and every tooth-germ. A common English lizard may be taken as our example from the class of reptiles. I have never had an opportunity of observing the development of the first-formed tooth-germs, but the manner in which the successional teeth are formed may be very completely worked out. In man the tooth-germs (even of the permanent teeth) are formed during the foetal period, and they are at first very close to the epithelial surface. But in reptiles, where the development of new teeth is going on throughout the creature’s life, the new germs are formed at a considerable distance from the surface. Hence the inflection of epithe- lium penetrates to a far greater depth before it reaches the spot where the dentine papilla is to be formed (see c in fig. 3), so that it plays an even more conspicuous part than ina mam- malian jaw. When it has reached through the whole thick- ness of the soft parts, nearly to the bone, a set of changes closely similar to those previously described takes place ; fig. 2 represents a young tooth-germ of a lizard. In its further development some points of difference which would serve to distinguish it from mammalian tooth-sacs arise. In the first place a less definite capsule is formed; the surround- ing connective tissue becomes pushed on one side and so forms a slight adventitious capsule, but this is all. The enamel organ also in its further development becomes differentiated into an internal epithelium (enamel cells) and an external epithelium, but these are at no period separated from one another by any loose network of stellate cells (the reticulum). The dentine papilla presents no marked diver- gence from the characters met with in those of mammalia. The tooth when completed moves up into position, tooth- sac and all; but it becomes fixed to the bone by a rapid de- velopment of coarse bone which takes place outside the limits of the sac, and not by a calcification of a part of its own capsule, as has been generally supposed. 46 CHARLES S. FOMES. In the order Ophidia an interesting and eminently charac- teristic arrangement is met with. A very large number of successional tooth-germs are always in course of preparation ; thus there are usually about eight or nine successional poison fangs in various stages, and six or seven sacs at the base of the ordinary teeth (see é,, @., e, in fig. 4). These successional sacs are arranged together in a sort of nest, within a connective-tissue capsule, an arrangement likely to prove advantageous when the mouth is dilated to swallow prey; each one consist of an enamel organ, a dentine pulp, and a feeble connective-tissue investment. The youngest tooth-germ is to be found at the bottom of the series (e, in fig. 4); it originates from a budding down of an ‘enamel germ” from the neck of the enamel organ of the tooth-germ next above it, and the subsequent or coinci- dent formation of a dentine papilla (see z in figure) ; not only do the tooth-germs of all Ophidia possess enamel organs, but these actually form a thin coating of enamel; and Professor Owen was mistaken in supposing that enamel was absent, but that the teeth of ‘all Ophidia consist of dentine and cement.” No Ophidian tooth, so far as the examination of a very large number of specimens can decide, possesses cementum at all. In Batrachia the process is in all essentials similar, though it is subject to modification according to position. In the newt the chain of successional tooth-sacs is very beautifully seen (see fig. 6), an enamel-germ, which as yet has no cor- responding dentine-pulp, extending beyond the youngest of the series (c in fig. 6). The tooth-sacs of the newt have the peculiarity, first noted by Dr. Lionel Beale, that they have no capsular investment whatever. They consist solely of aggregations of cells, and under pressure readily break up; the enamel or gans form a spear-like point of enamel upon the teeth, but the enamel does not extend down upon the sides of the teeth. In the frog the edentulous lower jaw closes within the upper teeth, and so little space is left that an arrangement like that seen in the newt becomes impossible. In fig. 5 an enamel germ is seen at the inner side of the base of the tooth in place; as the new tooth-sac attains to any consider- able size, space is made for it by the absorption of the base of the older tooth, into the pulp cavity of which it finally passes, recalling in some measure the manner of succession met with in the crocodiles. It is also a question whether the new enamel germs really do spring from the necks of the enamel organs of older tooth-sacs, or whether they originate de novo ON THE DEVELOPMENT OF TEETH. A.7 from the oral epithelium ; though analogy would lead one to infer the former to be the case, the small amount of avail- able space and the speedy migration of the growing sac render it difficult to positively decide this point. The sharks, in which Professor Owen was of opinion that tooth-development stopped short, as it were, at the papillary stage, present an instructive parallel with the condition of things seen in either the newt or the axolotl. The series of dentine papille, save that they originate from a continuation of the same sheet of mucous membrane, have no genetic conection with one another; on the other hand, the enamel organs of the teeth of successive ages form a chain the continuity of which is never broken by that sub- sequent individualisation of the several tooth-germs which takes place in reptiles and mammals. The teeth of sharks are seen, ina feetus just on the point of being hatched, to be merely highly-developed dermal spines, with which they are directly continuous, as there is no defi- nite lip at this period, and the spinous skin is seen turning in over the jaws. Hertwig has worked out, more carefully than I, the development of these dermal spines, which he declares to be in all respects similar to that of the teeth ; he considers the excitation of greater use to be adequate to account for the greater development of the dermal appendages where the skin passes over the jaws. In the development of the shark’s tooth we find, just as in every other creature, that there is a production of an enamel germ extending beyond the youngest one already existing (e in fig. 7); that opposite to this the submucous tissue becomes elevated into a dentine papilla. But there is nothing like a production of free papille, which become subsequently and secondarily enclosed or encapsulated ; and the production of the enamel organ is in no sense secondary to that of the dentine papilla. When the parts are undis- turbed the flap of mucous membrane (hf in fig. 7) which covers in the teeth is in slight connection with the jaw and teeth; it may be very readily torn away, but it is only so displaced by violence, and it is not, as was generally supposed, a free flap, which merely fitted against the developing teeth. In some of the elasmobranchs a distinct coat of enamel is deposited ; in others it amounts to a mere glaze, but from the appearance of the enamel cells, which in other cases proves to be a tolerably reliable guide, I should infer that they do discharge a function, and that the outer structureless film on the tooth, of Lamna for example, is to be regarded as enamel. 48 CHARLES 8, TOMES. If one were desirous of representing diagrammatically the development of the teeth of a shark and a newt, one would draw almost the same thing, save only that in the former the several tooth-sacs are somewhat less individualised. Hertwig entertains peculiar views as to the homologies of the parts of placoid scales and sharks’ teeth, regarding their basal portions as cementum ; his paper has been in my hands so short a time that I am not prepared to express an opinion upon this matter, but if we admit Hertwig’s views as to the basal part of a selachian tooth, we must probably extend the term cementum to a large number of structures never hitherto ranked as dental tissues at all. Passing from the sharks and rays to the osseous fish, I believe that a broad distinction may be laid down. In all mammals, reptiles, batrachia (the frog?), and elasmobranch fishes, successional teeth are derived, though the medium of their enamel organs, from their predecessors. In all osseous fish which I have examined, successional teeth appear to be produced de novo, i.e. from new inflections of the oral epithelium (see fig. 8, representing a young tooth-sac of a mackerel). With this exception the process is the same; there is the same formation of the enamel germ from the oral epithelium, which is transformed into an enamel organ over the uprising dental pulp; the latter becoming always calicfied, the former sometimes dwindling away without any enamel formation taking place. There are many fish (e.g. eels) which have spear-like ter- minal points of enamel, but no enamel on the sides of the teeth ; in the tooth-germ the enamel organ is, nevertheless, co- extensive with the dentine pulp, though its internal epithelium or enamel cells are only largely developed over the point where enamel is to be formed, and are small and dwarfed over the rest of the tooth (see fig. 10). From what has already been said it will be seen that there is considerable uniformity in the manner in which tooth-germs are developed, and that it is quite impossible to maintain the generalisations that the teeth of fish and reptiles typify any certain transitory stages in the develop- ment of mammalian teeth, &c. And it will also be seen that Goodsir’s original error of observation, small though it was, becomes vastly magnified when his views are stretched to make them apply, for instance, to the common snake (see fig. 4). Although, morphologically, as Professor Huxley reminds me, an inflection of epithelium is not a very different thing ON THE DEVELOPMENT OF TEETH, 49 from a groove lined by epithelium, yet any description which spoke of the successional teeth of a snake, or yet more of its poison fangs, as being developed from free papille in a groove, would be somewhat wide of the mark. My observations have led me towards certain other ques- tions of interest. In the first place, are we still to regard dentine as the most, cementum as the next, and enamel as the least constant of dental tissues? According to the older accounts of development such must almost necessarily have been the case ; but is it really so? The Ophidia, at all events, amongst whom cemeutum has been supposed to be of uni- versal occurrence, have none, but have enamel instead (see ‘Phil. Trans.,’ 1875), and I am inclined to think enamel at least as constant, if not more so, than cementum. The true homologies of the whole enamel organ were first pointed out by Professor Huxley in the valuable paper re- ferred to; with regard to the several parts of the enamel organ, the reticulum appears to be peculiar to the mammalia ; at all events it is certainly not an essential part, as enamel is formed in countless teeth in which it was never present. Indeed, some recent observations of my own upon the develop- ment of the poison fangs of snakes would seem to point to the inference that it is essentially a retrograde metamorphosis of a part of the enamel organ. It has already been mentioned that the concidence between the position of the large enamel cells in an enamel organ such as that of the eel (see fig. 10) with that of the enamel cap afterwards formed would, so far as it goes, support the conversion theory of the development of enamel; the more so as it is generally possible to judge from the extent to which this internal epithelium of the enamel organ is developed, whether enamel will or will not be deposited upon any tooth ; Hertwig, however, believes that enamel is not formed by direct calcification of the enamel cells, but by a secretion from them; he also reasserts the existence of the membrana preformativa,! but not, in my opinion, upon satisfactory grounds. The development of cementum is a subject by no means satisfactorily worked out; how far it has relation to the tooth-sac capsule, and how far to the periosteum and connec- tive tissue outside the sac, I hope to be able to determine ; but it appears to me that its presence is almost always asso- 1 After a careful review of the literature of the subject, and not a few attempts to demonstrate it, I cannot think that the presence of a basement membrane (M. preformativa) has been established, and the appearances de- scribed are, to my thinking, capable of other interpretations more in accord, with what I have myself seen. Waldeyer also denies its existence. VOL, XVI. D 50 CHARLES S. TOMES. ciated with implantation in a socket, though it may be in a very incomplete one. ‘To sum up the results of the foregoing observations : 1. All tooth-germs whatever consist of at least two parts— an enamel organ and a dentine pulp; a capsule may or may not be present. 2. The first step recognisable is invariably the ingrowth of epithelium, i.e. an enamel germ. 3. Enamel germs of successional teeth in mammals, rep- tiles, batrachia, and elasmobranch fish are derived from the necks of the enamel organs of their predecessors; the- respective dentine pulps originating independently, the enamel germs constitute the sole connection between dental germs of various ages. 4. In all the osseous fish examined by me the several enamel germs originate de novo from the oral epithelium ; this may, perhaps, be the case in some other creatures, but I have never seen it. 5. The enamel organ, invariably present, consists primarily of external and internal layers of epithelium ; the former atro- phies, the latter grows largely if the tooth is to be capped with enamel ; otherwise it also atrophies, but at a later period. 6. The stellate tissue of the mammalian enamel organ is obviously non-essential; indeed, it is probably a retrograde metamorphosis. 7. Dentine pulps originate independently of each other, at points in the submucous connective tissue apparently de- termined by the position attained by the ingrowing enamel germs ; in structure they are singularly uniform in all animals, 8. There is no example of a dentine pulp originating as a free papilla upon the surface; on the other hand, as in the snake, the process often takes place at agreat depth below the surface. 9. The terms “ papillary,” “ follicular,” and “eruptive stage” should be quite abandoned, as tending to perpetuate a somewhat mistaken hypothesis. 10. Enamel is obviously a metamorphosed epithelium ;! dentine belongs to the submucous connective tissue; the homologies of cementum have yet to be determined ; but the basement membrane known as membrana preformativa has probably no significance in the process, if indeed it has any existence. 1 This is particularly obvious in Fish and Amphibia, and Hertwig has given figures of developing placoid scales in which the rete Malpighi, but little specialised into an enamel organ, appears to become calcified into enamel. HAECKEL’S RECENT ADDITIONS TO GASTRHA-THEORY. 51 An Account of PRrorEssor HAECKEL’s RECENT ADDITIONS to the GASTR@#A-THEORY. By Professor E. Ray Lan- KESTER, M.A., F.R.S. With Plates VII, VIII, IX, X. In the last numbers of the ‘ Jenaische Zeitschrift’ Pro- fessor Haeckel has continued his important discussion! of the facts of development in connection with the significance of the primitive germ-layers as the key to animal genealogy. Some useful terms are introduced by him to designate phenomena which have been more or less clearly recognised during the development of this subject in the past three years, and further, some original observations on the develop- ment of particular animals are given. By the courtesy of Professor Haeckel I am able to place before the readers of this Journal four of the instructive plates which illustrate his recent contributions to the Gastrea-theory ; and I pro- pose to give some account of his views, without, however, committing myself to complete agreement with them. Palingeny and Cenogeny.—'Lhese terms were introduced by Haeckel in his Anthropogeny, and will no doubt be found useful. In the ontogeny, or individual development of living forms, we have been accustomed under the new régime of Darwinian embryology to distinguish those forms and structural dispositions occurring in the course of develop- ment from the egg, which appear to be due to heredity, from those which appear to be due to adaptation. It has been recognised? that there are two distinct tendencies to be estimated in the process of ontogeny: the tendency to recapitulation of the complete series of ancestral forms, the effects of which Haeckel terms Palingenesis, and the tendency to adaptation to present conditions resulting in the suppression and hurrying over of steps in the recapitula- tion and the development of special larval or embryonic organs. The effects of this tendency are what Haeckel sums up as Cenogenesis. Cenogenetic phenomena have hitherto been spoken of as ‘ falsifications ’ of the recapitulation, as ‘ abbrevia- tions’ and larval ‘adaptations.’ In the series of ontogenetic phenomena presented by an organism, it is clear that it is the palingenetic portion of them which have most value for the morphologist, whilst to the physiologist the cenogenetic are especially interesting. Heterochrony and Heterotopy.—Ontogeny being essentially 1 See this Journal, 1874, for a translation of Haeckel’s essay, “The Gastreea-theory.” 2 See “ Primitive Cell-layers of the Embryo,” ‘ Annals and Mag. Nat. History,’ May, 1873, p. 322. 52 PROFESSOR E. RAY LANKESTER. a series of phenomena of space-relations succeeding one another in definite order, it admits of variation (and conse- quently of appropriate discussion) in relation to the two elements of space and time. ‘The perturbations in the palin- genetic evolution of an organism which may be classed as cenogenetic, are necessarily either dislocations of the palin- genetic phenomena,.in so far as regards ¢ime or in so far as regards space. Hence we may with Haeckel very con- veniently speak of such phenomena as heterochronous or heterotopous as the case may be. The appearance of an organ in an embryo at an earlier period (that is to say, at a period of lower development of its other organs) than that at which we have reason to believe this organ made its appearance in the Phylogenesis or an- cestral development of the form in question, is a frequent example of heterochrony. As examples, Haeckel cites the early appearance of the notochord, of brain and eyes, of the gill-slits, and of the heart (before the vessels) in Vertebrata ; the early development of the limbs, and of the segments (of the primitive stripe) in Arthropods generally ; of the trachee of the Tracheata, and the liver of the Crustacea. The Mollusca furnish instances in their early developing otocysts, and, I may add, in the early appearance of the ‘ shell-gland’ or invagination of the mantle, the Gasteropods in their lingual rasp, and the Echinoderms in their calcareous skeleton. Similarly we have examples of heterochrony which consist not in the early but in the /ate appearance of inherited ancestral traits of organisation. This kind of dislocation is very generally seen in the late development of the sexual glands in all Metazoa, and in the postponement of the com- pletion of the alimentary canal, even though its rough materials take up their position at the normal palingenetic period. A striking example is, according to Haeckel, the late formation of the septum of the auricles in the embryonic heart of the higher Vertebrata, which is subsequent to that of the septum of the ventricles. In the phylogeny of the Vertebrata, on the contrary, it is clear that the two septa originated in the reverse order, as the Dipneusta, the Amphibia, and the Reptiles demonstrate. Heterotopy is even a more important element of the cenogenetic modification of the primary palingenetic pheno- mena. I have myself insisted on the fundamental importance of this common process in the comparison of the delaminate and invaginate forms of Gastrula or Planula, and in the comparison again of the epibolic and embolic forms of invagination (see ‘Ann. Nat. Hist.” May, 1873, p. 328, HAECKEL’S RECENT ADDITIONS TO GASTRHA-THEORY. 53 and ‘ Proceedings of the Royal Soc., 1874), and further, in relation to the development of organs in the middle layer of the germ which are palingenetically hypoblastic or epiblastic, as, for instance, the notochord of Vertebrates (shown by Mr. Balfour to develop from the hypoblast in the sharks, probably its true palingenetic position), and the nerve-ganglia of Loligo (shown not to develop from epiblast as nerye-centres should do in undisturbed palingenesis ; see ‘Quart. Journ. Micr. Sci.,’ 1875, p. 46). As Haeckel observes, first and foremost in this connection are the ‘wandering’ of cells and shifting of groups of cells in the earliest stages of development—small shiftings taking place in an early stage—such, for example, as the interpene- tration in limited points of the two primitive cell layers, the ectoderm and endoderm, or epiblast and hypoblast which constitute the critical developmental form of all higher animals, the Gastrulaor Planula. To this heterotopic move- ment of cells in an early phase of development, possibly occurring evenin the first stages of cell-formation or cleavage of the egg, it is very possible that large dislocations in the seat of development of organs, manifesting themselves at a later period, should be ascribed. Haeckel would thus account for the assumed mesoblastic origin of the sexual glands in certain of the higher animals, holding, as do most embryologists, that these glands are palingenetically not part of the middle layer of the germ, but part either of the epiblast or of the hypoblast. Considerations of the same kind enable us to explain in a general way the diversity of origin which the middle layer presents in different groups of animals. At the same time it must be admitted that accurate information as to even the apparent origin of the mesoblast in a large number of cases is not to hand, and the statements of authors worthy of credence vary in reference to one and the same animal. Haeckel, as is well known, holds that the mesoblast is phylogenetically formed of two layers, respectively the deep layer of the ectoderm and the deep (i. e. sub-epithelial) layer of the endo- derm. its apparent development entirely from hypoblast or endodermin the Vertebrates he regards as due tothe heterotopy of the ectodermal portion,’ and the splitting which occurs 1 A possible explanation of such a case as the disappearance of the ecto- dermal factor of the mesoblast is suggested (I venture to submit) by a comparison with the phenomena of atrophy, such as we observe in the cases of rudimentary organs and the final suppression ef such organs. Atropby very generally is accompanied by a ¢ransference of the nutri- tion proper to the atrophied part to a neighbouring or substituted structure, and when the transference reaches its full development and extends back into the period of early embryonic life, even the primitive cells which formed the first outlines of an organ or of a germ-layer may he totally ob PROFESSOR E. RAY LANKESTER, in the Vertebrate’s mesoblast giving rise to the ccelom of that group (pleuroperitoneal cavity) he regards not as the primitive separation of the two deep layers (of ectoderm and endoderm respectively) by which he supposes the ccelom to have originated phylogenetically, but as a secondary splitting following upon their heterotopous fusion. In this connection it is important to observe that we have not much evidence in favour of the view that the celom did phylogenetically arise by the splitting of the two factors of the mesoblast, that is to say, was a schizoceel (Huxley). Possibly it was in origin an outgrowth of the alimentary canal, a gastro- vascular apparatus or enteroccel (Huxley), as its development in Echinoderms, Terebratula, and Sagitta tends to prove. As the important general result of the two classes of ceno- genetic phenomena, which Haeckel distinguishes, viz., Hetero- chrony and Heterotopy he points out that in the progress of time those organs are more and more strikingly brought into the foreground of an ontogeny which are especially charac- teristic and important for the stem, class, or order to which suppressed. From one point of view, which is not here touched on by Haeckel, the most important developmental phenomena are those of Hypertrophy and Atrophy. In organic evolution (phylogeny) organs do not arise de novo, but are produced by the moulding influence of gradually increasing hypertrophy of this part and atrophy of that. In ontogeny such hypertrophies or atrophies may present themselves in the course of the developmental recapitulation in their due order, the order in which they appeared in the series of ancestors. They then fall under the head of ‘palingenetic’ phenomena. But if they occur earlier or later than is their proper place they fall under the head of heterochronous cenogenesis. It is clear then that cenogenesis in divisible into heterotopy, heterochronous hypertrophy (positive growth) and heterochronous atrophy (negative growth). Thus the atrophy of organs which have made their appearance in the pe but disappear in the adult is very generally part of the palingenesis. On the other hand, the total or very early suppression of organs in the embryo such as the notochord absent from most Tunicate ontogenies, the gill tufts absent from the ontogenies of Amnionate Vertebrata, which should appear if the palingenesis were carried out though to disappear before the adult form is attained, are examples of the heterochronous atrophy of cenogenesis. It is to be noted with refereuce to the terms palingenesis and cenogenesis, that they can only be used in relation to specified tracts of the organic pedigree. The cenogenetic phenomena of an early ancestor of this or that organism necessarily decome part of the palingenesis of its descendants. For instance, the development of the mesoblast from the hypoblast exclusively may be as Haeckel supposes, a cenogenetic phenomenon in vertebrata relatively to the ancestors of an earlier grade of development. Once, however, acquired by the earliest Vertebrates, it becomes in other Vertebrates relatively to them a palingenetic phenomenon. Thus in birds as represen- tatives of the Vertebrata, we may speak of it as cenogenetic, but in birds as compared with Vertebrates of the grade of development of the sharks, it can only be spoken of as palingenetic. With regard to atrophy see my paper on ‘ Development of the Cephalopoda,’ this Journal, 1875, p. 46. HAECKEL’S RECENT ADDITIONS TO GASTRAA-THEORY. 55 the developing organism belongs. Thus in the Vertebrata the notochord and the branchial slits are out of all proportion early in their appearance, and large in relation to their adult size and the size of other organs; whilst, on the other hand, those organs are more and more pushed into the background which have the most general significance for all Metazoa. Hence above all other organs we find the primitive alimentary canal,’ or archenteron, and the primitive mouth? suffering in this respect in so far as their primitive form is concerned. Hence too the simple, primordial Gastrula, which arises by the invagination of a perfectly simple sac, the wall of which consists of a single layer of cells (Blastula), is especially preserved in the most faithful way by the lowest, most indif- ferent, and oldest forms of the different groups (for example, in the ontogeny of Gastrophysema, Monoxenia, Sagitta, Phoronis, Argiope, Terebratula, Uraster, Toxopneustes, Ascidia, Amphioxus).? Palingenetic plastic yelk and cenogenetic food-material. —tThe great importance of distinguishing these two elements and their share in early developmental phenomena is insisted upon by Haeckel. He holds that (as I have maintained in relation to the developmental phenomena of the Mollusca) the food-material thrown in to the primitive egg-cell dis- turbs and clouds the subsequent development in the most profound manner, hindering and concealing the full unfolding of the palingenesis. In all cases Haeckel maintains that the egg is palingenetically a single cell and that the food-material is a cenogenetic addition or adaptation.* 1 Since we have no words equivalent to the German Darm and Urdarm, we may use the Greek equivalents enteron and archenteron. 2 Or ‘ blastopore.’ 3 The examples cited by Haeckel do not appear to me altogether to warrant this conclusion. Sagitta, Terebratula, and Toxopneustes are not generally admitted to be either indifferent or archaic types of the groupsto which they belong. And even if they were it is not easy to follow out the reasoning by which it is concluded that they should therefore be expected to exhibit the simplest form .of gastrula, for although assumed to be low in their respective groups, yet there is a very long distance between any of them (except the Sponges) and the ancestral gastrula, a distance sufficient to allow of the development of countless heterotopisms and _hetero- chronisms, and without doubt sufficient to allow of the development of that particular cenogenetic phenomenon which beyond all others is efficient in obscuring the gastrula form, viz. the addition to the egg-cell of food-yelk, deutoplasm, or ‘ food-material.’ I pointed out the important influence of this accessory yelk in my original paper on this subject, ‘ Ann. Nat. Hist.,’ May, 1873, p. 328, and in subsequent embryological writings. ‘From one point of view the admixture of food-material with the proto- plasmic egg-cell may be regarded as a palingenetic survival of the ‘amceboid form of nutrition,’ in which solid food is taken bodily into the living sub- 56 PROFESSOR E. RAY LANKESTER,. The four chief types of Egg-cleavage and of Gastrula-forma- tion.—According to Haeckel we may distinguish in the processes of egg-cleavage and the immediately subsequent phenomena of development which result in the assumption of the essential gastrula-form—four types, one primitive (the true palingenetic series of changes), whilst the other three are cenogenetic modifications of the process brought about by variations in the quantity and mode of admixture of the food-material. In each type five stages of development may be distinguished, leading as far as the completion of the Gastrula form—namely, the Monerula or fertilised egg after the loss of its ovarian nucleus or germinal vesicle; 2, the Cytula or egg with newly formed cell-nucleus; 38, the Morula or mulberry form (Polyplast), a spherical agglomera- tion of simple equi-formal cleavage-cells ; 4, the Blastula or blastosphere, a hollow one-cell-layered vesicle, formed by the accumulation of fluid within the Morula; 5, the Gastrula, a simple two-cell-layered sac, whose cavity, bounded by the two primary germ-layers, opens to the exterior by the Archistom (or blastopore). To the five stages belonging to the simplest type (that which he considers strictly palingenetic) Haeckel gives the prefix ‘archi.’ We thus have a series designated as fol- lows :—Archimonerula, Archicytula, Archimorula, Archi- blastula, Archigastrula. To the second type the prefix ‘amphi’ is assigned, and we thus have a corresponding series leading to the Amphigastrula. The third type have ‘ disco’ as their distinctive name-component, whilst ‘ peri’ marks the fourth group. We shall take these into consideration one by one, merely remarking to begin with that the Archi- blastic and Amphiblastic series correspond to two modifica- tions of what have long been called Holoblastic eggs ; whilst the Discoblastic and Periblastic series are two extreme forms of Meroblastie eggs. Haeckel is careful to point out that though these four types can be conveniently erected as a means for arranging our conceptions, yet that they really are not sharply cut off from one another, but as we should expect there really are transitions in blastoderm-formation stance of the sarcode mass. It is, however, no doubt most conducive to a clear appreciation of the facts of the case to regard it as belonging to a group of structural phenomena which are to be distinguished from the ulti- mately resulting embryonic phenomena classed as heterotopy, heterochronous hypertrophy and heterochronous atrophy, and may be designated ‘ matri- ficial’ (like artificial). These matrifacts are often clearly,the antecedents of embryonic cenogenesis. Such are besides the added food-material, the ege- envelopes. Structural phenomena proper to the protoplasm of the egg-cell itself may be called ‘ ovificial’ or ‘ ovifacts,’ HAECKEL S RECENT ADDITIONS TO GASTRHA-THEORY. 57 connecting the Archiblastic with the Amphiblastic and with the Periblastic, and the Amphiblastic with the Discoblastic and Periblastic. ‘To the student of Embryology it will be clear enough in a general way that these terms are meant toimply, lst, ARcHIBLASTIC, the primitive egg-segmentation into equi-formal cleavage spheres, and the subsequent assump- tion of the simple two-cell-layered gastula-form with or with- out apical orifice, either by invagination or delamination ; 2nd. AMPHIBLASTIC, the egg-segmentation into inequi-formal cleavage-spheres, one portion of which is charged with the matrificial food-material, and becomes overgrown by the smaller cells free from food-material (epibolé, Selenka). 3rd. DiscosBLastic, the segregation of a small disc or klastic patch from the rest of the egg, in which disc alone the cleavage process is carried on. 4th, PERIBLASTIC, the segre- gation not of a disc, but of a complete superficial layer of klastic material around the inert food-material. Tue ARcHIBLASTIC TypE.—Examples of this are met with among the Celenterata in Sponges, Hydroids, Meduse, and Corals ; among Worms, as in the case of Sagitta and Phoronis ; among Molluscs, as in the Brachiopoda and some Lamel- libranchs and Gastropods; among Echinoderms generally ; among certain low forms of Arthropoda, as for instance some Branchiopoda and perhaps the Pteromalina (Tracheata) ; among Vertebrates, in the case of Amphioxus and some Ascidians.' Haeckel gives the development of a very simple form of polyp, as typical of the Archiblastic type. This very curious little polyp, which he names Gastro- physema, is similar to the Haliphysema of Bowerbank, and appears even in its adult condition to get no further than the Gastrula stage: it is a true Gastrea. It was originally described by Carter as Squamulina scopula, that observer having taken it for a Polythalamian. In the plates which we reproduce no figure is given of the Archimonerula, Archicytula, Archimorula, which the reader can easily picture to himself; but in fig. 20 we have the Archiblastula of an Actinia, in fig. 21 its Archigastrula formed by invagination; in fig. 29 the Archiblastula of Limneus is given, and in figs. 30 and 31 its Archigastrula. Fig. 17 gives the Archigastrula of a Calcareous Sponge (Asculmis), fig. 22 that of a Medusa (Pelagia), fig. 23 that of a worm, Sagitta, fig. 25 that of a Brachiopod (Argiope), fig. 33 that of an Echinoderm (Asteracanthion). Haeckel holds (and this is an important point for theoretical 1 Professor Haeckcl is not responsible for the classification here adopted of the Tunicata under the great group of Vertebrata, 58 PROFESSOR E, RAY LANKESTER. consideration) that the Archigastrula is always what I have termed an ‘ invaginate Gastrula or Planula, and, of course, since the cells are of equal size, and the invagination is a real pushing-in, it corresponds with my ‘embolic invaginate’ type; the ‘ epibolic invaginate’ type, where the smaller cells of the blastosphere grow over the larger cells being classed under the Amphiblastic type. Haeckel is inclined to doubt whether such a thing as a ‘ Delaminate Gastrula or Planula’ ever is formed ; that is to say, he doesnot think that the observa- tions are satisfactory in which the endodermal layer of cells have been stated to arise by cell-division on the inner surface of the blastosphere.! Very probably careful observations would show in such cases an invagination at a very early period. We arein want of observations (by means of section-cutting) on the development of the Hydroid polyps. But even if delamina- tion does occur it can only be regarded as a heterochrony. The fate of the ‘ Urmund,’ as Haeckel calls it, the orifice of invagination or ‘blastopore,’ as I prefer to say, is not discussed by him. It is one of the points upon which the whole superstructure of the Gastreea theory rests, as I have pointed out (this Journal, April, 1875). Haeckel, however, insists on the importance of recognising the rim or margin of the blastopore, which he calls Properistoma. It is, he holds, identical with the much discussed and highly im- portant ‘ Randwulst’ or ‘Keimwulst’ of the discoblastic type of development (Chick, Shark, Cuttle-fish). It is here, in the annular space between Entoderm and Exoderm, that the first cells are separated from the two primary germ-layers to give rise to the mesoderm, or mesoblast. The Amphiblastic type(P1. X).—The most familiar instance of this type of development, which consists essentially in the unequal size of the cleavage-cells, a portion of which are large and charged with food-material, is seen in the frog and other Amphibia, also in Petromyzon and Accipenser. Probably the Dipneusta and placental Mammals belong to this category. The greater number of the Mollusca are amphiblastic, probably even some Cephalopods (the commoner forms of which are discoblastic) and some Brachiopods (others being archiblastic). Among the Arthropoda amphiblastic develop- ment appears to occur only in some of the lower Crustacea and Tracheata. Amongst the Echinodermata only certain Asterida and Holothurida, with a so-called ‘ direct’ or ‘abbre- viated * development, present the amphiblastictype. A large majority of the Worms are to be reckoned here, and amongst 1 Tt does not seem possible to explain Fol’s observations on the develop- ment of Geryonide as indicating anything but a true ‘ delamination.’ HAECKEL’S RECENT ADDITIONS TO GASTRHA-THEORY, 59 the Celenterata, or Zoophytes, there are some excellent examples of the type; for instance, among the Ctenophora and Siphonophora, as well as in Corals and Sponges. In Plate X two examples of the amphiblastic mode of development as far as the Gastrula stage are given; the upper series relates to the Annelid Fabricia, one of the Sabellide, and the lower to a Gasteropod, probably a Trochus. They were both studied by Haeckel at Ajaccio. He recommends the use of carmine and hematoxylin as staining agents, the em- bryo being rendered transparent by glycerine. The reader is referred to the explanation of the plates for further details. The amphiblastic type of development presents a great range of variation in respect of the amount of food-material incorporated with that portion of the Monerula which is to give rise to the endoderm-cells, and an accompanying varia- tion in the number of cells produced by its cleavage and the rate at which it cleaves. In this;way heterochronies are brought about, the unencumbered ectoderm proceeding more rapidly on its palingenetic development than does the endoderm. All amphiblastic Gastrulze are not epibolic, that is to say, brought about by the growth or circumcrescence by the ectodermal cells over the larger entodermal cells. In some (for instance, Unio, Figs. 26, 27, 28) the relative growth is such as to have the same appearance of embolé (or entobolé), which is observed in the Archiblastic series. As I pointed out (loc. cit.) there is no sharp line to be drawn between embolic and epibolic developments, but they are connected by transition forms. Haeckel endorses this view, and further, draws attention to the variation, which I have also insisted on, in the share which the primitive endoderm cells take in the production of the permanent alimentary canal. It is impossible to say, with the observations at present before us, to what extent these variations are exhibited in the various groups of animals. In some cases endoderm-cells are absorbed in the process of development, never taking | part in the formation of the wall of the primitive enteron (archenteron). Such cells may be inclosed within the enteron or lie outside its walls. Further, it appears (e.g.in Limnzus and Pisidium) that the archenteron is sometimes not directly conyerted into the alimentary canal of the adult, but that large portions of it are absorbed, or, as in Echinoderms, Sagitta, Terebratula, &c.,}give rise to a body-cavity. Haeckel distinguishes the permanent enteron as metagaster. We may call it the ‘ metenteron.’ The problems here pressing for solution are of the greatest consequence, The whole question of the nature of the coelom 60 PROFESSOR E. RAY LANKESTER, or body-cavity is involved, and on this as on the equally important question of the persistence of the blastopore Haeckel maintains a discreet though disappointing silence. The Discoblastic type —The essential feature in this type is in the overloading of the egg with food-material. It is led up to by many cases among Mollusca, which present the amphiblastic type'so far, that the portion of the egg-cell which contains the unassimilated food-material cleaves once, or possibly twice, but ceases after that to take part in the process of cell-formation and serves only as a reservoir of nutriment. Rarely (as I have suggested in the case of Aplysia, in a paper received January, 1874, and published in 1875 in the ‘ Phil. Trans.’ by the Royal Society, and as Fol has since recorded in the case of Pteropods) the large seg- ments charged with food-yelk segregate at a later period of development a certain number of cells which give rise to the hypoblast, or as appears from Fol’s observations, sometimes to part of the epiblast. ‘This late segregation may be compared to the Periblastic formation of the blastoderm to be described below. Haeckel points out that in the Discoblastic type we have cases (especially among osseous Fish, one of which, a marine Gadoid in all probability, he describes and figures in detail) in which the discoidal cap of egg protoplasm or formative-yelk separates entirely from the food-material, which remains perfectly homogeneous, and never gives rise to any cells, after the separation of the cleavage-patch or disc. The growth of this over the relatively huge mass of food-material he describes minutely, and considers that an ingrowth of the first cap of cells takes place all round its periphery—the well-known ‘ Randwulst’ of the osseous fish’s and chick’s development. ‘The result is the formation of a Discogastrula perched on a mass of food-material, which mass blocks or fills up as it were the blastopore or ‘ Urmund.’ The Discogastrula separated from its mass of food-material is seen in fig.50. Intermediate stages are admitted by Haeckel in which the food-material is not pure, but still contains formative material, which it separates during the growth of the Discogastrula. The cells thus formed are, he says, partly converted into conneetive tissue, partly into blood- cells. He regards them as belonging to the endoderm. Such are the cells described by Goette in 1874 in the development of the chick, by Balfour in 1874 in the Shark, and by myself in 1878 in Cephalopods (‘ Ann. Nat. Hist.,’ February, 1873, p- 82). To these I subsequently applied the name of ‘auto- plasts ’ as opposed to the ‘ klastoplasts,’ which are the pro- ducts of the segmentation of the cleavage-disc or cap, HAECKEL 'S RECENT ADDITIONS TO GASTR#HA-THEORY. 61 Haeckel is very careful to insist on the unicellular character of the Discomonerula and Discocytula, even where it attains the large size seen in Birds. In his support of Gegenbaur’s view advanced so long ago as 1861, he will be joined by most embryologists of to-day. He takes occasion to express his objections to the ‘ parablastic theory’ of His, which I may say does not appear to me to be really supported by the fact which His recently quotes from my observations on the ovarian egg of Loligo and Sepia (‘ Phil. Trans.,’ loc. cit.). The cells which are to be found in the ovarian egg of these Cephalopods, and which have passed into it from the cell- lining of the egg-capsule (granulosa), are not in such a state as to lead one to suppose that they are capable of development. They are, as Haeckel insists, passive nutritional material. The invagination of the disc-like morula in the discoblastic type, to give rise to the Randwulst or Properistoma (blasto- pore-margin) is a fact of great importance, which Haeckel attempts to illustrate in other vertebrate forms besides the osseous fish, in which he observed it with care. The diagrams (figs. 41 to 54) serve to explain these views, which the reader may compare with those advanced by Mr. Balfour in this Journal, July, 1875. The origin of the mesoblast in his marine osseous fish Haeckel did not satisfactorily determine. It appears, how- ever—a fact on the general importance of which, as above mentioned, Haeckel insists—that it first makes its appearance in the Randwulst (blastopore-margin), and is, he believes, traceable to delamination from the ectoderm (epiblast), and also to delamination from the endoderm (hypoblast). The first portion (Hautfaserblatt') has a dilateral symmetry, gives rise to the so-called ‘ prctovertebre,’ and corresponds to the bilaterally symmetrical commencements of mesoblast, which Carl Rabl has observed in Limnezeus, and which are charac- teristic, according to him, of all the Bilateria (that is to say, the Vermes and the four great types connected with them). The second portion, which comes from Hypoblast, consists of a layer of hypoblast cells and of ameeboid cells of very active movement, which wander between spaces in the hypoblast and spread themselves partly on the surface of the yelk and partly in the embryo itself. They give rise to blood cells, connective-tissue cells, and pigment cells. The outer layer of hypoblast cells and these amceboid cells Haeckel considers as the Darmfaserblatt.” He lays especial emphasis on the fact that this set of cells do not have a bilateral arrangement pri- 1 Hypoderon or hypoderic cell-layer. * Hypenteron or hypenteric cell-layer. 62 PROFESSOR E RAY LANKESTER. mitively, but arise equally and simultaneously over the whole periphery of the Discogastrula. The Discoblastic type of development is found in Birds, Reptiles, Teleostean and Selachian fish, in Cephalopods, some Tsopods and Copepods, Scorpions, some Spiders, and a num- ber of Flies. Probably it will be found in Monotremata and Didelphia. The Periblastic type (Plate IX).—This is, perhaps, the most important of any one of the four types distinguished by Haeckel, for here the palingenetic phenomena are most com- pletely disguised. Those embryonic histories which were formerly said to present no yelk-cleavage, or a superficial yelk-cleavage, belong here. The periblastic mode of develop- ment is most common in the Arthropods, in Tracheata as well as Crustacea ; it also appears from Kowalewsky’s observations to occur in some Celenterata (Alcyonians). Like the amphi- blastic and discoblastic types, the periblastic type exhibits variations, and these throw light on some of the secondary developmental phenomena in the other types. The essential point about the periblastic type is this, that the food-material collects at an early stage of development centrally, so as to be completely enveloped by the formative protoplasm. In many Arthropods this segregation of the two constituents occurs at the time of fertilisation, and we then have what has been called a blastema surrounding granular yelk. This is really the Perimonerula. Later this Perimonerula becomes a Peri- morula by the conversion of its superficial ‘ blastema’ into a number of cells. This is considered by some authorities (Weissmann, &c.) to be due in the case of the Hexapoda to the free formation of nuclei. Good observations on a number of examples are as yet wanting, but we have the excellent researches of Edouard Van Beneden and Emile Bessels, which show that in many Crustacea the many-celled condition of the Perimorula is due to a regular cleavage process.1 Haeckel is inclined from this to assert as a general rule that the Perimonerula acquires a nucleus, becomes a Pericytula, and then cleaves into two, four, &c., cells, to form the Peri- morula. This process is exhibited in Plate IX, in the case of a Decapod Crustacean (Peneus), studied by Haeckel at Ajaccio. It does not seem, however, at all certain that cases such as that of the Gammarus fluviatilis, described by Ed. 1 T cannot let this notice pass through the press without alluding to the importance of Ed. Van Beneden’s excellent work on the composition and signification of the egg, for the whole of the questions which turn on the presence or absence of his (deutoplasm). HAECKEL S RECENT ADDITIONS TO GASTRAA=THEORY. 63 Van Beneden, may not be largely diffused. In this case the separation of food-material from formative protoplasm does not take place at an early period, nor so as to produce a superficial homogeneous layer of protoplasm, covering-in a granular yelk. But the segregation takes place at separate points, so that a number of isolated cells (exactly comparable in origin to the Cephalopod’s autoplasts) rise to the surface of the yelk and then proceed to divide, and so form a complete Perimornla. Now, it is not possible to say (in the absence of observations) how those cells arose, but it seems probable, from the analogy of the Cephalopods, that there was not a primitive division into two, and then into four, and so on, which took place deeply in amongst the granular mass of food- material, but, on the contrary, it would appear most likely that the network of protoplasm spread in and out amongst the food-granules segregated simultaneously at many points, and gave rise to autoplasts. Just as, in the Cephalopod, the segregation to form the cleavage-disc is incomplete, and leaves material behind which independently segregates at many points as autoplasts—so in Gammarus fluviatilis, and probably other periblastic species, the segregation to form the super- ficial cleavage-tunic or rind never comes off, but is reduced to a simultaneous formation of many autoplasts. In such cases the fertilised egg passes at once to the Periblastula stage, and cannot be said to exhibit either a Peri-monerula or Peri- cytula, or Per-morula stage. The food-material in the cells of the Perimorula is so placed as to occupy the inner face of each cell, and so lie centrally, but it is still disposed in the substance of the cells. By the gradual separation of the cells from it, it comes to occupy a central cavity, and is now surrounded by the cells which have freed themselves from it. This is the stage of the Periblas- tula (Fig. 86). The invagination of the Periblastula to form the Perigas- trula, a fundamentallyi mportant fact for the whole of the ‘germ-layer theory, was first observed by Bobretzky in Astacus and Palemon in 1873. It is exceedingly curious to find that in the periblastic type the invagination is so well marked, and the observation lends a certain probability to the hypothesis that invagination is the primary mode of forma- tion of the Gastrula. It is also exceedingly important and interesting to find that here, as elsewhere, the orifice of invagination or blastopore (Urmund of Haeckel) does not become the animal’s mouth. Haeckel is not able to say, in the case of Peneus, whether it becomes the anus or not (see Plate IX, and its explanation). 64: PROFESSOR E, RAY LANKESTER, A second invagination forms the true mouth and the stomo- dzeum! (so-called pharynx or Vorderdarm). The hypoblastic archenteron or gastrula’s stomach formed by the first invagi- nation gives rise only to the middle portion of the alimentary canal (Mitteldarm or mesenteron) and the liver. Here, too, as in other cases pointed out by Haeckel, the first development of the mesoblast is in the region of the blastopore-margin, but here too no certain information can be given as to the origin of all its elements, in regard to the question of their derivation from ectoderm or endoderm. The formation of the Gastrula of the Calcareous Sponges.— Metschnikoff, Oscar Schmidt, and F. E. Schulze have re- cently written on the development of the Calcareous Sponges, and have shown that Haeckel’s statements on this matter were not entirely correct. In the first place the formation of the Gastrula of sponges by delamination, and the subse- quent breaking through of the mouth, has to be given up. As Haeckel observes, this is so much the better for the Gastrza-theory, for the delaminate mode of development, though, as I have pointed out, reducible to the same ultimate significance as the invaginate mode of development, is yet a cenogenesis. Haeckel doubts whether delaminate Gastrule really exist at all, and thinks that early invagination. will be found to occur even in the Hydroids on careful study. This point should be at once investigated. Haeckel admits in reviewing the work of recent observers his fundamental error as to the mode of origin of the Gastrula of Calcareous Sponges, but rightly enough maintains that in the most essential features they have proved his statements to be jus- tified. The observations of Metschnikoff are shown to be erroneous, as well as his conclusions by the later work of Schmidt and Schulze; in fact, he takes endoderm for ecto- derm. Oscar Schmidt’s observations are correct in most points, but incomplete, and his inferences quite erroneous, as appears from Schulze’s work. Schulze confirms Haeckel in the most important point, namely, that a true gastrula with its two primary germ-layers does make its appearance in the development of the sponges. The four works on the development of Calcareous Sponges furnish an exceedingly interesting series illustrative of the genesis of error in such studies. Haeckel seized upon the really great and fundamental fact that the Sponges are no Protozoa, but that, like those of Celenterata, their eggs give rise to a sac—the Gastrula—with a wall of two cell-layers, en- 1 This term and its correlative ‘proctodeum’ I propose for the oral and anal invaginations. HAECKEL’S RECENT ADDITIONS TO GASTRHA-THEORY. 65 doderm and ectoderm. Haeckel fell into pardonable error as to the mode of formation of this Gastrula of the Calcispongie. Metschnikoff, following him, had got an inkling of the true mode of formation, namely, that there was a process of inva- gation taking place in a biastosphere or one-cell-layered sac, but his observations were most incomplete, and his reason- ing from them so much influenced by animus against Haeckel, that he has given the most erroneous account of the matter, which has been published. Oscar Schmidt failed to interpret correctly what he saw. The truth is that the Gastrula of the Calcareous Sponges, or at any rate of the species which all these observers have more especially studied, namely, Sycandra raphanus, is formed on the amphiblastic type. Oscar Schmidt saw the Amphiblastula (fig. 19), as did also Metschnikoff, but strangely enough, Oscar Schmidt failed to trace the invagination of this Amphiblastula to form the Amphigastrula (fig. 18), as he should have done. Consequently he mistook the whole process, declared that the Sponges had no gastrula-form at all, and concluded that all Haeckel’s ‘‘sch6nen theoretischen Folgerungen ” drawn from the fundamental fact of the Sponges possessing a Gastrula, must be abandoned. Oscar Schmidt must have said this with some pain, for he has proved himself a very fair and well-disposed critic of the Gastrea-theory. He will therefore probably be well pleased that Franz Eilhard Schulze, working at Trieste, has made most complete observations on the egg of Sycandru raphanus and its development to the gastrula-form, and published them in the ‘ Zeitschr. fur Wissensch. Zoologie,’ Bd. xxv, Supplement. Schulze has followed with absolute precision the cleavage-formation of an Amphimorula, of an Amphiblastula, and, by invagination of the large non-ciliated granular cells of the last, of an Amphigastrula in Sycandra raphanus. His drawings are executed with the greatest care and are obviously ‘ nature-true.’ Thus the attacks upon the germ-layer- or Gastrula- theory from the point of view of the Calcareous Sponges are definitely put anend to. Haeckel holds, as is clearly very probable, that the Archiblastic type also occurs among Sponges, and it is possible enough that the Periblastic type also presents itself. NEW SER.—VOL. XVI. 66 PROFESSOR E. RAY LANKESTER. Summary of the facts known as to Gastrula-formation in Loological order." Sroner®. Archiblastic, amphiblastic. Hypromepus#. Archiblastic (Cassiopeia, Rhizostoma, Pelagia, Kowalew- sky). Delaminate, probably disguised archiblastic and amphiblastic (Hydroid polyps). Amphiblastic (many Siphonophora, Haeckel). Discoblastie (perhaps some Siphouophora). CrpenopHora. Amphiblastic (many or all. Kowalewsky, Fol, Agassiz) possibly some discoblastic (Haeckel). Actinozoa. Archiblastic (Actinia, Caryophyllia, Gorgonia, and Cereanthus, Kowalewsky, Monoxenia, Haeckel). Periblastic (Aleyonians, Kowa- lewsky). Delaminate, perhaps disguised archiblastic (some species of Actinia, Kowalewsky). Vermes. Archiblastic (Nemertines, Metschnikoff, Dieck. Balanoglossus, Sagitta, Kowalewsky. Cucullanus, Butschii. Phoronis, Kowalewsky). Amphiblastic (various epibolic and entobolic varieties among Platy- elminthes, Nematoda, and Annelida, described by Leuchkart, Cla- parede, Kowalewsky, &c., among Rotifera by Salensky, in the Gephyrean Phascolosoma by Se/enxka). Discoblastic (not known, but Euaxes furnishes a transition from amphiblastic to this type, Kowalewsky). Periblastic, not known. Moxtusca. Archiblastic (Argiope, Terebratula, Kowalewsky. Pisidium, Lankester). Amphiblastic (Thecidium, Kowalewsky, Unio, Rabi, Limneus, Lan- kester (not archiblastic), Purpura, Selexta, Trochus, Haeckel, Aplysia, Neritina, Lankester, Pterpods, Fol). Discoblastic (Cephalopods, Kod/iker). Ecuinoperma. Archiblastic (Holothurida, Kowalewsky, Asterida, Agassiz, Kehinida, Haectel, Crinoida, Wyv. Thomson). Amphiblastie (Uraster Mulleri, Echinaster Sarsii, Pteraster militaris, Amphiura squamata, and other viviparous Asterida, Anochanus chinensis and allied Echiniaa, Thelenota tremula, Phyllophorus urna, Synaptula vivipara, Cucumaria doliolum among Holothurida), Discoblastic and Periblastic type not known. Arturovopa. Archiblastic (some Branchiopoda and Copepoda, Van Bene- den, Vardigrada, Kaufmann, the Pteromalina, Ganz). Amphiblastic (some lower crustacea, Van Benedeu and Bessels, some lower Tracheata). Discoblastic (some larger forms of crustacea, Van Beneden and Bessels, Oniscus, Bobretzky, Scorpio (fig. 40), Metschnikoff). Periblastic (most Crustacea, Limulus, Packard, most Myriapods, Arachnida and Insects, according to observations of Bobretzky, Van Beneden and Bessels, Weismann, Kowalewsky, Claparéde, and Metsch- nikoff). Verteprata. Archiblastic (some Ascidians, Kowalewsky, Amphioxus, Kowalewsky). Amphiblastic (some Ascidians, Kowalewsky, Cyclostoma, Schultze, Amphibia, Ganoids, Owwsjanmikow, Marsupials ?, Placentals). Discoblastic (Pyrosoma, Hualey, Kowalewsky, Selachiaus, Balfour, Teleosteans, Birds, and Monotrema ?) Periblastic. This type has not been recognised in any Vertebrata. 1 This list and its zoological arrangement is modified and extended from the references given by Haeckel. NOTE ON STENOGRAMMA INTERRUPTA, 67 Nofe on StENoGRAMMA INTERRUPTA (Agardh). By E. PercevaL Wricut, M.D. In the number of ‘ Grevillea’ for December, 1874, and at p. 88, there will be found a notice, by E. M. Holmes, of Steno- gramma interrupta, in which the author states—l. That “‘the tetrasporic fruit of this rare and beautiful Alga has not hitherto been recorded as occurring in Britain.” 2. That it “is not described in any of the more recent works on Marine Algz published in England.” 3. That so far as the author is aware, no figure of the tetraspores has ever been published. 4. That though Miss Gifford forwarded a speci- men having tetraspores to Dr. W. H. Harvey in 1848, no further notice of the plant was taken by him and that it was probably lost. At the time when [ read this notice I had fully intended to send to my friend the editor of ‘ Grevillea,’ a reference to the ‘ Phycologia Australica’ of Dr. Harvey, vol. iv, Plate 220, published during 1862, in which a very full account of this interesting form will be found. I omitted to do so, and | would not have thought more of the subject, but that my attention has again been called to it, partly by a communi- cation from Mr. W. G. Farlow, assistant to Professor Asa Gray of Cambridge, U.S., and partly by a reminder from a friend to the effect that it is but an act of justice to the memory of my former master (whose post as Professor of Botany and Keeper of the Herbarium in Trinity College, Dublin, I for the present occupy), to show that he did not only receive but noticed Miss Gifford’s letter. For this reason the members of the Club will be pleased to pardon the enumeration of the following to them perhaps familiar facts : 1. The “ tetrasporic fruit” is described by Harvey “as occurring in British specimens”? in ‘Nereis Boreali- Americana,’ Part ii (March, 1853). (a) In specimens sent to Dr. Harvey in 1848 by Miss Gifford, from Somersetshire. (4) In speci- mens sent by Mr. Isaac Carroll in 1851 from Cork Harbour. 2. It is true that Harvey’s ‘ Nereis Boreali-Americana ’ was not published in England, yet it is a very accessible and most necessary work for the student of British Algw. Harvey’s ‘Phycologia Australica’ was however published by Lovell Reeve, at Henrietta Street, Covent Garden, London, and in 68 DR. E. PERCEVAL WRIGHT. it, as well as in the North American ‘ Nereis,’ a description of the “ tetrasporic fruit ” will be found. 3. On Plate ccexx, Figures 2, 3, 4, 5 of volume iv of Harvey’s great work on the ‘ Australian Algee,’ illustrations of the tetraspores and nemathecia will be found. This is scarcely the place to criticise the former. 4. Miss Gifford’s'letter and specimens are in the Herbarium of Trinity College, Dublin. Her letter is dated from Minehead, Somerset, September 21 [1848]. I cannot say if it was answered, though there is proof that she was in cor- respondence at the time with Dr. Harvey, and that the specimens she sent were at the time registered in the Herbarium. The priority of Miss Gifford’s discovery of the tetraspores is fully acknowledged in page 169, of Part ii, of the ‘ Nereis Boreali-Americana.’ I would further remark that Dr. Montagne’s letter to the Rev. M. J. Berkeley is alluded to by Dr. Harvey in the work last quoted; also that J. G. Agardh’s ‘ Species Genera et Ordines Floridearum,’ which forms the second volume of the ‘Species Genera et Ordines Algarum,’ was published not only in parts but in fasciculi, and that vol. 11, pars. u, 1, pp. 1 to 504, containing the description of Stencgramma, was published in 1851. (Pars. 11, 2, was published in 1852.) As this fasciculus was probably printed early in 1891, it is not likely that Agardh could have seen Dr. Montagne’s letter in the June number for that year of the ‘ Annals and Magazme of Natural History.’ The Trinity College Herbarium possesses specimens from the following localities : Evrore.—Strangford Lough; in six to eight fathoms, adhering to small stones in Castle Ward Bay, between Portaferry and the Old Castle, Dr. Dickie, 1858. Cork Harbour, Isaac Carroll, 1851. Minehead, Somerset, Miss Gifford, 1847-48. Plymouth, Dr. Cocks, 1846; Rev. W. Hore, 1847; Mr. Gilbert Sanders 1847. Lisbon, “In Tago salso, lecta mense September, 1842, F. Welwitsch.” Gibraltar, Mrs. Craige. ‘Toulon, M. Giraudry. America.—Florida, Key West, Dr. Harvey. California, San Francisco, Dr. Sinclair. AusTRALASIA.—New Zealand, Colenso. Tasmania, F. Mueller; W. H. Harvey. In many specimens, especially in luxuriant ones from New — Zealand, with fronds over a foot in length, the nemathecia are distinctly to be seen on both surfaces of the frond; but the shape of the nemathecia varies much, and so do the terminal edges of the lacinie, being sometimes broadly obtuse SECTION OF CEREBRAL AND CEREBELLAR CORTEX. 69 and at other times almost pointed; often the edges of the frond are “entire,” but sometimes they are clothed with dense tufts and fringes of “ proliferous forked leaflets.” The specimen with tetraspores first examined by E. M. Holmes was from Mr. Isaac Carroll’s collection, and was doubtless gathered in Cork Harbour. The one from which the figure on Plate xxxvui of Grevillea was drawn is stated to have been gathered in Scotland. It would be interesting to know from what part, as this will be the most northern habitat as yet known.—Eztract from Minutes of Dublin Microscopical Club for November 18, 1875. PREPARATION of Sections of CEREBRAL and CEREBELLAR Cortex for Microscopic Examination. By W. Bevan Lewis. Tue histological analysis of animal tissues, so far as it relates to processes of preparation and staining for the better differentiation of the elementary constituents under micro- scopic examination, has long been regarded as an important item in physiological and pathological research. Much has already been accomplished in this sphere of labour, yet much still remains to be done, especially in the histology of the central nervous system. New statements in regard to the staining of brain sections which have recently been advanced require mature consideration and experimental verification before they become standard facts for the guidance of the physiological inquirer. Until this sifting of evidence is accomplished we cannot hope to find any substantial basis for the establishment of histological science. The constituents of the cerebral cortex differ much zntéer se in the facility with which they acquire the coloration neces- - sary for a good delineation of their outline and structure. Thus, while it is but a trivial task to stain equably and deeply the smaller cells of the different layers of a con- volution or the granular layer of the cerebellum, or even to bring distinctly into view the nuclei of the blood- vessels or the contour of the nerve-fibres, it is a far more difficult matter to effect a good staining of the pyramidal cells or the process of the cells of Purkinje. By a good stain- ing I mean a deep staining, which yet leaves the cell con- tents as unaltered as possible, the nucleus and nucleoli clearly exposed to view, and the numerous minute extensions of these bodies well defined. Undoubtedly a really good section 70 WwW. BEVAN LEWIS. of the cortical layer of the brain should embrace all these qualities, together with a fair view of the vascular channels, whilst the delicate reticular arrangement of the surrounding matrix should be unaltered by the reagents employed. The difficulties in our way are great, but yet of late patient and laborious investigation has rewarded the inquirer with fre- quent triumphs of a most substantial character. Of the numerous colouring agents used by the microscopist, the more valuable for purposes of studying cerebral histology are the following: carmine, logwood, picric acid, aniline blue, aniline black, magenta or rosaniline, aniline red or fuchsin, and the various Judson’s dyes. We owe import- ant assistance also to the reduction of certain metallic com- pounds by the agency of the organic germinal centres of the tissues, e.g., osmic acid, chlorides of gold and palladium, oxide of uranium and nitrate of silver. The latter series I shall not refer to further in this article, as my attention is to be given entirely to the members of the first series, which give their own natural colour to the objects exposed to their action. I may, however, in passing, recommend solutions of each of the latter class of the ordinary strength of one per cent. to be kept in hand. _ For actual use the solutions of gold and silver may then be diluted to the extent of 0°25 to 0°5 per cent., the chloride of palladium to 0-1 per cent., the chlorides of gold and potassium combined should represent in every 100 cc. of the solution ;1,th of a gramme of the salt, whilst the osmic acid solution should vary from 1 to 2 per cent. in strength. With this brief mention of the metallic dyes I pass on to the subject more immediately concerned and describe sertatim the several processes employed by myself for stain- ing the cortex of the cerebrum and cerebellum. First in the series is carmine, the earliest agent used for this purpose. Carmine dyeing and its modifications.—Gerlach, with whom originated the process of dyeing, used a solution of carmine which has since been modified by Thiersch, Beale, and Frey. The solution recommended by Beale (a carminate of am- monia) is, I believe, the one most generally adopted, and © possesses, with some drawbacks, several points of advantage over other media. The whole process of carmine-staining demands, however, great attention to details to secure perfect success. One essential feature is that the solution should contain but a minimum amount of ammonia, and this is of special importance when we are dealing with cerebral tissue. Again, the after process of washing should be conducted SECTIONS OF CEREBRAL AND CEREBELLAR CORTEX. 71 with a very weak acid solution, the best wash being a half to one per cent. solution of glacial acetic acid. Small por- tions of the cortex, a third of an inch in thickness, are pre- viously hardened in the bichromate of potash or in Muller’s fluid, using chromic acid solutions as accessories to hurry on the process near its completion, are then transferred to Stirling’s microtome and the finest possible sections made. They should now be placed for a few minutes in methylated spirit, and next passed into Beale’s carmine solution, diluted with seven times its bulk of distilled water. I find my sections are usually stained to the requisite depth of colour in six or eight hours, but variations in rapidity will constantly occur, the delay being frequently occasioned by the presence of free chromic acid, which should always be completely removed by immersion in spirit prior to section cutting ; and we can readily conceive with Beale that any changes undergone by commencing putrefaction in the nuclei will, by altering the acid reaction which is the natural post- mortem condition, interfere with the necessary decomposition of the carminate. Sections that have assumed the proper tint may now be removed to a capsule containing a half per cent. solution of glacial acetic acid. They are next washed from all acid, dehydrated by steeping in absolute alcohol or rectified spirit, cleared with oil of cloves or anise, and mounted in balsam. This is the usual method adopted by me in single staining with carmine, but the results, though good, are by no means so uniformly satisfactory as by log- wood and aniline processes. ‘There is one measure, however, which I adopt with this reagent with the best results; I may denominate my process as a mode of differentiation by peculiar alterations in the refractive indices of the structural elements. Changes which occur to the cell-contents and their processes —I refer to alterations in density by the agency of heat, spirit, coagulating media, and the essential oils —alter the refractive relationships previously held by them to the sur- rounding matrix, and thus bring into view what would otherwise be passed by unnoticed. It appears to me that this variety of histological analysis may, in regard to brain sections, be turned to very good account. On placing an unstained section of cerebrum or cerebellum in the field of the microscope, saturated with spirit, little or no structure is apparent, but if a drop of essential oil be now allowed to run over it there will be observed at a certain stage of the clear- ing up, and whilst the spirit is evaporating, a sudden start- ing out in bold relief of the cells, nerve- fibres, vessels, &c., which again disappear or partially fade on perfect clearing 72 W. BEVAN LEWIS. of the section. Now this appearance may be fixed by sud- denly dropping over its surface a httle balsam: and perma- nently mounting. Upon this fact depends the process now to be described. Sections treated by Beale’s carmine solution (1 to 7 in strength) and washed with the acid wash are placed, saturated with spirit, upon a slide. When the spirit has nearly all evaporated a drop of oil of anise is aliowed to flow over the section (not to float it up), and the clearing process is watched on the stage of the microscope; then, just when the appearance referred to above is presented to view, a drop of balsam is allowed to run over the section and a covering glass permanently fixed on. In lieu of the oil of anise I frequently employed glycerine with the same results, and mount ultimately in glycerine jelly. Still better effects may be obtained by the following method. Picro-carmine Staining.—I use the solution recommended by Ranvier, the sections being left in it for an hour. I then transfer them to a strong ammoniacal solution of carmine for the space of twenty minutes, and on removal wash freely and mount, as in the process described above. ‘The appear- ance of sections of cerebellum treated thus is very striking, and the cells of Purkinje and their processes are far better delineated than by the usual carmine method. Logwood Staining.— Formule for the carmine dyes are well known; not so, however, those for hematoxylin. Béhmer, who first employed logwood for these purposes, gives a formula which is quoted by Frey.' I may also refer the reader for an excellent formula for the logwood solutien to the pages of ‘The Lens’ for July, 1872. The solution usually sold under this name varies greatly in efficiency, and I am told by one of the most eminent authorities im cere- bral histology that this variability has proved a matter of great and constant annoyance to him. I must confess to a similar experience in my employment of this reagent. When, however, the proper solution can be obtained, it undoubtedly stands in the foremost ranks of staining fluids, its results being unrivalled for the major details in the cerebral cortex. Of the solution sold as logwood dye about ten drops should be added to each drachm of distilled water, carefully filtered and exposed for a short period to the air. The sections should remain in it until the required depth of colour is ob- tained, the logwood poured off, and the pieces well washed by allowing a stream of water to flow on to the inclined side of the porcelain vessels in which they are held. All diffuse 1 ©The Microscope and Microscopic Technology,’ translated by Cutten, page 158. SECTIONS OF CEREBRAL AND CEREBELLAR CORTEX. 73 staining may now be removed, by keeping them in a vessel containing methylated spirit for some hours. When suffi- ciently washed by this measure, as may be seen immediately by the removal of a section to the stage of the microscope, and examining it with a low power, the tint of the ger- minal centres dyed in this way may be darkened by exposure of the slide for a few seconds to the vapour of ammonia, or to a faintly alkaline spirit. They are next cleared in the usual way with oil of cloves, and mounted in balsam. The Aniline sertes of Dyes.—Magenta or rosaniline is a powerful and active dye, but its solubility in spirit is so great that its effects are not permanent, and the difficulties in fixing this reagent have proved insuperable obstacles in the way of its employment for these purposes. Judson’s purple dye also shares in this disadvantage, but to a less extent. The latter may be used in pyoducing the peculiar conditions of refractive power in the tissue elements which are described under the heading of carmine; but for these results the picro-carmine solution far surpasses any of the aniline or Judson’s series of dyes. To Mr. W. H. O. Sanky belongs the credit of introducing to our notice the aniline black as a staining reagent applicable to the microscopic investigation of cerebral tissues. A section of human cerebellum exhibited by him at the last annual conversazione, held at the West Riding Asylum, attracted great and deserved attention. The cells of Purkinje and their processes were shown with extra- ordinary beauty. His process has been described in the pages of the ‘ Lancet ;’ but for full details I would refer my reader to the ‘ West Riding Asylum Reports’ for this year, where an extremely interesting paper by Mr. Sanky will be found, together with most faithful and beautiful plates of the cerebellar cortex, &c. The process he adopts for drying fresh brain for sections I have not tried, but the aniline black as a dye I have used extensively and with the best results. My experience leads me to recommend two distinct methods as productive of most satisfactory slides. The solu- tions of aniline black employed by me are three, varying in strength as follows: 0:25, 0°5 and 1 per cent. It may be dissolved in water, alcohol, or even glycerine; but for most purposes the aqueous solution is the most useful. It is a matter of no difficulty with this reagent to dye to any depth, but the washing-out of diffuse staining is at first rather per- plexing. ‘The solvents of aniline which present themselves as useful accessories here are absolute alcohol, methylated or rectified spirit, glycerine, or glycerine and water. ‘hese, however, do not not succeed with me in demonstrating satis- 74: W. BEVAN LEWIS. factorily the processes of the cells of Purkinje, or the finest of twigs which arise from the pyramidal layers of the cerebral cortex. No reagent has found such claims upon our atten- tion for this purpose as chloral hydrate. For some time past I have employed this reagent for the demonstration of nervous elements, having been directed to its use by a paper by Victor Butzke.1 This writer states:—‘‘ Eine solution von chloralhydrat in wasser, von 1:1 bis 1: 10 pro cent. auf das frische Gehern angewandt, lost zwar das Hirnfett nur theilweise, versetzt es aber in einen Zustand, in welchem es molecular zerfallt, so das es leicht aus dem Zusammen hange mit den Formelementen herausgewaschen werden kann. Als ganz vorziiglich habe ich eine combination von Hyperosmiumsaure (1 per cent.) mit chloralhydrat gefunden, was nach einander angewandt, in der Schonheit der Isolation der Hirnelemente bald, so glaube, ich die alten Rivalen weit hinter sich lassen wird.” The latter statement I have not been able at present to confirm; but as to the full value of chloral hydrate for such purposes I can fully confirm the opinion of Butzke. Still another important quality belongs to this compound, namely, that it is a power- ful solvent of aniline black, so that we possess in it the very valuable combination _of qualities most likely to give us the best results. This assumption, put practically to the test, has answered my highest expectations. ‘The process I employ I can confidently recommend, especially for the demon- stration of the cerebellar cortex. ‘The sections are first deeply stained in aniline dye, gently washed from superfluous colour in distilled water, and removed to a porcelain vessel containing the solution of chloral, where they are allowed to remain from twenty to thirty minutes. ‘They are next trans- ferred to the following solution:—Of solution of chloral hydrate and oil of cloves equal parts. Alcohol enough to dissolve perfectly and form a perfectly clear solution. The alcohol must be added by degrees, the mixture being stirred with a glass rod, and care being taken to avoid any excess. During use this mixture must of course be kept carefully covered up to avoid evaporation. It will now be found that the sections are clearing up, whilst all diffuse staining is still further removed by the chloral. We should remove one occasionally to a slide, and examine under a low power the progress made, and when satisfactory results are obtained, the sections should be washed with a little alcohol, floated up by one or two drops of clove cil, and when perfectly clear mounted in balsam. In regard to the essential oil used I ! ¢ Archiv fiir Pyschiatiie und Nervenkrankheiten,’ 1872. SECTIONS OF CEREBRAL AND CEREBELLAR CORTEX. 75 employ oil of anise and oil of cloves indiscriminately, both being of high refractive powers. ‘The former has, however, the disadvantage of solidifying at ordinary temperatures. Its oxidized compound stearoptene, which forms the greater bulk of the oil, and which separates in a solid mass, may, however, by a very gentle warmth be kept in a fluid state during the process. ‘The beautiful effects of logwood stain- ing may also be obtained for the granular layer, whilst the layer of Purkinje’s cells and their antler-like processes may be produced with all the accurate details of aniline-staining by a process which I now proceed to describe. Double Staining with Logwood and Aniline.—It will soon be noticed by those who attempt a logwood staining of the cerebellar cortex, that whilst the granular layer nuclei of blood-vessels and nerve-fibres are beautifully stained, the cells of Purkinje and their processes remain very faintly or not at all affected by the colouring reagent; the aniline black, on the other hand, appears to possess special affinities for these latter structures. If, therefore, sections dyed with logwood are immersed for a few seconds only in a solution of aniline black of a strength of 0°25 to 0°5 per cent., it will be found that the logwood staining still remains, whilst the layer of Purkinje has taken up the aniline sufficiently to afford a decided and beautiful contrast. The chloral hydrate should now be used, but the alcoholic solution is not here advisable. They should be well washed from the chloral with a stream of water, rapidly dehydrated by alcohol, cleared with oil of cloves, and mounted in balsam. Summary.—Having so far dwelt on the more general methods employed for demonstrating the structure of the cineritious substance of the brain, I shall in conclusion briefly sum up their special advantages and disadvantages in individual cases. First, in regard to carmine stainings, there are two great faults which all microscopists will, I think, immediately recognise. One defect is the glare of a colour such as carmine, which is very tiring to the eye, and renders prolonged and steady working with the microscope a matter of great difficulty, not to say real injury, to the eye. Then, again, the want of definition given by a deeper, darker colour is felt by all experienced microscopists, and more especially by those who have employed the logwood and aniline dyes. On the other hand, it never clouds the cell, but leaves the nuclei and all minor details clearly exhibited. No one who has examined a really good pre- paration of the cerebral cortex with logwood staining will fail to acknowledge the great superiority of this dye over carmine 76 H. C. SORBY. in its general results. As regards the peculiar refractive qualities, however, which are impressed upon the tissue elements by the modified carmine process above described, I may state my belief that this will yet prove of most essential service in the estimation of the relative proportions of cell- processes in any individual section, and the most accurate tracing of any existing connections, for not by the deepest aniline staming have I yet succeeded in demonstrating the existence of so thick and numerous a series of processes diverging from the pyramidal layers of the cerebral cortex as by the methods described above. With regard to the smaller cells of the cerebrum and cerebellum the logwood staining is eminently successful ; but when it is our object to bring into view the larger cells and their extensions, I would recommend the aniline dyeing above described, or the double staining with logwood and aniline. On the Evouution of HamMocrosin. By H. C. Sorsy, F.R.S., F.L.S., F.Z.S., President of the Royal Micro- scopical Society. In a paper read before the Royal Microscopical Society last April, published in the ‘ Monthly Microscopical Journal ’ for May,! I endeavoured to show the very great importance of referring all the measurements of spectra to the wave- lengths of each part expressed in millionths of a millimeter ; and in another paper read before the same Society in November, published in the above-named journal for December,” I described a new form of apparatus by means of which the wave-lengths can easily be measured to a mil- lionth of a millimeter, and the difference in the wave- lengths of the centre of the bands in closely related spectra determined with still greater accuracy. In the former paper I also endeavoured to point out the very important con- clusions which may be drawn from those small differences in the position of the absorption-bands, which might easily be overlooked, and which in some cases I had myself over- looked until quite recently. Unfortunately the full meaning of some of these differences cannot yet be ascertained with I Vol) xmi, p. 196. 2 Vol. xiv, p. 269. cetacean tte iii ea eta ON THE EVOLUTION OF HAMOGLOBIN. 77 perfect certainty. The physical facts have altogether out- stripped chemical knowledge. They clearly point out what kind of investigation is necessary, but at the same time it would be very difficult, if not impossible, to obtain a suf- ficient quantity of material to decide some of the most important questions. In the present state of the subject the only course open to us is to draw the most probable analytical conclusions that we can by careful induction from facts already determined by synthetical methods. The best illustration of this kind that I can give is the difference in the spectra of hemoglobin, in which the oxygen is replaced by other gasses without there being any actual decomposition. ‘Thus, for example, on replacing the oxygen by nitric oxide, or by carbonic oxide, the general character of the spectrum of oxidized hemoglobin is only slightly changed, but the position of the bands is materially different. When expressed by wave-lengths in millionths of a millimeter, the centres of the absorption bands are as follows :1 - Nitric oxide hemoglobin . 583 : * 545 Oxygen ; - : JAbS E : : 545 Carbonic oxide. ‘ DOTS Ti ie F 542 It will thus be seen that the substitution of one gas for another in loose combination with the same very complex coloured radical hemoglobin alters the wave-lengths of the centres of the bands to an extent which can be easily measured. This change in the spectra is accompanied hy most decided differences in chemical characters, so decided in fact that, as is well known, the substitution of the oxygen in the blood by carbonic oxide makes ali the difference between life and death. In all these cases, however, it is easy to prove that there is one complex radical common to all, since they can all be changed into identical products. Now, supposing that these three substances were met with in nature, and that their spectra were known, and also the fact of their yielding common products, it is quite clear that we should be justified in concluding that they were closely related in containing a common radical, and at the same time differed by the substitution of one substance combined with this radical by another substance. I think that the strict accuracy of this reasoning cannot be denied in this 1 Since the wave-length method has not yet been adopted by all observers, it may be well to give the wave-lengths of a few of the Fraunhofer lines that occur near the absorption bands described in this paper. D. 589, EK, 527, F, 486. 78 H. C. SORBY. instance; and in applying it to less known facts, I think I may claim high probability in favour of the provisional con- clusions. It would indeed be premature to look upon the results as more than provisional, seeing that the whole sub- ject is in its infancy. At the present time it would be impossible to give a more striking illustration of this method of research than the various facts which seem to indicate the gradual evolution of hemoglobin. Very much remains to be learned, and I now publish what I have so far been able to learn, chiefly with the view of attracting attention to a most promising field for research, and to show the importance of the spectrum method in the investigation of the comparative physiology of the lower animals. Some few months ago I commenced the study of the different coloured substances found in the shells and soft parts of various species of Mollusca. Many of these have very striking characters, but on the present occasion I shall confine my remarks almost entirely to those which bear on the evolution of hemoglobin. If the common large snail, Helix aspersa, kept some time without food in autumn, be killed with chloroform and then immediately dissected, it will be found that the intestine near to the part where the secretion from the liver passes into it, is more or less distended by a reddish-brown liquid, which easily runs out when the intestine is cut. ‘here cannot, I think, be any doubt that this bile is a mixture of at least two coloured substances. One is probably related to the brown substance, quite unlike the chief coloured con- stituent of the bile of the higher vertebrata, which is the cause of the dark colour of the liver, especially in Helix nemoralis, and gives no well-marked or characteristic spectrum. ‘The other gives one very dark and well-defined absorption-band towards the yellow end of the green, and another much more faint towards the blue end of the green. That this substance is not a product of decomposition is proved by the fact that the bands can be well seen in light transmitted through a living Limazv. At first sight the spectrum might easily be mistaken for that of deoxidized hematin, but on comparing them together side by side, though the bands of this bile pigment are of exactly the same character as those of the deoxidized hematin from human blood, they are in a most decidedly different position. There is, in fact, the same kind of relation between these two spectra as that between the spectra of hemoglobin when loosely combined with different gases. ‘This bile pigment, ON THE EVOLUTION OF HAMOGLOBIN. 79 however, differs from normal deoxidized hematin in not uniting with loosely combined oxygen when exposed to the air. In order to raise it to an oxidized state, it is necessary to add a minute quantity of potassic permanganate. ‘The well-marked absorption bands then disappear, but are again developed by the addition of a deoxidizing reagent. ‘This spectrum is, however, not seen unless the solution be some- what alkaline; and when the natural bile is exposed some time to the air, so as to become somewhat acid, the bands may not be seen until a slight excess of ammonia has been added. Hence clearly enough, as in the case of normal hematin, the above-named bands in the spectrum are charac- teristic of the alkaline deoxidized solution, but this bile pigment of Helix has a less affinity for loosely combined oxygen. Such then being the case, it is necessary to inquire whether it be possible to change it into normal hematin, or to alter both into one common product. As far as I have yet been able to ascertain, there are at least three very distinctly different modifications of hematin, which, when in an alkaline solution and deoxidized, give spectra of almost exactly the same character, the bands are at the same distance apart, but their centres are situated in very materially different wave-length positions, as will be seen from the following table :— Very dark Much fainter band. band. Bile pigment hematin : : P , 5645 = > 532 Fresh made normal hematin from human blood 5614, : 529 The same kept several years sealed up ina tube 5562 F 5242 On taking a small quantity of an aqueous solution of human blood, adding ammonia, and boiling, we obtain a brown solution of what I regard as normal hematin. Adding a little potassic sodic double tartrate, and deoxidizing with a minute portion of ammonium ferrous sulphate, we obtain the well-known spectrum of deoxidized hematin. If, how- ever, the boiled ammonical solution of blood be sealed up in a glass tube and kept for several years, it becomes deoxidized by its own decomposition, and the spectrum, though very closely analogous, is not identical with that obtained on deoxidizing the fresh made solution. Both bands are raised towards the blue end to the extent of nearly five millionths of a millemeter, the upper one being also made somewhat broader. I may here say that these quantities are as dis- tinct and easily measured with suitable apparatus as an equal number of inches in the height of a man. According to the principle already named, such a relation between the spectra indicates some definite change of the 80 H. C. SORBY. hematin into a closely related product. Much light is thrown on this question by the action of potassic permanganate. On adding a small quantity to normal hematin formed by boiling human blood with dilute ammonia, it is first changed into a modification which, when deoxidized, gives similar bands in exactly the same situation as those in the solution kept long sealed up in atube; but yet there is such a decided dif- ference in their width as to make it probable that the two products are not exactly the same substance. The facts do, however, prove that normal hematin can be changed by slight oxidization into an analogous substance, giving the absorption bands somewhat nearer the blue end of the spectrum. On adding still more permanganate this is still further and very completely changed into an entirely new product, which when deoxidized gives a well-marked band in the yellow, having its centre at wave length 587. Now, on treating the hematin of the bile of Helw with potassium permanganate, it is in the first instance so changed as to give on deoxidization exactly the same spectrum as normal hematin, with the bands in exactly the same position ; and further action of the permanganate alters it into the other modification already named. I have not, however, succeeded in preparing the product giving the band in the yellow, probably on account of the presence of so much of more easily oxidized substances, which make the bile so thick and gummy. It thus appears almost certain that the hematin of the bile of Helix can be changed into normal hematin by a process of oxidization, and that this change modifies both the spectrum and the affinity for loosely combined oxygen. We may, therefore, very reasonably expect to meet with this normal modification in some of the organs of the living ani- mals, and, in fact, I think it very probably does occur in the muscles of the foot and elsewhere, since the absorption band is certainly nearer to the blue end and nearly corresponds with that of normal hematin. I have, however, not yet proved this to my entire satisfaction, for want of proper material and good direct sunlight since I perceived the importance of the question. The amount of hematin found in different species and genera of pulmoniferous molluscs differs very much. In Testicella, Pupa, and Clausilia, there was so little as to make its presence very doubtful. The amount in Limnea is small, but yet very decided, and is much greater in Helix, Zonites, Limax, and Cyclostoma. In all these its presence is usually most decided in the liver, but yet it is not confined to that organ. I have uot been able to detect it in the blood itself, ON THE EVOLUTION OF HAMOGLOBIN. 81 but if it is really formed in the liver, it seems probable that it must to some small extent pass into the blood, or else it could scarcely find its way into the other organs. I have also met with it in Planorbis corneus, in the liver, and in another glandular organ with a granular structure; but the most important coloured substance in the animal is a modi- fication of hemoglobin dissolved in the blood. This com- pound is of such a remarkable character as to claim very special attention. In its natural state this red blood of Planorbis gives so nearly the same spectrum as that of the hemoglobin of human blood, that it was regarded as the same substance by Ray Lankester.! There is, in fact, no difference in general character, and, like the normal kind, when deoxidized, the two bands disappear and are replaced by a broad band; but yet on careful comparison it may easily be seen that the two well-marked bands of the oxidized state lie nearer to the blue end than in the spectrum of normal hemoglobin, as will be seen from the following table :— Centres of the bands. Normal hemoglobin . 5 : : : 58, 3) 2545 iaeekinserarse Gi) te nor tif} «pei DRS 4 F,5405 Even if there bea little doubt as to the exact wave-lengths true to a millionth of a millemeter, there is none as to the fact of the bands being nearer to the blue end by a wave- length of 24 or 3 millionths. This difference cannot be due to any difference in the physical state of the substance, since in both cases it was perfectly dissolved in a relatively large quantity of water. It is rather of such a character as would result from the substitution of one kind of albumenoid for another in combination with the same modification of hematin, into which both are decomposed when boiled with ammonia or decomposed by acids. This conclusion is made almost certain by the difference in the temperature at which they are decomposd by the coagulation of the albumenoid. The red aqueous solution of Planorbis hemoglobin slowly turns brown, and is decomposed into hematin at the compara- tively low temperature of 45°C., and is rapidly changed and coagulated at 49°; whereas in the case of a similar solution of human hemoglobin, similar changes do not occur until the temperature is raised to 65° and 69°. There is thus a dif ference of about 20° C. in the temperature at which the albu- menoids coagulate; and this fact taken in conjunction with the character of the spectra appears to me very satisfactory evidence of a well-marked difference in the albumenous con- ' € Proc. of Roy. Soe.,’ vol. xxi, p. 70. NEW SER,—VOL, XVI. F 82 H. C. SORBY. stituent of these two modifications of hemoglobin. If this be so, we might reasonably expect to find some difference in their chemical relationships, whilst at the same time both might yield identical coloured products. This is most cer- tainly the case. When treated with citric acid both yield the same hematin, but the hemoglobin of human blood is far less stable and far more easily decomposed than that from Planorbis. Taking an equal quantity of both, and adding an equally small quantity of citric acid, the hemoglobin of human blood was changed almost at once into hematin, whereas that from Planorbis showed no such sudden decomposition ; and even after half an hour was not so much changed as the other in less than a minute. In another experiment, taking equal quantities of the two hemoglobins, I added twice as much citric acid to the Planorbis. ‘That of human blood was changed to hematin and lost the hemoglobin bands in ten minutes, whereas that from Planorbis was changed so much more slowly that the alteration was not complete until after two hours, though, as I have said, the amount of citric acid was double. Hence clearly enough the hemoglobin of Planorbis is a far more stable compound. I am also much inclined to believe that some of the secondary spectra do also differ in important characters. ‘Thus, when the hemo- globin of Planorbis was deoxidized, I could see a faint absorption band at about wave-length 504, not visible in the case of deoxidized human hemoglobin. This may be due to some other colouring matter mixed with the original, but the spectrum of the oxidized compound furnishes no evidence of any such second substance. Taking everything into con- sideration, it appears to me that the complete study of these two modifications of hemoglobin cannot fail to throw very much light on a number of interesting physical and bio- logical questions. It also appears to me very desirable that the hemoglobin of the various animals in which it has been detected by Ray Lankester and others should be carefully examined from this new point of view, in order to ascertain whether the two modifications are correlated to the existence or non-existence of blood discs, since perhaps it may be found that the modification of globulin met with in Planorbis is characteristic of blood coloured by a hemoglobin solution, and the other modification that which is essential to the forma- tion of coloured blood discs in a nearly colourless serum. According to the observations of Ray Lankester,' there is strong reason to believe that hematin is the coloured radical of his chlorocruorin, which plays the part of hemoglobin in 1 «Journal of Anatomy and Physiology,’ vol. iv, p. 125. ON THE EVOLUTION OF HEMOGLOBIN. 83 the green blood of Sadella ventilabrum, though the exact connection remains to be determined. Reviewing now the whole subject from the point of view of the theory of evolution, it should appear that the most stable form of hematin made its appearance as a bile pigment in a state totally unfit for the purposes of respiration, since it will not unite with loosely combined oxygen on exposure to the air. By a slight change in its constitution it might however perform this function, and perhaps to some extent does in some of the Mollusca, or at all events supplies the place of the hemoglobin which forms one of the constituents of the red muscles of higher animals, whilst in some of the Gasteropoda it is so combined as to give rise to a form of hemoglobin. According to Lankester,! Planorbis alone amongst this group of animals has a red blood, coloured by hemoglobin, perhaps, as he suggests, because the necessity for utilizing the whole of the small amount of oxygen ayail- able in the water of stagnant marshes, rendered the further development of the respiratory fluid very advantageous. This was brought about by the modified bile hematin combining with an albumenoid, so as to give rise to the modification of hemoglobin found in the blood, which is certainly a more suitable oxygen carrier than hematin, since it can be deoxi- dized and reoxidized without decomposition. Advancing to higher animals, it appears to have been very advantageous to have this oxygen carrier, not in solution, but in the form of discs suspended in a nearly colourless serum, so that these red discs might be filtered out, and it alone penetrate to some parts. This was perhaps brought about or accom- panied by the substitution of the original albumenoid by another, whilst at the same time hematin ceased to be formed as a secretion of the liver. ‘The green blood of Sabella per- haps resulted from an analogous development in another way, not yet fully understood. In all these changes, as we advance from the most rudimentary condition, we find that, though the fundamental coloured radical, which determines the oxygen-carrying properties, remains the same, the compound becomes more and more unstable, and is more and more easily decomposed, which is in all probability correlated to a greater and greater vital power, capable of counteracting stronger and stronger chemical affinities. This advance from a yery stable to a very unstable com- pound, is in many respects analogous to what I have found to occur during the growth of orange-coloured flowers exposed to varying light. When developed nearly in the dark the 1 Proc. of Roy. Soe.,’ vol. xxi, p. 78. 84 H. C. SORBY. petals are coloured mainly by the more stable compounds which absord only the blue end of the spectrum, whereas when developed under the the influence of bright sunlight other compounds are formed, which absorb light more towards the red end of the spectrum, and are much more readily decomposed. By exposing to the light the mixed solution of the colouring matters developed in the living plant under the influence of light, these less stable substances are soon de- stroyed, and we obtain a mixture closely corresponding to that met with in those petals which are developed in the dark. All this may be explained by supposing that the increased vitality due to the exposure of the plant to the sunlight suffices to counterbalance a much greater amount of mere chemical affinity. Such then is the best account Iam now able to give of what appears to me to be a most promising field for research. Of course I cannot but feel that it is very imperfect, but yet I trust that it will suffice to show the great value of this method of research in the study of comparative physiology, and the importance of attending to minute differences in spectra, since this may often lead to the discovery of very striking chemical differences which otherwise would never have been suspected. Itis much to be regretted that the amount of material usually available will make it very diffi- cult,if not impossible, to confirm such results by accurate chemical analyses, but at the same time this makes the value of the spectrum method still more apparent, since it is pos- sible by that means to learn a large number of most important facts, which in all probability would for ever have escaped detection by pure chemical analysis. Now that the gradual evolution of complex chemical compounds essential for the performance of physiological processes has been forced on my attention, I can clearly see that this view of the subject will throw much light on other cases; and it appears to me that for the future we must not confine our attention to evolution of mere form, but materially extend the scope of our inquiry, with the hope of being thereby able to better understand the manner in which has been brought about the wonderful correlation between structure and function. P.S.—Since writing the above my attention has been called to the following remarks made by Ray Lankester in his paper in ‘ Pfliiger’s Archives.”!_ He there says—‘‘ The 1 “Ueber das Vorkommen von Hemoglobin in den Muskeln der Mol- lusken, und die Verbreitung desselben in den lebendigen Organismen,” * Pfliiger’s Archiv fiir Physiologie,’ Bd. iv, s. 318. ON THE EVOLUTION OF HEMOGLOBIN. 85 chemical differences of different species and genera of animals and plants are certainly as significant for the history of their origin as the differences of form. If we could clearly grasp the difference of the molecular constitution and activities of different kinds of organisms, we should be able to form a clearer and better grounded judgment on the question how they have been developed one from the other than we now can from morphological considerations.” As will be seen, this view of the question agrees remarkably with my own, though he was led to it by more general considerations, before such a striking illustration of the principle had been discovered as almost to compel us to admit that there must have been a gradual evolution of such important and com- plex chemical compounds as hemoglobin, a substance which contains the same constituent radical in the various stages of its development. REVIEWS. The Anatomy of the Lymphatic System. By KE. Kuein, M.D., Assistant Professor at the Laboratory of the Brown Institution, London. II.—The Lung. London: Smith, Elder, & Co., 1875. In this handsome volume Dr. Klein gives a full account of his researches into the minute anatomy of the lymphatics of the lung and pleura, a short summary of which had already been communicated by him to the Royal Society.’ The work is divided into two sections, the normal con- ditions being described very fully in the first, while the second is occupied by a most interesting account of the pathological changes in acute and chronic inflammation, in the artificial tuberculosis of guinea-pigs, and in the acute miliary tuber- culosis of man. Commencing in the first section with the pleura, Dr. Klein points out a remarkable difference in the appearance of its endothelium in the distended and collapsed lung. In the former, in which it has to ‘cover a wider area, the endothelium is seen as flattened plates, rather thicker in the centre, with a flattened circular nucleus, and only faintly granular body ; whereas in the collapsed lung the cells are distinctly granular, and are moreover no longer flattened, but shortly columnar, with a spherical nucleus. ‘The tops of the cells are seen to be rounded, leaving a considerable space between neighbour- ing cells, the deeper portions only of which are cemented together. The endothelium of the costal pleura consists of flattened plates, so that it differs from that of the pulmonary pleura in the same manner as Waldeyer has shown that of the surrounding part of the peritoneum to differ from that of the upper part of the ovary, the cells in each case bearing a close resemblance toan epithelium. Passing on to the matrix of the pleura, Dr. Klein describes it as consisting of extremely delicate connective tissue with a few elastic 1 © Proc, Roy. Soc.,’ January, 1874, REVIEWS. 87 fibres, small spaces occupied by connective tissue corpuscles, and communicating more or less completely with each other, being left between the bundles, and representing the lymph- canalicular system. In guinea-pigs a meshwork of unstriped muscle-fibre was also found, more especially developed in those parts which move most freely in respiration, and show- ing in the meshes lymphatic lacune, which communicate with the rich subpleural lymphatic plexus. The vessels forming this arise in the superficial alveoli, receive branches from the deeper parts of the lung, and discharge themselves into trunks that run in the ligamenta pulmonis to the bronchial glands. Dr. Klein has satisfied himself of the existence of stomata forming a communication between the cavity of the pleura and the above-mentioned superficial lymphatic plexus and intermuscular lymph-spaces; so that when these stomata are dilated (as happens in inspiration) the lymphatic system of the lung may become filled with whatever matter may occupy the pleural cavity. The stomata are best seen in the lungs of animals suffering from chronic pleurisy, when their position becomes very clearly indicated by a germination of the endothelium at their margins. Dr. Klein next describes the lymphatic system of the bronchi. ‘This consists of a rich network in the adventitia, constituting the peribronchial lymphatics, which receive branches from the submucous tissue, and anastomose with the perivascular lymphatics which accompany the blood- vessels. In the guinea-pig’s lung (especially in animals suffering from artificial tuberculosis) there are spherical, oblong, or even cord-like accumulations of adenoid tissue in the wall of many peribronchial lymphatics. The larger ones are provided with a special network of capillary blood- vessels. ‘These were suspected by Burdon Sanderson, who first described them, to be connected with the lymphatics, and are shown by Dr. Klein to be what he has called “ peri- lymphangeal follicles,” consisting of adenoid tissue in direct connection with the lymphatic wall. These follicles were also found in the rabbit’s lung, but less numerous and not of so dense a structure. The rootlets of the peribronchial lymphatics consist chiefly of a system of communicating spaces in the mucosa, the muscularis, and the submucosa, which are interfascicular, and vary in size according to the amount of separation of the contiguous bundles. ‘They are smallest in the mucosa, where they consist of lacune and anastomosing canals, whereas in the adventitia they are elongated or rhombic spaces ; the former spaces are occupied by branched connectiye-tissue-corpuscles, while the latter are lined by 88 REVIEWS. rows of flattened cells closely resembling an endothelium, Interspersed among the epithelium of the bronchi, branched connective-tissue cells were found, communicating by their processes with those of the mucosa, and thus forming a pseudo-stomatous tissue by means of which the lymph- canalicular system may be brought into communication with the surface of the bronchial mucous membrane. That such a communication does exist was proved by Sikorsky, who found that coloured particles introduced into the bronchi penetrated into the lymph-spaces of the mucosa. Lastly, the perivascular lymphatics are described as origi- nating inthe walls of the alveoli by a lymph-canalicular system occupied by branched connective-tissue cells, whose processes often project between the epithelium lining the alveoli, and thus form pseudo-stomata, which permit of communication between the cavity of the alveoli and the lymph-canalicular system. The lymphatic trunks formed by the confluence of the capillaries which arise from the lymph-canaliculi accompany the branches of the pulmonary artery and vein, chiefly as distinct vessels running by their side, but often, especially around the smaller arterial branches, the lymphatic vessels are replaced by lymphatic lacune which communicate freely with eaeh other. The arterial or venous branch was sometimes seen to pass directly through a lacuna, in which it thus becomes invaginated. The pathological portion of the work begins with an account of the changes observed in the pleura pulmonum in inflammation. The endothelium was found to germinate around the stomata, more especially in chronic inflammation ; and in the course of chronic pyezmia and artificial tuber- culosis the changes found were, (a) thickening of the matrix of the pleura preceded by infiltration with lymphoid cells ; (6) hypertrophy of the muscular coat in guinea-pigs, so that the meshes between the muscular bundles become much narrower, and even a continuous muscular membrane may be found in some parts; (c) the intermuscular lymphatic spaces and many subpleural lymphatic vessels become filled with lymphoid cells, derived, in all probability, partly from the germinating endothelium around the stomata, and partly from emigration from blood-vessels. These plugged lymphatics share in the formation of the characteristic nodules of arti- ficial tuberculosis, so far as the superficial parts of the lung are concerned. The vessels at first only filled with lymphoid cells are converted into cords of adenoid tissue, by an out- growth of their endothelial walls in the form of fine fibres, forming a reticulum between the cells. The cords leave REVIEWS. 89 meshes corresponding to the superficial alveoli of the lung, which in early stages are filled with cells which are undoubtedly altered epithelium ; and later, by cells indistin- guishable from lymphoid cells. These nodules are to the naked eye at first rounded, grey, and transparent; later they increase in size, become of a more irregular shape, and their centre becomes opaque and caseous. ‘This central softening extends gradually in all directions to the circumference, and the nodule which first forms a distinct prominence on the pleural surface, becomes depressed in the centre when soften- ing is advanced. In the substance of the lung Dr. Klein distinguishes granulations of three kinds. (1.) More or less well-defined nodules in connection with the walls of small bronchi. These he regards as simply hyperplasz of the normal adenoid tissue of this part. They are found at a comparatively late stage and do not soften. (%.) Perivascular cords. These are developed earlier around the small arteries, at first as endolymphangeal follicles by plugging of lymphatics with lymphoid cells, and their subsequent conversion into cords of adenoid tissue, and then the cords become further thickened by a perilymphangeal growth. Changes also take place in the blood vessels themselves. The endothelium of the ultimate branches of the pulmonary artery is found to germinate so as very materially to diminish the lumen of the vessel. In larger branches the middle coat becomes laminated, and infil- trated by lymphoid cells which extend into the coats of the vessel from the perivascular cords; finally the capillary vessels in the alveolar walls become ultimately converted into nucleated threads. ‘This change takes place only in the later stages of the process, subsequent to a thickening of the alveolar septa by encroachment of the perivascular cords. In the perivascular cords no caseous degeneration (softening) was ever found. (3.) The last kind of granulations is due to catarrhal pneumonia. The alveolar septa become thickened as above described, and the alveoli themselves become blocked at first by alveolar epithelial cells and their derivatives, giant- cells forming a prominent feature. The largest of these Dr. Klein believes to originate from a fusion of several epithelial cells, as their substance shows an indication of being divided into territories. The catarrhal changes finally spread from the alveoli to the infundibula and small bronchi. It is these catarrhal pneumonic granulations which undergo caseation, a process which spreads at last to the thickened alveolar septa. The last chapter is devoted by Dr. Klein to an account of 90 REVIEWS. acute miliary tuberculosis in man, based upon the examina- tion of the lungs of seven children who died of this disease. He found that in early cases the tubercles were due to catarrhal pneumonia; the alveoli being found distended with a fibrinous material in which numerous lymphoid cells (emigrated colourless corpuscles) were imbedded, the struc- ture of the alveolar wall was barely discernible, and its capillaries obliterated. In later stages the fibrinous exudation which occupied the alveoli gradually disappears by absorption, and becomes replaced by groups of cells which are mostly derived from the alveolar epithelium, or by one large multi- nuclear mass or giant-cell. The giant-cell is connected by processes with a retiform tissue infiltrated with lymphoid cells, which represents the alveolar septa. ‘This is not true adenoid tissue, but is regarded by Dr. Klein, in agreement with Schiippel, as formed by cells derived from the giant-cells. The giant-cell finally degenerates into a mass of débris, some- times passing through a previous fibrous stage. Dr. Klein considers that, in the lung, giant-cells are formed from the alveolar epithelium, though he admits that it is possible that they may arise (according to the observations of Ziegler) from emigrated colourless blood-corpuscles. In still later stages, when the above tubercles already show necrotic changes, numerous blood-vessels are found surrounded by perivascular cords, and spherical collections of adenoid tissue are met with in the adventitia of the bronchi, so that the various processes take place in man in inverted order as compared with the artificial tubercle of guinea-pigs. The book is illustrated with six admirable double plates, and is certainly among the most valuable of Dr. Klein’s numerous contributions to normal and pathological histology. The Histology and Histochemistry of Man. By Hetnricu Frey, Professor of Medicine in Zurich. Translated from the fourth German edition by ArtHuR E. J. Barker. London, 1874. (Pp. 683 ; 604 woodcuts.) No working histologist who is acquainted with the German language needs any introduction to Professor Frey’s manual. Avowedly a compilation, it is yet a very satisfactory and valuable compilation, and, especially for students’ use, is perhaps the most useful text-book on the subject in any language. It is hardly necessary to say that it has gone through several editions, and as each successive issue has had to be brought up to the present day, as the phrase is, some parts present a curious patchwork of conflicting views, or REVIEWS. 91 rather, perhaps we should say, of views arranged in suc- cessive strata, like a geological formation. Sometimes the successive views are very contradictory, and sometimes the contradiction hardly seems to have been perceived by the author; the latter portion having been tacked on without rewriting the original text. But this is probably unavoidable in a book which has been patched and altered so many times. It is an unfortunate peculiarity of histology that so little appears to be positively established, and so much is a matter of “views.” The fact appears to be that observers do not, after all, differ so much as to the phenomenon itself as in their interpretations of it. If they confined themselves to stating precisely what can be seen, there would be little or no controversy. But there is little chance of this happy unanimity ever being attained, for several reasons. In the first place, many phenomena are in themselves, strictly speaking, ambiguous, with our present means of obser- vation. There is a certain limited number of possible explanations, each of which has certain points in its favour, but no one of which can be absolutely proved. Then, again, no one is content with a mere description of the phenomenon, because a mere appearance without any attempt at interpre- tation has no interest for us. We do not want to know about black shadows and dots, but about what the shadows and dots may be supposed to mean. So that histological contro- versy is not likely to come to an end just yet. A remarkable illustration of the ambiguity of appearances is afforded by the changes of opinion with respect to so well- known a tissue as voluntary muscle. These are well illustrated in Professor Frey’s work. He gives, first, a very clear statement of what were once two rival theories as to the ultimate composition of the muscular fibre (though the former of them can hardly be regarded as a rival now), viz. the theory that fibrille are the pre-existing essential elements of the fleshy mass; and Bowman’s view, which regards it as made up of “ sarcous elements.” The latter conception has very naturally the preference, since it has certainly commanded the suffrages of most recent observers ; and when compared with the fibrillar theory, it appears plain that only the authority of some popular teachers and writers of text-books could have given the latter so much acceptance as it had at one time, especially in Germany. Professor Frey gives a very good account of the further developments which the conception of sarcous elements has undergone in the hands of Krause, Hensen, and others, the history of which appears 92 REVIEWS, to us rather instructive. In the first place, we have the cross line of the transparent space, or “‘ the transverse plate of Krause,” as itis now named. Now this tranverse plate was not only referred to by “‘ the English observer Martyn and others,” as Professor Frey says, but described by almost every English authority from an early date. A very distinct figure is given in old editions (but omitted in later editions) of ‘Carpenter’s Human Physiology’ (we quote the fourth, of 1853) though certainly interpreted in a very different way ; the clear space is seen divided by a tranverse line, and a similar light space figured at the szde of each muscular element ; appearances which led Dr. Carpenter to regard the muscular elements as cells. This view of the structure of muscle, Dr. Carpenter states in a foot-note, was published simultaneously by himself and by Professor Sharpey, both statements being founded on some preparations made by Mr. Lealand, the optician. The same fact had also been previously recognised by Dr. Goodfellow and Mr. Erasmus Wilson. So that the “ transverse line ”’ itself was anything but a novelty when attention was drawn to it by Krause. The latter observer, however, not only described, he founded chiefly upon that an elaborate theory of the structure of muscle, which, whether accepted or not, has always been regarded as possessing in a high degree the merit of originality. In Professor Frey’s words : “Krause holds a very peculiar view in respect to the structure of muscle. He regards the dark cross line just mentioned as the optical expression of a delicate transverse partition springing from the sarcolemma, which divides the interior of the muscle fibre into a number of discoid compart- ments built up one over the other. The contents of such a compartment would consist from below upwards of (1) half of a transparent transverse zone; (2) of a dark zone occupying the middle (z.e. of a transverse disc of sarcous elements) ; and (3) of another half of a transparent cross zone. Krause believes also in the existence of a delicate lateral membrane, investing closely the sides of the sarcous elements and ends of its transparent appendages, and uniting with the transverse membrane. In this way he supposes the elementary structures of the striped fibres to be formed—the so-called muscle cas- kets [or compartments]. In longitudinal rows they constitute the fibrille. This author also believes the clear longitudinal and transverse cementing medium to be liquid, and that during contraction the layers of fluid flow from the end surfaces to the sides.” Now, strange to say, even this peculiar theory of Krause REVIEWS. 93 is not new. It has some resemblance to the old “ cell” theory before referred to, but it appears to be nearly identical with that referred to by Dr. Carpenter in the following extract (op. cit.): ** A similar delineation had previously (before 1846) been published, however, by Dr. Goodfellow (‘ Physiological Journal,’ No. iv), but his interpretation of the appearances was altogether different ; for he considered the dark spaces as the ‘ sarcous elements’ of Mr. Bowman, and regarded them as separately enclosed within partitions formed by internal prolongations of the external investing myolemma.”’ These are merely the “ Muskelkastchen.” Dr. Capentrer continues : “ By Mr. Erasmus Wilson, again, the appearances were described as leading to the belief that two kinds of cells exist in each fibrilla, a dark and a light; a parr of light cells, separated by the delicate transverse line just spoken of, being interposed between each pair of dark ones” (‘Manual of Anatomy,’ third edition, p. 162). Substituting the word “ disc” or “ body ”’ for “ cell,” this enumeration of the constituents of a muscle fibre agrees precisely with Krause’s. We do not quote these anticipations merely to show that * there is nothing new under the sun,” still less to deprecate the labours of the eminent professor of Gottingen, but to illustrate the assertion made just now that many phenomena are truly ambiguous, and that the number of possible explana- tions is, with given means of observation, limited. One explanation gains currency in preference to another not only by emphasizing certain features of the object, but by sup- pressing or ignoring others. Nevertheless, the suppressed points are certain to come to light again, and when they are emphasized the dominant theory is overturned. Thus the see-saw goes on, one side of the question alternately pre- dominating ; though it is true each explanation, when it is revived, is never revived quite in its original shape: it is always more complete than in its previous state of existence. Finally, some new method or improved instrument of research makes it possible to explain the true and the false in each of the contending theories. Whether this will be the case with regard to muscle, when the views of Engelmann and Schafer come to be thoroughly worked out, and whether sarcous elements are to disappear as a merely provisional hypothesis, we cannot now discuss, nor can we speak of Henson’s “ middle disc” dividing the dark zone. 94. REVIEWS. Another interesting point discussed in Frey’s work is the structure of the gastric glands and their peculiar forms of cells—the “‘ Hauptzellen,” or “chief cells” of Heidenhain (adelomorphous cells of Rollett); and “ Belegzellen,” or “investing cells,” delomorphous, of Rollett (strangely trans- lated ** overlaying cells ” by Mr. Barker). Heidenhain’s observations on the differences seen in these cells in periods of rest or activity of the gland, are one of the most important connecting links between physiology and pure histology. It is, of course, impossible to criticise a work of this kind in detail. We can only say that other parts seem to be as well brought up to the date of publication as those which we have mentioned. In conclusion, we must cougratulate Mr. Barker on the accuracy and fluency of his translation, and on the faithful reproduction of the German woodcuts. As we have before complained of the costliness of the American translation of ‘Frey ou the Microscope,’ we feel bound to state that this work, though very considerably larger and containing nearly twice as many woodcuts, is published at a lower price, and is at least equal, if not superior, in mechanical execution. Outlines of Practical Histology. By Witt1AM RuTHERFORD, M.D., Professor of the Institute in the University of Edinburgh. London, 1875. Since the first edition of this little work appeared in the form of notes in the number of this Journal for January, 1872, we are relieved from the necessity of subjecting them to any detailed criticism. It is right to say, however, that the original notes have been considerably expanded and im- proved, as well as made more useful by the introduction of figures. In its present state, Professor Rutherford’s bro- chure forms an excellent guide for class work, and will be useful even for the solitary student who is bent on seeing things for himself. It has the great merit, for a scientific text-book, of being useless in the absence of the objects de- scribed: a quality which will render it quite unfit for the crammer or the crammed. Se —_ NOTES AND MEMORANDA. Mr. Sankey, on a new Solution for Staining Sections of Hardened Animal Tissues.—Although the number of dyes recommended for use in _ histological investigation is already somewhat large, I cannot refrain from adding another to the list, which appears to present useful quali- ties in a combination not met with in any other dyeing material with which Iam acquainted. I have already spoken in favour of this dye in a paper in the last number of the ‘ West Riding Reports ;? but I had not at that time used it extensively for staining hardened sections, and was, therefore, not acquainted with the best method of usingit. In the dry state the substance, under the name of aniline blue-black, can be obtained from the manufacturers, Messrs. Read, Hol- liday, and Sons, of Huddersfield.! It is a blackish powder, something like gunpowder in aspect. It can be dissolved in alcohol, but it is much more solublein water. When dis- solved in alcohol it will be found to make a very useful dye for hardened sections of animal tissues. Owing to the great facility with which it dissolves in water, and the difficulty with which it dissolves in alcohol, the best way to get it in solution in the last-mentioned liquid is first to make a very concentrated aqueous solution, and then to pour it into strong alcohol. In this way it is very easy to get a solution strong enough to dye any texture in a few minutes. I find the following quantities yield a solution of con- venient strength :—'05 gramme of aniline black is to be dis- solved in 1 or 2 cubic centimétres of water; when solution is complete 99 cubic centimétres of methylated alcohol are to be added; the mixture must then be filtered, and is ready for use. ' Or at 15, Fenchurch Street, London. The price of the dye is about 6s. per lb. 56 NOTES AND MEMORANDA. By using an alcoholic dyeing fluid one of the steps in the process of mounting an ordinary hardened section in Canada balsam is avoided, and as the transference of a thin section from alcohol to an aqueous dye is always more or less hazardous the advantage seems to be of some moment. I am aware that an alcoholic solution of logwood has been prepared for dyeing sections ; but this fluid requires that the section should be immersed in it for a space of four or five hours before a sufficiently dark colour is obtained; whereas the fluid I recommend does not take more than a few minutes to stain the section darkly. With regard to its power of staining some structures in preference to others, I think that, speaking generally, it stands in this respect intermediate between logwood and carmine, for while it differentiates nuclei from their cells more than carmine does, it seems somewhat inferior to log- wood in this respect. It is, however, permanent, which logwood is not, and more- over its solution is not liable to decompose by keeping, and is not precipitated from its alcoholic solution by oil of cloves ; hence it is less necessary to rinse the dye from the prepara- tion before placing it in the essential oil. The dye, more- over, seems to have a special affinity for nervous cells, and on this account it will be found very useful in preparing sections of the spinal cord and brain. The colour produced by this material is a blue grey.— Hersert R. O. Sanxey, Undergraduate in Medicine of the London University. Reinsch’s Contributiones ad Algologiam et Fungologiam.— Those of the readers of this Journal who busy themselves with, or take an interest in, the broad range of Algology and Fungology will be possibly glad to have their attention directed to a descriptive work of comparatively recent ap- pearance and of considerable scope, under the title of ‘ Con- tributiones ad Algologiam et Fungologiam,’ by Professor Paul F. Reinsch, of Erlangen, in Franconia. Of this work the first volume only has as yet been pub- lished ; it is not a monograph, but descriptions with illustra- tions and explanatory details of new species of Melanophycee, Rhodophycee, and Chlorophyllophycew, as well as a few species of Fungi; the second forthcoming volume will be devoted to Phycochromophycee. Of Melanophycee 57 new species are described, distributed in 9 genera, of which 5 are new; of Rhodophycee 68 (with 4 new forms), dis- tributed in 22 genera, of which 7 are new ; of Chlorophyllo- phycee 51 new species (with 32 new forms), distributed in NOTES AND MEMORANDA. 97 24 genera, of which 4 are new; of fungi are described 15 new species, distributed in 13 genera, of which 8 are new. Amongst the most interesting of these new forms of alge are some belonging to each of the three great groups above mentioned, which are of a truly parasitic, that is, entophytic, habit, that is to say, they carry on their existence embedded, at least partially, within the tissues of larger forms. It is only a comparatively short time since algz of this nature were recognised, and a résumé of the knowledge of them at the time was given in the ‘ Quart. Journal of Micr. Science,’ n.s., vol. xii, p. 365, 1873. To the small number already known Professor Reinsch has, in the present work, added consider- ably, and what would seem somewhat surprising is that their distribution would appear to be pretty wide, though the forms are, perhaps, difficult of detection. Entonema (n. g., Reinsch), belonging to Melanophycee, embraces species inhabiting the tissues of both Rhodophycee and Melanophycee of larger and of lax structure, both super- ficially and in the internal parenchyme of the infected plant ; sometimes occupying the intercellular spaces, sometimes penetrating into the interior of the cells. The parasite itself forms jointed filaments with fructification of Ectocarpee. Species referable to Ectocarpus itself are described as having rhizomatoid radicles penetrating into the living substratum. Entonema intestinum (Reinsch) produces zoosporangia, the zoospores standing in transverse series. Actinema(n. g. Reinsch) is a new epiphytic Melanophyceous genus—its fructification unknown; the minute plant forms a disc of parenchymatously conjoined cells, to some extent com- parable to Phyllactidium (Kiitz.),or Coleochete (Bréb.), minus bristles, flatly adhering to the surface of larger alge. In Plectoderma (n. g. Reinsch), referable to Rhodophycee, of which the fructification is also unknown, we havea struc- ture morphologically quite comparable, and apparently of wide distribution. Again, amongst Rhodophycee we have anew truly parasitic genus in Choreocolax (n. g., Reinsch,) described with several species. The frond is composed of two parts or portions— one an irregularly filamentous system, becoming expanded in the parenchyme of the infected plant; the other rising above its surface, and forming externally a convex or hemi- spherical, or nearly spherical, or irregularly-lobed cellular body; the cells of the immersed portion are more slender than those of the external outgrowth, or equal to them ; those of the external part are variously figured, disposed more or less without order, or in subramose densely intricate VOL. XVI,—=NEW SER. G 98 NOTES AND MEMORANDA. threads ; sometimes the most external cells are the longest. Fructification not known. These curious Rhodophyceans parasite, living half in and half out of the host-plant, occur on Delleserieze, Polysi- phonia, Spherococcus, Gigartina, Rhodomela, Laurencia, Ceramium, the various examples coming from far-distant localities. The first observed representative of this genus was that named by the author Choreocolax Polysyphonia, found by him on P. fastigiata, taken on the northern coast of North America. Examples may possibly be found on our own coasts of these remarkable growths, if sought for by our marine algologists. Sometimes the mass of the parasite ex- pands out considerably larger than the stem of the infected plant on which it grows, and causes a good deal of distortion of the constituent cells of the latter. These, for instance, in the form named Choreocolax mirabilis, growing in the stems and branches of Rhodomela subfusca, may become dis- located in some numbers, and in section can be seen more or less displaced and commingled with and surrounded by the cells of the parasite. The cells of the host embraced by the parasite are described as becoming much altered, their con- tents deprived of colour, and completely filled by a granu- lose matter, assuming, on application of sulphuric acid and tincture of iodine, a bluish-violet, the cuticular stratum becoming dissolved, but the primary cellulose membrane remaining unchanged ; at the same time the contents of the cells of the parasite becoming deeply coloured a dusky purple, the primary membrane unchanged. The contents of the cells of the parasites appear, for the most part, as composed of a nitrogenous substance (albumen), those of the host ofan amylaceous matter. The author describes also a further allied genus—Syrin- gocolax (n. g., Reinsch)—also strictly parasitic, and, as to habit, like the preceding, living partly within and partly without the host-plant. Here, as in Choreocolax, the plant is made up of two portions, one immersed in the parenchyma of the infected plant, and composed of very slender threads, passing in amongst the cells of the host-plant, the external portion (as in Choreocolax) rising above the surface of the infected plant, here by a short pedicel, is irregularly figured. The substance of this external portion is formed of hetero- morphous threads, the lower densely intricate, subcontorted, very slender, the outer oval, disposed in thicker tubular threads, forming a regular cortical stratum, externally bounded by a rather thick continuous covering. Fructifica- tion seems to be Polysporangia produced by the cortical NOTES AND MEMORANDA, 99 layer of cells, and containing twelve to twenty broadly oval spores. Of this genus but one species is described, found on Gelidium cartilaginum. Entocolax (n. g., Reinsch) contains but one described form; here the whole plant is entophytic, itself seemingly composed of slender cells radiantly disposed in series, and spreading in cavities of certain monstrous curiously-lobed or horned cellular excrescences of the host-plant (here Bo- sirychia growing on Gelidium cartilagineum) , its fructification wholly unknown. ‘This curious production, the author seems to think, may take origin within the cavity ofa cell of the host- plant, but this is surely very questionable. At any rate the young and very minute cellular plant is wholly included by the adjacent parenchym of the infected plant. More advanced, the expanded cavity seems to be covered by a laminated lining, in which nidus is seated the parasite. The curious horned excrescences on the host-plant are similar to those sometimes induced by and accompanying certain other para- sites appertaining to Choreocolax, and are more minutely cellular than the normal tissue. How propagation of this remarkable parasite (referable, like Choreocolax and Syrin- gocolax, to Rhodophycee, but of uncertain position therein) can be effected remains a marvel. Pseudoblaste is the name of a new genus (of three species) formed by Reinsch for certain obscure little productions erowing on the surface of other Rhodophycean alge, and forming little convexities composed of homomorphous cells, arranged in vertical or radiate series or irregularly scattered, and enclosed in a sharply bounded, nearly hemispherical or lobed, colourless matrix, closely adherent to the plant on which they live, but wholly without any organic cohesion therewith. Coming to Chlorophyllophyce, a new form, amongst some others, referred to Chroolepus, is found to be a true parasite living in certain Jungermanniee. In many cases the author observed the parasite, not only in the intercellular spaces, but within the cavity of the cells. The cells become some- times wholly embraced by the growth of the parasite and dis- joined, such being mostly without chlorophyllaceous granules, and the plasma densely and finely granular. The author establishes a new genus under the name of Chromopeltis, for certain forms living attached to the leaves of mosses; these are disciform, subcircular, or irregularly lobed, composed of irregularly-figured cells, sometimes running into radiant series; contents finely granular, and colour dusky green. The figures convey the idea of plants 100 NOTES AND MEMORANDA, not unlike young examples of such a form as Coleochete scu- tata without bristles (Phyllactidum, Kiitz.), as does possibly the unnamed ‘ nov. genus Ulvacearum” described on p. 76. Certain forms of Polyedrium, Sorastrum, Gongrosira, Chlorotylium, and Ulothrix are described, as well as Microthamnion, Gidogonium, and Bulbochete. An alganamed Spirogyra annularisis described, which seems remarkable. In it the filament tapers towards the (upper) extremity. The cells of the cylindrical (lower) portion are very short, being, in fact, nearly one half broader than long, the cell-wall rather thick ; the endochrome forms a complete ring, slightly twisted. To judge from the figure, the endo- chrome of the upper or tapering cells is different, and forms a longitudinal axile band. Quéere, is this truly a Spirogyra ?) A puzzling little, very slender, filamentous form is described and figured, but not named, believed by the author to come under Cymatonema (by an error printed “ Cymatopleura’’), but having, he thinks, also possibly as much right to be placed to Zygnemez. The cells are elongate, comparatively rather thick-walled, more or less regularly hexagonal, colour bright green. Actidesmum:Hookeri (n. s., Reinsch) is a curious little alga, somewhat calling to mind Sciadium arbuscula (Al. Br.). The thallus is formed of families of a few spherical cells, each single cell at the summit of a short slender hyaline pedicel, several of which radiate in a whorl from the summits of a number of longer and thicker hyaline pedicels, which latter themselves radiate from a common centre ; propagation by zoogonidia. It thus seems to differ, so far as can be judged, from Sciadium by the aggregate family not standing on a substratum by a common pedicel, and by the ultimate cells being comparatively large and spherical, not elongate. Celastrum (Spherastrum) verrucosum (n.s., Reinsch) is a well-marked form, characterised by the external subacute verruce. Characitum Dyervi (n.s., Reinsch) has a singular habitat —on the backs of entomostraca—cells elliptico-oval, contents granular, amylaceous, colour pale green. Of Desmidiez several new and well-marked species are described, a few of which, however, are not named. Cos- marium pseudomargaritiferum (Reinsch) appears to be the same as C. Portianum (Archer). It has a very singular zygospore, globose, thick-walled, and covered by pits, that is, scrobiculate ; hence de Bary’s figure (‘ Untersuchungen ueber die Familie der Conjugaten, t. ,f. ) cannot refer to this species; in fact, to that extent the zygospore of the NOTES AND MEMORANDA, 101 Cosmarium in question agrees with the zygospore of the otherwise very different Xanthidium armatum. Cosmarium auriculatum (Reinsch) seems to be, in part at all events, equal to C. perforatum (Lundell). A new genus, under the name of Schizospora, is founded by the author for two forms, which, however, were previously described and named by Lundell—Cylindrocystis diplospora (Lundell) and Penium didymocarpum (Lundell). Viewed apart from the zygospore, and having regard only to the arrangement of contents of the parent-cells, these two forms seem generically distinct, but they have in common, no doubt, the double (or twin) zygospore, upon which circum- stance the new genus is founded by Reinsch. But the far more common form, Cylindrocystis Brébissonii, it would appear, sometimes produces double zygospores (see ‘ Quart. Journ. Micr. Science,’ n. s., vol. xiv, p. 423), though single zy- gospores are characteristic, and frequently enough met with in that species. Again, that well-marked species, Closterium lineatum, produces double zygospores, like those of the two species referred to Schizopora by Reinsch, very closely ap- posed, so much so as to become mostly mutually flattened by the pressure, but also, like them, readily separable when mature. Again, Spirotenia condensata has double zygo- spores ; but during formation they stand somewhat apart, not closely apposed. ‘The conjugation, in such cases, seems to be effected by the union of the half of the contents of one of the parent-cells with that of the corresponding half of the opposite cell; that is to say (supposing the parent-cells to stand in a vertical position), the contents of the two upper halves passing across to become mutually combined, the con- tents of the two lower halves simultaneously and mutually doing quite the same thing, If, as we think, this is really the process, the resulting spores are truly twin or double spores, not a single spore subsequently subdivided. Seeing, then, that at least four forms appertaining to the diverse and well-recognised genera, Closterium, Spirotenia, Cylindro- cystis, and Penium, produce these double spores, it would seem as if the proposed genus Schizospora were untenable, or, at least, unnecessary. The form recorded as Ewastrum gemmatum (Bréb.) forma, appears a very well-marked one, and should seemingly rank as autonomous. Onichonema leave (Nordst.), ‘Symb. Fl. Bras.,’ found by that author in a collection from Brazil, is an interesting addition by Dr. Reinsch to the European list. This is an allusion to a few only of numerous very pretty forms described, 102 NOTES AND MEMORANDA. The author describes and figures a few abnormalities in some Desmidian species, consisting of the interposition of a monstrous growth between the semicells, without any sub- sequent constriction or division, and of misshapen lobules in the wrong place, mimicking those that occur normally in other situations. Such monstrosities now and again turn up. : ‘ In Fungi several new genera are established for curious forms occurring on and in the stems and leaves of mosses, ou alge, &c. eT The author adds not less than 131 plates, containing figures of all the new species described in the work. ‘These, if not finely done, are graphic and expressive, and, together with over 100 quarto pages of letter-press, containing the various descriptions, form a valuable record of long and patient and successful labour; and the work will be no doubt welcome to cryptogamic botanists in this country.— Wn. ARCHER. . Change of Editorship.—Professor W. T. Thiselton Dyer has found it necessary to retire from the joint editorship of this Journal in consequence of his appointment as Assistant Director of the Royal Gardens, Kew. His place on the edi- torial staff is taken by Mr. William Archer, F.B.S.,,. at Dublin, well known to the readers of this Journal. Professor Cohn’s Beitrage ;—Bacterium rubescens.— We have received the third part of the contributions to botanical physi- ology, published under Professor Cohn’s direction by Kern, at Breslau, 1875. At present we are not able to give an account of the contents, except to mention that it contains several important communications relating to the Bacteria, besides other papers on Volvox, Utricularia, and Aldrovanda by Professor Cohn. Professor Lankester wishes to state that he is not convinced by the evidence adduced by Cohn of the correctness of a separation of the forms included under Bac- terium rubescens into the two species Clathrocystis roseoper- sicina and Monas okeni. ‘The flagellum ascribed by Cohn to Monas okeni (the large Bacterioid plastids of B. rubescens) is parallelled by the flagella of B. termo, described by Dallinger and Drysdale. Professor Lankester had observed the appear- ance of a flagellum as described by Cohn in this form on treatment with iodine or magenta solution, but had not been (and is not now) able to satisfy himself that the apparent flagellum is really part of the plastid to which it is attached, since in the fluid, together with the red-coloured plastids, are always found very numerous filamentous forms (Bacillus and Vibrio) of great delicacy and length, which by acci- NOTES AND MEMORANDA. 103 dental contact with the red-coloured plastids may simulate flagella. Mr. Worthington Smith’s paper on the Resting Spores of Peronospora infestans.— By an unfortunate blunder on the part of the Woodbury-Type Company both the photographs on Plate XIX were reversed, and the upper one was also in- verted in the mounting. The key (Plate XX), of course, fails in consequence to correspond with the photographs. PROCEEDINGS OF SOCIETIES. Dustin Microscopical CLups. July 15th, 1875. Leaf-Structure in Sphagnum Austini, Sullivant, and Sph. papillosum, Lindberg.—Dr. Moore drew attention to the im- portance of minute microscopic characters in the discrimination of species, as exemplified in two related mosses, Sphagnum dustini, Sullivant, and Sph. papillosum, Lindberg, and he exhibited speci- mens of their leaves, pointing out the fringe-like marginal de- velopment of processes around the spiral cells of the former, as distinguished from the dot-like papille occupying a similar position in the latter. The former examples he had gathered at the Island of Lewes. Differential characters found in the minute Structure of the Leaves in Pinus Nordmanniana, P. pectinata, and another pro- bably new species—Dr. McNab exhibited sections of the leaves of three Pines, viz. Pinus (Abies) Nordmanniana, Pinus pecti- nata, and a species from the Himalayas. The plant of the last is growing in the Botanic Garden, Glasnevin, and was raised from seeds received from the East India Company by Dr. Moore; it resembles P. pectinata in general appearance, but differs in the shape of the leaf and in the quantity of hypoderm present. Pinus pectinata differs from P. Nordmanniana in having the two resin- canals in the parenchyma of the leaf, and not (as in P. Nord- manniana) in contact with the inferior epidermis. The other form, —which, if it turn out to be a new species, might be called Pinus Mooreana—has the resin-canal in the parenchyma, as in P. pecti- nata, but has a much-interrupted hypoderm, instead of the almost continous layer met with in P. pectinata. Marasmis Hudsoni exhibited—Mr. G. Pim showed Marasmis Hudsoni, the long sharp bristles with which the pileus is beset causing it to form a very pretty low-power object. Navicula divergens, form, exhibited—Rev. E. O’Meara showed a form of Navicula similar to V. divergens, but differing in being regularly elliptical in outline. He had found this form occa- sionally in the Lough Mourne deposit, and recently in great abundance on a slide kindly supplied by Rev. George Davidson, of Logie-Coldstone, the material having been gathered in a lake in his neighbourhood, called Lough Canmore, DUBLIN MICROSCOPICAL CLUB. 105 Passalurus ambiguus was exhibited by Mr. Booker. A minute Rhizopod belonging to the genus Microgromia, Hertwig and Lesser, probably distinct from WM. socialis (Archer), Hertwig and Lesser.—Mr. Archer desired to mention that, in connection with the plant he had formerly described (now as is proven erroneously) as a species of Dictyospherium, Niig., under the name of D. con- strietwm, and of which he had shown the conjugated state at the meeting of the Club in May last, he had now discovered that the little pear-shaped colourless body he had so long noticed accompanying that alga and imbedded in its mucous matrix, was not, as he had once conjectured, in any way necessarily belonging to the alga, but was a veritable Rhizopod, and in fact, as it would seem, appertaining to the genus Microgromia, a genus founded by Hertwig and Lesser on his own Gromia socialis. This little organism, found along with the little alga referred to, had long puzzled him ; it might or might not have some genetic con- nection with the alga—it might be anything, in fact. But there it was, a little pyriform, nearly ‘‘ colourless ”’ (but yet showing a very slight bluish tint) inert body, a frequent concomitant of the alga. In the very last example he had seen (strange to say, very shortly after Mr. Archer had taken the gathering yielding the conju- gated specimens of the alga, it had abruptly disappeared from its site!) he had, however, noticed its possession of pseudopodia and a whitish nucleus (like that of Wicrogromia socialis), which latter took a high tint on the application of Beale’s carmine fluid. Its linear dimensions are scarcely more than one third those of I- erog. socialis, its pseudopodia very slender, branched, granulifer- ous, their movements very slow. Ifthe alga with which this little Rhizopod consorts should again make its appearance, Mr. Archer hoped to obtain more examples and give it a closer study than he was able to do with so limited material. Bacteria (Bacillus) habitually forming a Nidus in Mucous In- vestments of Alge.—Mr. Archer directed attention to an alga of which he had wished for specimens at the May meeting in order to have contrasted it with the so-called Dictyospherium, referred to above, but he then had no examples at command: this was Cos- mocladium Saxonicum, de Bary, a plant, though widely distributed, very rare in its occurrence. ‘This species, however, he had before shown to the Club, and he now merely brought it forward as a good example of a fact he had found pretty generally noticeable in alge surrounded by a mucous envelope, but he likewise thought not generally (if at all) noticed. This was that im- bedded in a vertical or radiant position in the matrix occurred numerous bacteria (Bacillus-form), which from their rather regular disposition imparted a radiantly striate appearance to the sur- rounding halo of mucus. Similar occur tov in the mucous envelope of very many Desmidiex as well as other alge, and give that striate appearance often noticeable ; in fact, the depth and density of this mucous investment seems to a certain extent specific in various Desmidiex, and even the amount of striation due to the 106 PROCEEDINGS OF SOCIETIES, presence of these Bacterian forms might be regarded as character- istic in certain species ; so much so that a Cosmarium had been described by Mr. Lobb as “ spinous,” and possessing “rays,” and named Cosmarium radiatum (‘ Quart. Journ. Mier. Sci.,’ vol. xiv, p- 56), but Mr. Archer ventured to suspect erroneously so, and fancied the so-called “rays”? might in reality be simply these striz in the mucus. Sometimes these bacteria near the periphery become dislodged and “ wriggle off” with themselves, but their movement is “ lazy,” comparatively speaking ; sometimesa little pressure facilitates their removal and their becoming free. Similar bacteria also sometimes occur in the periphery of Volvox globator, but there they lie in a tangental position. Chlorophyllaceous alge are seemingly more prone by a good deal than Phycochro- maceous forms to thus harbour bacteria (themselves doubtless to be regarded as Phycochromaceous alge), but the former, as a rule, have more mucous envelopes, and thus form a better nidus. It must not for a moment be supposed the alge are in any decaying or abnormal condition ; on the contrary, the presence of the “bacteria”? is most marked in the most healthy and vigorous examples, and in various forms (Desmidiex and others) they are pretty constantly and more or less characteristically present. As to how these “parasitic” bacteria become deve- loped in the position adverted to, Mr. Archer could not hazard a conjecture, but their comparatively regular radiant, though interrupted, linear disposition (around some globular forms, almost calling to mind under a moderate power some “ Actino- phryan”) and their habitual presence seemed sufficiently re- markable; at any rate such a peculiar nidus for these indeed, in some form or other, omnipresent existences, Mr. Archer believed had hardly been noticed. August 19th, 1875. New Species of Synedra.—Rey. Eugene O’Meara brought for- ward a new species of Synedra, lately found by him in a swampy place on banks of Royal Canal, near Kilcock, Co. Kildare, and which he named Synedra spathulata :—Frustules very large, length 0:0130", in front view wider at the ends than at the middle; greatest breadth 00012", ends straight; in side view wider at the middle and gradually attenuated towards the ends, at some distance from which (0:0025”), bending inwards and then outwards, then suddenly constricted towards the broadly capitate rounded extremities. Young of Water Snail exhibited—Mr. Crowe showed specimens moving by their cilia stillin ovo, a pretty example of “* pond life.” Peculiar globular problematic enlargements of Mycelium of Peni- cillium glaucum hitherto unobserved.—Dr. McNab showed ex- amples of the Common Blue Mould, Penicilliwm glaucum, which he had cultivated for fifteen days on moist bread. The mycelium had produced an abundance of conidia-spores, but in addition peculiar swellings of the mycelium had been observed. These DUBLIN MICROSCOPICAL CLUB, 107 swellings are not noticed by Brefeldin his memoir on Penicillium and are therefore of interest. Sometimes they occurred singly, but in general there were necklace-like rows of them—six to eight together ; the contents consisted of colourless protoplasm, with one or two nuclei. The swollen portions were generally sur- rounded by a thick mass of mycelium-threads, so that they became obscured ; the swellings were globular, about 3,/55” in diameter, their size bringing them at once into marked contrast with the slender mycelium-threads which produce them. Dr. McNab did not hazard any conjecture as to their nature or function, but stated that they seemed somewhat to resemble the “ Macrogonidia’’ of Penicillium figured by Hallier in his ‘ Phytopathologie,’ Pl. v, fig. 31. Per angie pores of Peronospora infestans exhibited.—On the part of Prof. Thiselton Dyer, who kindly sent the material, Mr. Archer exhibited some of the original diseased potato-specimens in which Mr. Worthington Smith had made his recent very inter- esting discovery of the resting-spores of Peronospora infestans. On looking over the slide, only one example of the “antheri- dium” was to be noticed in actual contact with an oogonium, but even in Mr. Smith’s experience this was rare to find. Fructification in Polysiphonia.—Dr. McNab showed a minute branch of Polystphonia fastigiata, gathered at Seapoint in July, which showed the young cystocarps, and in two of them the minute lateral structure was developed, which he identified as the trichogyne. On the same portion of Fueus nodosus he had met with a Polysiphonia bearing antheridia on same plant and tetraspores on another. Structure of Spines in Echinothrix Desorii—Mr. Mackintosh exhibited transverse sections of the spine of Hchinothrix Desorii, Peters. The spines of this species approach those of EF. calamaris much more closely in structure than Z. turcarwm, both of which have been described in these Minutes. In ZH. Desoriz there is a wide central cavity surrounded by a narrow zone of network through which pass the stems of the solid pieces, which are very elongated, with concave sides, and usually globular terminations. The stems are joined by broad transverse bars, and in some cases there are the remains of a network which extended over the central cavity. Mr. Mackintosh was indebted to Prof. Peters, of Berlin, for opportunities of examining the spine of this species. Sections of Obsidian from Ascension Island were exhibited by Prof. Hull. New Species of Euglypha.—Mr, Archer exhibited the test only, as the sarcodic portion had ceased to live, of what seemed doubt- less a new species of Euglypha:—Test minute, but of variable size, ovoid, compressed ; when old, brown, or reddish in colour (quite as highly tinted as Arcella vulgaris), sometimes seeming to incline a little to a purplish tint, when young, colourless; in the highly coloured examples paler near the opening; hexagonal facets extremely minute, elongate, test not prolonged into a “ neck,” its 108 PROCEEDINGS OF SOCIETIES. opening bordered by indistinct teeth, but sometimes indefinitely terminated, even as if somewhat torn (with, as it were, an “ un- finished”’ appearance there) ; no spines whatever ; nucleus, with nucleolus, large, well-marked, posterior (as usual in the genus) ; a band of darkish granules across the middle portion. Its small size, the ovate compressed form of the test, without any neck, the reddish colour and very minute facets, seemed to render the form now shown a very distinct Euglypha. It occurs not rarely on Bray Head, but Mr. Archer had not noticed it from any other locality. In reference to its high tint, as compared to the other always colourless and hyaline species, Mr. Archer thought this form might stand as ELuglypha tineta. September 16th, 1875. Sections of leaf of Pinus (Tsuga) Sieboldii, var. nana, exhibited.— Dr. McNab showed sections of the leafof the foregoing species— the Finie—of the Japanese. It differs considerably from the typical form in having the young shoots hairy, a character not mentioned by Parlatore, the leaves much smaller and having well-developed hypoderm cells. The type-form has the shoots perfectly glabrous and developes no hypoderm except at the margins of the leaf. The characters observed seem to warrant the foregoing being considered a distinct species, bearing the same relation to Sieboldiu that Mertensiana does to Canadensis, and Hookeriana to Pat- toniana. Peculiar globular problematical bodies occurring in diseased potato-leaves—Mr. Greenwood Pim showed preparation of dis- eased potato-leaf infected with Peronospora infestans treated after the manner recommended by Mr. Worthington Smith, viz. maceration in water. There were to be seen a number of spherical bodies with somewhat rough or tuberculated exterior, the external coat of which showed a certain amount of tendency to split into three valves, and this circumstance, coupled with the fact that no direct connection of these with the hyphx, which abundantly permeated the cell-substance of the leaf, could be detected, rendered the nature of these bodies problematical. They were at first taken for the resting-spores of the Peronospora, but that ceemed to be at best but doubtful. This indeed imparted to them probably even a more considerable interest. It was seemingly highly unlikely they could be any kind of pollen-grain, so tho- roughly interspersed as they were in the substance of the leaf. Mr. Pim detected some much smaller colourless bodies, possibly the antheridia, alluded to by Smith, but could not satisfy himself on the point. Nectria peziza exhibited—Mr. Pim likewise showed a specimem of Nectria peziza in which the white spores were oozing in a globule from the orange spherical stromata, the whole forming a very pretty object for a low-power and reflected light. Sections of Petioles of Nymphea, species exhibited.—Mr. Mack- DUBLIN MICROSCOPICAL CLUB. 109 intosh exhibited cross sections of the petioles of Nymphea alba and J. dentata, and called attention to the differences of structure which obtained in the two. In M™. alba the central part of the petiole is occupied by four moderately large lacune, which are nearly equal in size, one pair being somewhat larger than the other, and surrounding these are a series of smaller lacune; the whole of the passages are thickly studded with stellate cells. In NV. dentata the central part is occupied by two very large lacune, with a pair of much smaller ones at each end of the septum which separates them; other still smaller lacune occupy the rest of the circumference ; there are no stellate cells. Mr. Mackintosh hoped to be able to work out these interesting points more in detail, in order to determine if each species has a characteristic structure in its petiole. He also showed sections of the petiole of Villarsia nympheoides, in which the lacune are provided with stellate cells somewhat like those of Nympheza, but devoid of the peculiar tubercles which roughen the surface of the cells of Nymphea. Micrasterias angulosa, Hantzsch, exhibited for the first time in Treland.—Mr. Archer showed Micrasterias angulosa, Hantzsch, for the first time, though he had long been acquainted with this species ; the specimens were from Co. Westmeath. He showed also a specimen of this species from Professor Reinsch’s collec- tion, gathered in Germany, drawing attention to the complete identy of the examples. Mr. Crowe showed, for comparison’s sake, several species of Micrasterias taken lately in North Wales, such as WM. rotata, M. denticulata, M. papilifera.—He also showed Actinospherium Eichhornii occurring in the same gathering. CosmariumReinschii, n. s., echibited.—Mr. Archer further showed a Cosmarium which he had no doubt was identical with that re- corded and figured by Prof. Reinsch in his ‘ Contributiones ad Algologiam et Fungologiam,.’ t. xviii, f. 4, but not named by that author. This Mr. Archer had also some time known, but it is quite rare ; the present examples were from Co. Westmeath, and he was able to place side by side therewith, under another micro- scope, aspecimen found on a slide in Reinsch’s collection. These were perfectly identical, nor could he doubt that the form found in the Reinschian collection was that the author had in view (loc. cit.), but Mr. Archer thought his figure showed the lateral projections as too angular: they are rather undulately rounded. This species, which seems abundantly distinct, might well stand as Cosmarium Reinschit. A Glimpse of a Vampyrella-form.—Mr. Archer spoke of what he would call but a glimpse of a Vampyrella-form and its doings; though time would not admit of his fully exhibiting some examples of the encysted condition merely (which he had{put up temporarily with a little glycerine) of this so energetic little particle of sar- code, as well as so choice apparently in the selection of its “ prey.” The active “animal ” was of a reddish hue, vacuolate (not unlike 110 PROCBEDINGS OF SOCIETIES. Hertwig and Lesser’s Leptophrys, ‘Archiv fiir Mikrosk. Anatomie,’ Band x, t. ii, f. iii), and its food the moving examples of a Chlamy- doccus swimming about in pairs or fours. The little vampire— which was always altering its figure, and throwing out and with- drawing its slender, slightly branched pseudopodia—when it came in contact with one of these colonies, lay around one of the cells, embraced it, sunk into its outer coat, wrapped up the inner green cell with its own body-substance, then became itself encysted (in order to enjoy its dinner, unmolested, in comfortable and quiet seclusion, one would suppose!), and so remained for a number of hours (seemingly some twenty-four) ; by and by, the sarcode contents, “in the twinkling of an eye,” suddenly burst the cyst and reappeared separated into four to five or six small Vampyrelle, with the same characters of vacuolar, reddish (but paler), granulated sarcode, and the characteristic pseudopodia and movements. In the encysted condition, especially if indeed four Vamppyrelle (which was rare) hadeach “got hold of’ the four com- ponent cells of a colony of the alga, the group would call to mind Cienkowski’s description of his “Tetraplastic” forms, but this would apparently be a deception, for by far more frequently one only (sometimes two) of the component cells of the alga became the passive victim of this little vampire ; such a partially attacked colony would still move about and carry its destructive visitor with it. When the wall of the encysted parasite lay empty the remains of the vegetable portion could be seen as a condensed black little body, as if so much of it was not capable of being “ di- gested,” and was thus left behind—as unassimilated fecal matter. Indeed within the encysted examples the alga substance could be seen in different degrees of alteration, from bright green to brown and black, and gradually becoming smaller in size. Mr. Archer could. not dare to name this “ rhizopod” or make sure of its individuality ; it might just be a small representative of Cienkowski’s own form. It was but scarce in the gathering and the supply soon ran out, and he regretted to say that all he could communicate was this brief record of his “ glimpse.” MEDICAL MICROSCOPICAL SOCIETY. Wt Mepicat Miscroscorican Sociery. October 15th, 1875. JaBEZ Hoaa, Esq., Vice-President, in the Chair. The Cochlea in Birds.—Dr. Pritchard explained and exhibited specimens illustrative of the structure of the cochlea in birds. Artificial Fibrillation of Hyaline Cartilage —Mr. Cresswell Baber exhibited specimens illustrating his paper in the ‘Journal of Anatomy and Physiology’ on the above subject. His observa- tions were based upon a statement by Tillmanns, in Max Schultze’s ‘Archives,’ to the effect that fresh hyaline cartilage can be fibrillated by macerating it for several days in a solution of permanganate of potash, or in 10 per cent. solution of chloride of sodium. Mr. Baber showed that fibrillation of the matrix can be produced by macerating sections of hyaline cartilage in solu- tion of chloride of sodium (both 103 per cent.) in lime water or in baryta water, and in each case after the maceration applying momentary pressure to the glass covering the section before examining it. The fluid that acted most rapidly was baryta water, which produced the fibrillation in half an hour, while permanganate of potash, that Tilmanns prefers, he had found un- certain in its action. Mr. Baber had found the fibrillation of the cartilage matrix in all cases in which he had searched for it, and concluded therefore with Tillmanns that the hyaline matrix is composed of fine fibres held together by an interfibrillar cement substance that can be dissolved by certain coagents. A dis- cussion followed, and the meeting then resolved itself into a conyersazione. November 19th, Friday. Dr. Pritcuarp, Vice-President, in the Chair. Micro-photography..—Dr. George Giles read a paper on, and exhibited an instrument for, quickly connecting an ordinary microscope with an ordinary camera, and obviating the use of the heleostal. The latter difficulty is met by using as a con- denser an achromatic lens of large diameter, but long focus, say from ten to twelve inches. The image produced by such a lens is so large in comparison with the field of the microscope that one has simply to change the focussing screen for the dark slide, and to expose, before the earth has moved sufficiently to throw the light off the object. Ifthe condenser has a diameter of three inches it will condense quite sufficient light for any power that one may want to use, say up to 1000 diameter. It consists of a firm base board, at one end of which is a stage to which a camera can be fixed by means of binding screws. In front of this stage is a sort of tramway, in which slide first a piece of wood, with 112 PROCEEDINGS OF SOCIETIES. cavities to fit the feet of the microscope ; next a stand to support the condenser and alum cell; finally a mirror moveable in all directions, the motion being performed by means of pulleys at the back of the instrument. The camera is connected with the microscope by means of a loosely-fitting metal tube, made proof against light by a lining of thick washleather. The instrument is used by placing it across a table in front of a window, so that the mirror projects out of it clear of all shadow. The condenser is focussed by sliding it along the tramway, the light thrown on by pulling with the string, and the exposure conducted as in al! photographie manipulations, the time of exposure being as instantaneous as possible. The process that answers best when the sun is tolerably constant is the ordinary “ wet collodion ;” but when it is being often obscured by clouds the “ gelatin-bromide,” in the form of “ Kennet’s patent sensitised gelatin-pellicle,” a dry process, and by which micro-photographs can be obtained by the light of an ordinary paraffin lamp. The Chairman suggested the use of a frosted silver mirror to supply a white light. Dr. Matthews proposed using a paraffin lamp with double flame, and stated that, in order to save daylight, plates need not be developed at once, but could be put aside safely for some hours if melted with treacle and water. The yellow colour of the specimens, if it were requisite, might be corrected with hematoxylin. Mr. Giles, in reply, preferred sunlight direct if it could be obtained, and did not find the cclonr of the slides any drawback. Differential Warm Stage.—Mr. Golding-Bird explained and ex- hibited in action a simple form of hot stage, heated by a spirit lamp, capable of being kept in action for any length of time, its temperature being regulated according to the condition of pieces of solid paraffin placed on it, and on a copper tongue connected with it. He had found it extremely useful for purposes of de- monstration, and its simplicity allowed of its being used in the wards of an hospital when examining blood in a morbid state. A discussion followed, and the meeting then resolved itself into a conyersazione. 1 This stage has been already described in ‘ Quarterly Microscopical Journal’ for Oct.,’ 1875, and is made by Millikin, of St. Thomas’s Street, Southwark, 8.E. MEMOIRS. OsBsERVATIONS on the EARLY DEVELOPMENT of the Common Trout (Salmo fario). By Dr. E. Kurtin, F.RS. (With Plate VI.) In March, 1872, a paper was read before the Royal Micro- scopical Society, in which I described and illustrated the segmentation and spontaneous movement of the germ of the fertilized trout’s ovum, as observed in the living condition. These observations, as far as the segmentation is concerned, were in accordance with the older assertions of Rusconi, Vogt,1 Lereboullet,? and Coste, on Teleostean fishes, and were opposed to those of Stricker.* In that paper (which was printed in the ‘Monthly Microscopical Journal,’ May, 1872) I have stated that in the germ of the trout’s ovum segmentation, at least in its earlier stages, is of the same regular type as in other Teleostean fishes, and that only in the more advanced stages the blastoderm presents the appear- ance as if the elements of cleavage—z. e. the embryo-cells— originated by being constricted off from the main body of the blastoderm, in a manner similar to that described by Stricker. Independently and almost simultaneously,* Oellacher’® de- scribed the movement .and segmentation of the fertilized blastoderm of the trout’s ovum, in a manner which is in complete accordance with the description given by myself. In that paper I have also mentioned the spontaneous movements and the appearances of division presented by the embryo-cells, while in a living condition, between the fourth and tenth days. The same has been afterwards noticed also * The Reporter on Embryology, in ‘Jahresbericht iiber die Leistungen und Fortschritte in der Anatomie und Physiologie’ for the year 1872, edited by Virchow and Hirsch, while making an imperfect abstract of an abstract that had appeared in this journal, leads the reader to assume—of course incorrectly—that my observations were made after those of Oellacher. VOL. XVI.—-NEW SER. H 114 DR. E. KLEIN, by Weil®* and Romiti.® The appearance of the segmentation cavity, the differentiation of the blastoderm, while growing round the yolk-sphere, into a thinner central and a thicker peripheral part, the foundation of the embryo, and the de- velopment of the embyonal layers, were described in accord- ance with Stricker and Rieneck.® The development of the central nervous system was mentioned in conformity with the assertions of Kupfer‘ on this subject. Oellacher, in his paper!! on the development of the embryo and the embryonal layers, has made some very minute and detailed observations, which to some extent tend to modify the previous accounts of these matters. Thus, Oellacher shows that the segmentation or subgerminal cavity does not appear, at the outset, between the central part of the blastoderm and the yolk, but is founded excentrically, in consequence, as it were, of the peripheral thickening of the blastoderm being much greater and broader on one side than on the other. This statement I can fully confirm (see fig. 4). As this, however, does not form the chief subject of my present inquiry, I will not pursue it further. Comparing thin sections of the blastoderm of different Stages of early development which have been hardened in dilute chromic acid (one sixth per cent.), and then prepared in the ordinary way, I find that besides the blastoderm proper in its different aspects during early segmentation (compare figs. 1—4), the subgerminal and paragerminal substance deserves equally close attention, for this substance bears at all stages of development an important genetic relation to the blastoderm and the embryo. As is well known, the blastoderm of the trout and of Teleos- tean fishes in general lies closely upon the surface of the yolk- sphere in a slight depression of the latter, round which most of the oil or fat globules accumulate immediately after fertilization. As segmentation proceeds, the blastoderm becomes gradually separated from the surface of the yolk- sphere by the appearance of a cleft, 2.e. the subgerminal or segmentation cavity. From the earliest stages of segmenta- tion—say, from a period when the blastoderm appears on section to be a finely granular mass, which, by the presence of a few more or less distinct perpendicular and horizontal fissures is divided intoa certain limited number of contiguous * The Reporter in the ‘ Jahresbericht,’ while mentioning this observation of Weil, states, axticipando, that Romiti, whose researches were conducted in the Reporter’s laboratory, arrived at similar results ; while writing so the Reporter is altogether unaware of my own description of the same pheno- mena. OBSERVATIONS ON DEVELOPMENT OF COMMON TROUT. 11D masses, of which those nearest to the yolk are largest, the fissures being distributed chiefly over the superficial part of the blastoderm—there is a uniformly granular substance to be noticed around the blastoderm, which, although connected with its (the blastoderm’s) peripheral edge, forms no integral part of the blastoderm itself, and does not participate directly in the process of segmentation. It is this substance and its changes which I am going to describe in the present paper. Let us then commence with the examination of a thin sec- tion through the ovum of a trout of a very early stage of development. [ Note.—The ova which formed the material for the present research developed somewhat slowly, for only after nine days’ incubation did the subgerminal cavity make its appearance. | A vertical section through the blastoderm of the third day, which when viewed from the surface has the appearance of being divided into about twelve or sixteen elements, presents the following features :—The blastoderm is of the shape of a plano-convex disc, the free surface being plane or rathervery slightly depressed, the lower, 7. e. that resting on the surface of the yolk, being convex ; the largest vertical diameter amount- ing toabout0‘4mm. The external margin of the blastoderm does not rest on the yolk, but is overhanging, as it were, like a lip, owing to the presence of a relatively deep groove (see fig. 1). Asection comprising the largest horizontal diameter —which is about 1°15 mm.—would therefore cut the blasto- derm in the external half of its largest vertical diameter, as is shown in fig. 1; the blastoderm is divided into a number of large elements, which are separated from each other by more or less distinct fissures. These latter, which in my specimens hardened with thin chromic acid, present the appearance of thin septa, are seen to penetrate only to a certain depth, the deepest mass of the blastoderm being free from them, 7. e. being as yet undivided. This corresponds to what I have figured in my first paper (l.c., fig. 9), and what is termed by Oellacher!® (I. c., p. 23) ‘‘ basale masse.” The blastoderm appears as a granular mass ; the granules, however, are not uniformly distributed through it, for in the depth they are much larger than in the superficial parts, and they gradually shade off into those contained in the superficial parts of the yolk. Nevertheless, the boundary by which the lower surface of the blastoderm is separated from the yolk of the saucer-like depression on which the blastoderm rests, is tolerably sharp (see fig. 1); the yolk of the saucer-like depression contains, besides large oil drops—occasionally, 116 DR. E. KLEIN, but rarely, I have seen one or the other oil drop included also in the deepest part of the blastoderm—solid granules of a yel- lowish colour and of very various sizes (see fig. 1). Some of the last-mentioned elements appear in the specimens under consideration, as if they contained perfectly black specks (fig. 1), the number. of which varies according to the size of the yolk-granules they are contained in; in the large yolk- granules the black specks are grouped together in the centre. In the blastoderm itself are found numerous yolk-granules containing these black specks ; in fact, we are able to detect, with great ease, in the blastoderm the former by the presence of the latter, and in the same way we are able to state that towards the surface of the blastoderm the yolk-granules and their black specks decrease in size. Similar yolk-granules, containing these black specks, may be detected in the blastoderm also in later stages (see figs. 2,3,4,5). Already Rieneck (1. c., p. 360) mentions the presence of yellowish yolk-granules in the cells of the blastoderm of the more superficial layers, and Oellacher’® (1. c., p. 26) speaks of the elements of the blastoderm as if feeding (on the yolk- granules) ‘from mouth to mouth.” I have mentioned above that the extreme marginal portion of the germ does not rest on the surface of the yolk-sphere. By this I mean that portion only which is, so to speak, over- hanging the paragerminal groove, for I have to add now that the substance of the germ extends below that groove out- wards on the surface of the yolk. This extension of the germ is, from the circumstance above mentioned, only a con- tinuation of the deeper part of the germ. It consists of the same granular mass, and includes also smaller or larger yoik-granules ; it is thickest where it is attached to the germ, and becomes thinner in proportion as it becomes more distant from it (see fig. 1). This quasi-extraneous portion of the germ I will call paradlast, in contradistinction to the segmented part or blastoderm of the authors, which I will term archi- blast. These terms I do not use, however, in the sense in which they are applied by His (‘ Entwicklungs geschichte der Wirbelthiere ; Entwicklung der Hiihnchens,’ Leipzig, 1868) to the ovum of hen, for, according to His, parablast 1 is not a portion of the same substance of which the blastoderm consists, but is a part of the white yolk. Parablast and archi- blast in the trout’s ovum, however, is one continuous mass, 2. e. one and the same substance, of which only one portion —blastoderm (Auct.) or archiblast, ce. that lying in the saucer-like depression of the yolk—undergoes segmentation, whereas the second portion, parablast, not participating in OBSERVATIONS ON DEVELOPMENT OF COMMON TROUT. 117 the process of segmentation, extends as a thin crust on the surface of the yolk outside the saucer-like depression, and, as will be shown afterwards, is important for the development of the layers of the embryo and some of its tissues. Oellacher,! who first correctly described (l.c., p. 12) that part which corresponds to our parablast—but who unfortunately calls it vitelline membrane, not knowing its great importance for the future development of the embryo and embryo-cells—mentions that itis a direct continuation of the germ, already before the germ undergoes segmentation. Although I have not been able to ascertain how far the parablast extends on the yolk- sphere, still I am inclined to accept Oellacher’s very tenable proposition, that the ovum of trout is comparable to a fat cell, thus: the protoplasmic mantle of the fat cell consists of a thickened portion which includes the nucleus, and of the rest which is only a thin crust of protoplasm; the thickened nucleated part corresponds to the unsegmented archiblast or blastoderm (Auct.) with its germinal vesicle, while the rest of the mantle is represented by the parablast; to the oil drop of the fat cell included in that protoplasmic mantle, corresponds the yolk (food-yolk) of the ovum. [Vogt! speaks of a vitelline membrane in the ovum of Coregonus palea, which in the fertilized ovum is continuous with the blastoderm. He says, p. 29: “Le renflement (blastoderma Auct.) occupe invariablement le milieu du disque huileux ....et ses bords passent insensiblement a la membrane vitillaire qui a lair de le recouvrir.” Lereboullet? (p. 127) says that an ovum which imme- diately after fecundation is coagulated by acidulated water contains an amorphous, granular, membranous pellicle which surrounds a third or half of the ovum, and includes also oil drops ; it lines also the saucer-like depression and represents the future “ feuillet organique ” or “ feuillet muqueux ” of the embryonal germ, ?. e. of the blastoderm, Auct., which lies above it, and which is very adherent to that pellicle. The “ feuillet muqueux ” does not participate in the segmentation (1.c., p. 128); it consists (1. ¢., p. 184) of a thickened peri- phery and a thin central lamella; while the blastoderm grows around the yolk it is accompanied by the “ feuillet muqueux,” which precedes, however, the former (l.c., p. 136). Later on the “ feuillet muqueux”’ contains also cells, and enters into the formation of the wall of the intestine. Kupfer* (p. 217) mentions the appearance of nucleated cells around the blastoderm on the surface of the yolk in the ovum of Gasterosteus and Spinachia at a somewhat late stage of segmentation, These cells are not derived from the 118 DR. E. KLEIN, blastoderm, but develop, according to Kupfer, after the type of “ free-cell formation,” probably (p. 218) out of a fluid or semifluid blastema, comparable to that of insects (Musca, Chironomus) ; this blastema is situated in the periphery of the yolk, and in it appear at first nuclei—larger than those of the embryo-cells of the segmented blastoderm—around which the substance of that blastema gradually accumulates as “ cell- substance.” Kupfer believes (p. 220) that this blastema is not only limited to that part where it was seen by him (Kupfer did not examine sections, but observed only fresh ova in surface and profile views), 7.¢. at the periphery around the blastoderm, but that it extends over the whole surface of the ovum, and is concealed by the blastoderm which gradually spreads (grows) over it. Thus Kupfer maintains that, independently of the blastoderm, a layer of cells appears which covers the yolk. Kupfer is unable to say whether this layer has anything to do with the formation of the hypo- blast (* feuillet muqueux ” of Lereboullet) or not. Van Bambeke,’ in examining vertical sections through the meridian of the ovum of osseous fish at the end of segmen- tation, finds (Il. c., p. 1057): “la calotte blastodermique se compose de deux parties parfaitement distinctes, une supérieure, représentée par les cellules issues du fractionne- ment du disque, et qui entourent la cavité de segmentation .... 3 Pautre partie de la calotte blastodermique est formée par une couche .... qui ne prend point part au fractionne- ment.” On account of this latter portion being situated between the segmented blastoderm and the yolk, Bambeke calls it “ couche intermédiaire,” and distinguishes in this “ couche intermédiaire” a peripheral thick portion (bourrelet périphé- rique) from a central thin part (1. ¢., p. 1058) ; the former pos- sesses in vertical section a triangular shape, and is therefore to be regarded asa prism bent into an annular mass and resting with one surface on the upper segment of the yolk-sphere ; its external surface, ¢. e. that directed outwards, is free, and the upper surface supports the peripheral part of the seg- mented germ. The central portion of the “couche intermédiaire” is a very thin lamella uniting the inner angle of the prismatic ring, and thus separating the segmented germ from the yolk. Bambeke thinks that this latter, 2. e. the central thin lamella, has probably grown from the periphery, as it is not present in earlier stages; it forms later on the hypoblast or * feuillet muqueux ” of the blastoderma, and accompanies this latter while growing around the yolk. OBSERVATIONS ON DEVELOPMENT OF COMMON TROUT. 119 Bambeke describes the “‘ couche intermédiaire”’as consisting of a granular protoplasm, the granules being much larger than in the embryo-cells. The thick portion of the ‘ couche intermédiaire ” includes constantly a certain number of nuclei and cells, which Bambeke believes to develop by endogenous cell-formation. Bambeke finally concludes that the ‘couche intermédiaire” is the lower portion of the germ-disce, split off from the latter in consequence of the fecundation; it does not undergo cleavage like the upper portion, 2. e. the blasto- derma (Auct.).* From what has been said previously we can modify already now a portion of these statements. Another portion, that referring to the hypoblast, we shall have to discuss hereafter. The only mention of cells outside the blastoderm made by Oellacher!" refers to a “‘ not inconsiderable numbers of cells ” (1. ¢., p. 12), which having become separated from the lower surface of the blastoderm during the formation of the seg- mentation cavity remain on the floor of this latter (compare on this subject Rieneck’s paper, my first communication on the development of the trout’s ovum, and Stricker’s article “On Development of Simple Tissues,” in Stricker’s ‘ His- tology’), and “ graben sich in die oberflachlichsten Schichten des Dotters ein.” Besides this there are found cells also out- side the region of the segmentation cavity; these Oellacher thinks may have migrated'there from the former place, or may have become separated from the lower surface of the blasto- derm where this is in contact with the yolk. These cells remain fora very long time in the yolk. Oellacher found them (1. c., p. 13) very numerous, even after the heart was formed and while the vessels of the yolk-sac are being developed; espe- cially in the hind part of the embryo they are very numerous. They enlarge while the embryo proceeds in its development, and are of very various shapes. In longitudinal sections through the embryo these cells form long strips below the former ; they also undergo multiplication. Oellacher further mentions (1. c., p. 17) that in certain stages of the development - of the subgerminal cavity the most superficial layer of cells of the blastoderm extends for a short distance on the surface of the yolk beyond the blastoderm; this, Oellacher goes on * This “couche intermédiaire” of Van Bambeke cannot be compared, as is done in the Reports in the ‘Jahresbericht fiir Anatom. und Phy- siol.,” for 1872, p. 78, with the “basale masse” of Oellacher (see above), for this is only the deep part of the blastoderm, which undoubtedly undergoes segmentation though somewhat later than the part situated more superficially. The “couche intermédiaire” of Van Bambeke does not segment, 120 DR. E. KLEIN. to say, may correspond to the layer of cells observed by Kupffer (see above) around the blastoderm of Gasterosteus and Spinachia. As we have mentioned before, Kupfer makes these cells originate after the mode of endogenous cell-formation. Balfour, in speaking of the later stages of segmentation of the ovum of Elasmobranch fishes, says (I. c., p. 4): “* The limits of the blastoderm are not defined by the already com- pleted segments, but outside these new segments continue to be formed around nuclei which appear in the yolk. At this stage there is, therefore, no line of demarcation between the germ and the yolk; but the yolk is being bored into, so to speak, by a continuous process of fresh segmentation.” Balfour then goes on: “‘ Intimately connected with the seg- mentation is the appearance and history of a number of nuclei which arise in the yolk surrounding the blastoderm. ey 2G The blastoderm thus rests upon a mass of finely granular material, from which, however, it is sharply sepa- rated. At this time there appear in this finely granular material a number of nuclei of a rather peculiar character.” These nuclei vary, according to Balfour, very much in size. «“They are rather irregular in shape, with a tendency when small to be roundish, and are divided by a number of lines into distinct areas, in each of which a nucleolus is to be seen. The lines dividing them have a tendency to radiate from the centre.” These very peculiar nuclei are scattered through the subgerminal granular matter ; they are distributed in a special manner under the floor of the segmentation cavity, on which new cells are continually appearing, and are identical with those present in the cells of the blastoderm. From these facts Balfour concludes that the ‘‘ nuclei of the yolk” become actually the nuclei of cells which enter the blastoderm; they are later on concerned partly in the formation of the vascular system, and still more in that of “the walls of the digestive canal and of other parts.” Haeckel,1 in his studies of the development of Teleostean fishes (probably Lota), finds that only the cells resulting from the segmentation of the blastoderm participate in the forma- tion of the developing body of the fish, all the rest of the ovum, 7. e. food yolk, is homogeneous and amorphous, and has nothing to do with the development of the blastoderm. “ Thus,’ Haeckel goes on to say (I. ¢., p. 96), “ the false ‘ Parablast theory of His,’ and all similar theories, according to which in discoblastic vertebrate ova histogenetic embryo- cells are said to develop out of the separate food yolk, zde- pendently of the primary embryonal layers, are hereby ‘ biindig A OBSERVATIONS ON DEVELOPMENT OF COMMON TROUT. 121 widerlegt.’”? But only a few pages later (p. 102) he says, - that there exist, however, other discoblastic ova in which that portion of the food yolk which lies next to the blasto- derm participates also in the segmentation, and produces cells which become partly blood-cells, partly connective-tissue cells ; in doing so Haeckel maintains that Goette “has proved” (Max Schultzse’s ‘ Archiv,’ vol. x, 1872) that in the dis- coblastic bird’s ovum segmentation extends also into a por- tion of food yolk; in this are produced cells—* yolk-cells,” which become used “ partly as blood-cells, partly as food for the developing embryo;” likewise Haeckel takes it that Balfour “has shown” that, in Elasmobranch fish, a large portion of food yolk undergoes segmentation ; and, finally, E. Ray Lankester (‘‘ Development of the Cephalopoda:” see this Journal, 1875, No. LVII) “has seen” in the discoblastic ova of Cephalopods numerous cells originate in the food yolk, which therefore undergoes secondary cleavage. ] We proceed now with our own observations. The next stage I wish to describe is that represented in fig. 2. The archiblast consists of nucleated embryo-cells, the most superficial layer of which contains large cells of an opaque substance. The segmentation has proceeded (as is seen in fig. 2) to a considerable extent; the shape of the archiblast is that of a biconvex disc, thicker, however, at one edge than at the other; its thickness in the centre is over 0°5 mm., its breadth over 14 mm. The archiblast is in its whole extent resting on, but at the same time very sharply defined from, the yolk, which latter appears in hardened preparations as if limited by a membrane. Under- neath the archiblast the yolk contains large oil drops and yellowish granules; the latter become larger in size, the farther away from the surface of the yolk. The paragerminal groove above mentioned is still distinctly seen. Next to this groove, and in immediate contact with the archiblast (see fig. 2) extends the parablast outwards on the surface of the yolk, gra- dually becoming thinner as it becomes more distant from the archiblast. The most conspicuous part is the thickened por- tion of it (parablast), which, as is shown in fig. 2, presents in vertical section a more or less triangular shape ; that is, it cor- responds to a prismatic body, of which Van Bambeke (see above) correctly observes that it surrounds the archiblast like a ring-shaped rim. But Van Bambeke is not correct in saying that this mass has an outer, inner, and lower surface, for it has an outer, inner, and upper surface; nor that it extends as a thin layer over the saucer-like depression of the yolk; and 122 DR, E. KLEIN. likewise he is not correct in making it stop at an outer angle, for this prismatic annular mass is only the thickened portion of the original parablast. For the sake of simplicity, however, we will henceforth consider under the name of parablast only this prismatic rim. Its greatest thickness is about 0°12 mm.; its lower surfaces, z.e. its internal and external surface (contrary to the assertion of Bambeke) rest on the yolk, with which they are in very close connection, the parablast being, as it were, moulded into the jagged surface of the yolk; the upper surface of the parablast is free, and appears in hardened sections bordered by a very sharp out- line as if by a membrane. The substance of the parablast is a finely granular material, containing isolated, minute yolk granules, which become larger nearer to the yolk; on account of this the line of boundary between parablast and yolk cannot be at all points made out with precision. In the very superficial parts, and also at some places of the deepest parts, I think I can recognise faint outlines of what corresponds to nucleus-like bodies. In a later stage (fig. 3) the archiblast is seen to be still biconvex (slightly thicker at one edge), and much broader than in the former case (over 1°09 mm.); its thickest diameter being, however, smaller (0-4 mm.). The most superficial layer of its cells is very conspicuous, and appears almost separated from the rest by a thin cleft. This sepa- ‘ration, as we shall see, remains persistent in the following stages, and to it is due the differentiation of the corneous layer from the nervous layer of the epiblast (see Rieneck’s paper, my first communication, and Stricker’s article, men- tioned above). The archiblast is very well defined from the yolk of the saucer-like depression. ‘The yolk possesses the same morpho- logical characters as in the former case. The parablast, however, has changed considerably : (@) it has become more finely granular and transparent, thinner, and at the same time broader; it resembles now a mass of a more placoid shape, which is wedged in for a certain distance (see fig. 3) between the archiblast and yolk ; at the same time bearing the same close relation to the yolk as in the former case. That this wedge-like process of the parablast is due to an active ingrowth of the latter, and not merely to the circum- stance that the archiblast, while growing in breadth, spreads over and beyond the inner edge of the parablast, is clearly proved by comparative measurements ; (6) in many sections its superficial layers are, over greater or smaller areas, of a OBSERVATIONS ON DEVELOPMENT OF COMMON TROUT. 123 more or less distinctly fibrillar structure especially well shown in the part next to the archiblast; (¢) it contains a great number of well-defined vesicular nuclei, many of which con- tain a nucleolus; they (nuclei) are most numerous in the parts near the edge of the archiblast, and are of different sizes and shapes—chiefly elongated in the superficial parts—and appear generally to be disposed in groups, forming chains of bodies more or less pressed against each other. ‘lo these nuclei we shall return more minutely hereafter. [ Note.—The fibrillar structure just referred to is of such a character that the apparent “fibrils”’ run in a direction parallel to the free surface. Balfour mentions the presence, in osmic acid specimens, of a fine network of fibrils “ in any part of the fine granular yolk around the blastoderm,”’ that is, in the portion comprising “ the nuclei of the yolk.’’] Next in order is a blastoderm twelve days old, which in vertical section exhibits a distinct subgerminal or segmenta- tion cavity. ‘This is seen to be situated excentrically in con- formity with the assertion of Oellacher. The archiblast is thicker in the periphery than in the centre. The former is not of uniform thickness, for at one side it is much thicker than at the other (see fig. 4) ; this latter corresponds to that part of the periphery towards which the segmentation cavity extends. The central thinner part of the archiblast is several cells deep; they are more or less polyhedral, and contain one or two nuclei. At the periphery there exist underneath these, in addition, layers of cells, which are more opaque, containing numerous yolk granules; they are of different sizes, and possess not unusually a nucleus in the state of division or two or more nuclei; also to the lower surface of the thin part of the blastoderm is attached one or the other cell of this character. The parablast is seen to have increased considerably in extent (see fig. 4). Although under a low power only the thicker portions of it are discernible, yet the examination with a higher power proves that it not only underlies the peripheral part of the archiblast, but that it extends as a thin lamella on the surface of the yolk for a considerable distance towards the zone of the segmentation cavity. Thus, in the preparation which is represented in the present figure 4, I can discern, under a moderately high power (Hartnack, fig. 7), a thin layer of parablast extending on the right side beyond the peripheral thickening of the archiblast for some distance underneath the segmentation cavity ; whereas on the left side a thin layer of para- - blast may be followed on the surface of the yolk, close to the 124 DR. E. KLEIN. edge of the segmentation cavity. The peripheral thickening of the archiblast rests on the surface of the thickened para- blast, both being in’ very close contact. Sometimes (see the left side of fig. 4:) the boundary between the two is indistinct, and then it appears as if the deepest elements of this part of the archiblast were directly developed out of the parablast, especially if one bears in mind that numerous nuclei are contained in this portion of the parablast, which (nuclei), as will be more minutely described hereafter, are similar to those of the cells of the archiblast. Whether this definite boundary between tie two structures (parablast and thickened part of archiblast) is a sufficient reason for saying that the deepest cells of the archiblast of this stage develop out of the parablast—the substance of the latter becoming differentiated round its nuclei into cell-terri- tories—is a matter which I am not in a position to decide definitely ; it is, however, very probably so, inasmuch as in only a little later stage cells may be followed with certainty to develop out of the parablast. This will be stated hereafter. From the inspection of fig. 4 it is also clear that the thickened part of the parablast is not situated symmetrically in all parts in relation to the archiblast, e.g. on the right side it is situated more externally than on the left. This thickened portion of the parablast contains very numerous nuclei; they are more numerous in the superficial than in the deeper parts. In a somewhat later stage (thirteen days) the archiblast is seen to be separated, for nearly its whole extent, from the yolk by the segmentation cleft (see fig. 5). The elements of which the archiblast now consists are little smaller than those in thé former stage, except the most superficial layer, which is composed of slightly flattened cells: all the other © layers are made up of polyhedral cells. At and next to the peripheral thickening of the archiblast, there are present on its lower surface numerous more or less spherical elements loosely connected with each other; some of them are much larger than the ordinary embryo-cells, and possess two, three, and more nuclei, besides yolk granules. I have seen some of these cells contain as many as six nuclei, each with a nucleolus. These nuclei were arranged like the sections of one large lobed nucleus. But also in other parts, on the lower surface of the archiblast, isolated cells may be found of the same character as those just mentioned (see figs. 5, 6, 7, and 8). Except the difference of the most superficial layer of cells, the archiblast does not present as yet any differentiation of its elements into strata. . The parablast is seen to have assumed much greate OBSERVATIONS ON DEVELOPMENT OF COMMON TROUT. 125 dimensions. First of all it is found to possess a considerable thickness, not only around the peripheral thickening of the archiblast, but also at the peripheral part of the floor of the segmentation cavity; and, secondly, with a high power the parablast may be traced to extend for a considerable distance towards the centre on the surface of the yolk as a thin granu- lar lamella, including nuclei, and possessing here and there swellings, which appear to be wedged in between the large oil globules of this part of the yolk. There is little to be added at present with regard to the nuclei of the parablast, except that in the part surrounding the edge of the archiblast they (nuclei) are elongated and form extensive rows. A section taken at a right angle through the peripheral part of the specimen represented in fig. 5 naturaily shows the segmentation cavity included between the archiblast and the thickened parablast, the lower surface of the former showing the loosely connected elements of various sizes, and the latter being conspicuous by its uniformly granular sub- stance and the larger or smaller groups of its nuclei. It may be mentioned already here, that amongst the ele- ments lying loosely on the floor of the segmentation cleft (see above), there are seen here and there apparently free nuclei, some of considerable dimensions, and_ resembling nuclei contained in the parablast. At a somewhat later stage (fourteen days) the thin part of the archiblast consists only of two layers of cells, very dis- tinctly separated from each other (see fig. 6), the upper layer (corneous layer) being continuous with a similar layer at the thickened periphery of the archiblast, and the lower with what corresponds to the nervous or second layer (see my first com- munication on this subject). In the peripheral thickening (of course, including that portion of it which becomes trans- formed into the embryo) there are present underneath the two strata just mentioned several layers of cells, which, as has been stated already, form a less continuous mass than the superficial cells. The parablast is found to have increased considerably, not only in thickness, but also in breadth, as it may now be traced very distinctly over almost the whole surface of the yolk, 7.e. almost along the whole extent of the floor of the segmentation cleft. As in the preceding stage so also in this, the greatest thickness of the parablast is no more to be found externally to or directly underneath the edge of the archiblast, but internally to it, i.e. on the peripheral portion of the floor of the segmentation cleft. ‘Towards the centre the parablast becomes thinner, and in some places it is just possible to trace it from one 126 DR. E. KLEIN. cuneiform accumulation to the other, wedged in between the fat globules of the yolk. As regards the later stages, I may now be very brief. In all stages I found the parablast present as more or less con- tinuous membranous masses, forming at numerous places cuneiform or prismatic or irregular shaped accumulations, not only underneath the embryonal portion, but also in all other parts of the archiblast. Its (parablast) aspect remains always the same, 2.e. a finely granular material containing isolated, or, as is more commonly the case, groups of nuclei. We have thus far been able to ascertain that one part of the germ, viz. the parablast at first situated outside the area of the archiblast (blastoderma, Auct.), gradually encroaches on the surface of the subblastodermic yolk, due partly to the growth of the blastoderm over the parablast, and partly due to an active growth of this latter itself, so that after a certain time the subblastodermic or segmentation cleft becomes lined on its floor with parablast. There remain now two other points to be discussed: (A), the substance of which the parablast is composed and the nuclear elements found in it; and (8) the relation of the parablast to the cells on the floor of the segmentation cavity and to the elements of the archiblast in general. (A) The substance of the parablast is, as has been men- tioned on several previous occasions, a finely and uniformly granular substance, showing this slight difference in the earlier and later stages, that it is more transparent in the latter than in the former. (In this, as in the preceding, we mean by “ parablast” only that portion which is at first situated in the vicinity of the edge of the archiblast, and, as has been shown, gradually grows inwards underneath the archiblast, so as to line the floor of the segmentation cavity.) Whether the appearance of fibrillation which is to be observed in some parts of the surface of the parablast is due to a real fibrillar arrangement of its substance, or is only produced by the hardening reagent, must remain undecided. The outlines of the parablast are not well defined towards the yolk, especially when the parablast has grown inwards on the yolk of the saucer-like depression; this is chiefly due to the fact that the yellowish granules which are contained in the yolk—yolk granules—are gradually shading off into the smaller granules contained in the parablast. To suppose that on account of the deepest part of the parablast con- taining granules which insensibly pass into the larger granules of the yolk, the two substances, 7. e. parablast and OBSERVATIONS ON DEVELOPMENT OF COMMON TROUT, 127 yolk, pass into each other, would be no more or less than to admit that the “granules” form the decisive character of the structures in question, and that the “substance of the matrix ” plays only a secondary part. The parablast we are dealing with is a living organism; it grows from an incon- siderably small body into a structure of very great dimen- sions—dimensions which relatively are not very inferior to those of the archiblast; and that therefore this living struc- ture (parablast), which is almost semifluid and has no limiting membrane, should, while growing on the surface of the likewise soft yolk, which has no limiting membrane either, take up the granules contained in the latter and thus feed itself, is just what we should expect. Under these circum- stances it 1s clear that the line of boundary between the two structures must be lost, 7. e. obscured by the granules; for the granules contained in what belongs to yolk pass directly into the granules contained in what belongs to parablast. The fact that the parablast has, at the outset, been forming one unit with what represents the archiblast (blastoderm, Auct.), and while increasing, has spread, i.e. grown over the yolk which underlies the segmentation.cavity, is, I think, the most absolute proof that the yolk is as much different from the parablast as it is from the archiblast. The nuclei contained in the parablast are different as re- gards shape and size. In the sections of hardened specimens which we are dealing with, only very few appear quite circular ; many are roundish, with their sides more or less flattened on account of being pressed together in small groups; others are oblong, especially those about the periphery of the archi- blast; and others again are either hour-glass shaped or kidney shaped, or Jobed, furrows and notches extending for various depths into their substance; that these nuclei, therefore, multiply by division into two or more smaller nuclei is, no doubt, to be inferred from the frequent occurrence of chains of nuclei and of groups of nuclei of different sizes, in which the individual elements appear as sections of one large lobed nucleus. The largest nuclei which deviated least from the circular form were measured in a preparation represented in fig. 6; they were found to be 0°012 mm. in diameter. From nuclei of this size down to the smallest bodies which by their outline and connection with a distinct nucleus could be just recognised as such, all intermediate sizes could be detected. The substance of these nuclei is different from that of the parablast itself: being more transparent, the granules contained in the nuclei irregular and of very different sizes ; besides this, each nucleus possesses a sharp outline as if 128 DR. E. KLEIN. bordered by a definite, though delicate, membrane. There are, however, present in the parablast also true cells, 2. e. granular corpuscles (raore transparent than the parablast itself), containing two, three, or more bodies, which, either all or only some, possess a complete similarity with the nuclei of the neighbouring parts of the parablast. That masses of substance of the parablast separate themselves from the rest and collect round the nuclei so as to form “ nucleated cells ” is, I think, the simplest way of explaining the facts just stated. ‘The question at present of greatest importance is, how do the first nuclei of the parablast develop? The cir- cumstance that nuclei are not perceptible in the parablast in the earlier stages of segmentation, as mentioned on a former page, although not absolutely proving, renders it at least very probable that they originate 7 situ, as development in general proceeds. Besides, there is one definite fact I have been able to ascertain in somewhat later periods (e.g. in preparatious like the one represented in fig. 5), which is apt to throw some light on the origin of these nuclei. It is this: searching carefully through the substance of the parablast with a moderately high power (Hartnack’s No. 8), we detect numerous zsolated, small, transparent bodies very faintly outlined, so as to be rendered just perceptible ; between these and distinct nuclei all intermediate forms may be met with as regards general aspect, outline, and size. This obviously means new formation of nuclei. It therefore stands to reason to assume that, inasmuch as at a period when nuclei may be seen to multiply by division the formation of nuclei de novo, as it were, still takes place in the parablast, the first nuclei of the parablast have also originated in the same manner, i.e. de novo. From the fact that some of the nuclei of the parablast possess elongated or knob-like processes of different sizes, which in some instances are more or less constricted off from the main mass of the nucleus, it may be said that the multi- plication of nuclei takes place also by gemmation or budding. The appearances of multiplication described by Balfour of the “nuclei of the yolk” in elasmobranch fishes (I. ¢., p. 4; compare also fig. 1 of his paper), as quoted on a former page, are not identical with those of the parablast of our case; whereas the appearances described and figured by Oellacher” (figs. 30—36) of the nuclei of the archiblast during early segmentation more closely correspond with those of the nuclei of our parablast. (8) On a previous occasion we mentioned that on account of the intimate connection, at certain points, between the OBSERVATIONS ON DEVELOPMENT OF COMMON TROUT. 129 peripheral thickening of the archiblast and the parablast, it appears probable that the deepest cells of the peripheral thick- ening of the archiblast owe their origin to that part of the para- | blast. In examining preparations like those represented in figs. 5, 6, 7, and 8, it may be easily ascertained that many of the cells lying on the floor of the segmentation cavity, especially many of those forming a loosely connected deep stratum of the peripheral thickening of the archiblast, are formed out of the parablast. Looking, for instance, at a preparation represented in figs. 7 and 8, it requires no difficulty, I think, to find not only nuclei perfectly identical with those of the parablast, but also larger masses, which in all characters denote their origin from the parablast. In fig. 8, especially, these facts come out with certainty; that huge element represented in the figure as attached to the lower layer of cells of the archiblast cannot conceal its place of birth ; and the same may be found on close inspection with many large multinuclear elements, not only on the floor of the segmentation cavity, but also on the lower surface of the archiblast. It thus follows that cells of the deeper layer of the archiblast—im the peripheral thickening (including, of course, the embryonal shield), as well as in the central thinner parts—are derived, directly or indirectly, from the parablast. The assertions, therefore, of Rienek,’ myself,’ and Stricker (Stricker’s ‘ Histology,’ see the chapter on development of simple tissues), that the peripheral thickening of the archiblast owes its origin, to a great extent, to a displacement of elements at first situated on the floor of the segmentation cavity, but originally derived from the archiblast ; and likewise the assertion of Oellacher,! that all the elements of the peripheral thickening owe their origin to the continuous reproduction of its cells, 7. e. those contained in the peripheral thickening ; and, finally, the assertion of Gétte!? and Haeckel,’ to the effect that the deeper layers of the archiblast owe their origin entirely to an ingrowth of the edge of the archiblast (Gotte, l. c., p. 684: “Der Rand des Keimes schlagt sich auf einer Seite um und breitet sich an der unteren Flache des Kenines aus”) ; all these asser- tions are but partially correct. I cannot express myself with anything like certainty on this supposed ingrowth of the edge of the archiblast as maintained by Gotte, and to some extent accepted also by Balfour; but this much I have seen, that the appearances presented by the peripheral thickening in general, and the embryonal shield especial, do not strictly necessitate the assumption of an ingrowth of the archiblast. On the other hand it is certain, as we have shown, that the deeper layers of cells of the archiblast are in VOL, XVI,——NEW SER. I 130 DR. E, KLEIN; a considerable measure genetically derivatives from the para- blast. This last fact is also insisted upon by Balfour, who, however, incorrectly, as we have proved, considers the para- blast as a part of the yolk. Now, the deeper layers of the peripheral thickening of the archiblast are, as is well known, in the embryo—which is developed out of a portion of that peripheral thickening— used for the formation of the hypoblast ; from this and from what I have shown with reference to the parablast, the asser- tions of Lereboullet (feuillet muqueux), Kupfer, and Van Bambeke, with reference to the alimentary canal, receive their due significance. From the presence of parablast, after a certain stage, beneath the whole extent of the archiblast—embryo and yolk-sac—it is only natural to draw the inference that within the area of the latter, ¢.e. the yolk-sac, notorious for the development of blood and blood-vessels, the parablast is concerned in the formation of blood and blood-vessels. But whether the elements derived from the parablast and contained in the deeper layers of the embryo are destined to be also material for the formation of connective and kindred tissues, is a conjecture which requires further consideration.* Bibliography. 1. Carl Vogt—* Embryologie des Salmones,” in ‘ Histoire Naturelle des Poissons de l'Europe Centrale.’ Par L. Agassiz. 1842. 2. M. Lereboullet.—“ Recherches d’Embryologie comparée sur le Developpe- ment de la Truite, du Lézard et du Limnéc,” in ‘ Annales des Sciences Naturelles,’ quatriéme série, Zoologie. Tome xvi. . S. Stricker.—*“ Untersuchungen iiber die Entwickiung der Bachforelle,” ‘ Sitzungsberichte der Wiener Kais. Academie d. Wissenschaften.’ 1865. Bd. 51, ii. 4. C, Kupfer.—‘ Beobachtungen iiber die Entwicklung der Knochenfische,” ‘Max Schultze’s Archiv.’ Bad. iv. 5. Rieneck.—‘* Ueber die Schichtung des Forellenkeims,” ‘Max Schultze’s Archiv.’ Bad. v. 6. Goette-—* Zur Entwickelungsgeschichte der Wirbelthiere,” ‘ Vorlaufige Mittheilung in Centralblatt f. Medicin. Wissenschaften.’ 1869. No. 26. 7. E. Klein.—* Researches on the First Stages of the Development of the Common Trout (Salmo fario),” read before the Royal Microscopical Society, March 6, 1872. ‘Monthly Microscopical Journal.’ 8. Van Bambeke—‘Premiers effets de la Fécondation sur les cufs de Poissons: sur lorigine et la significatione du feuillet muqueux ou glandulaire chez les Poissons Osseux ; Séance de |’Académie des Sciences, 15 Avril, 1872,” ‘Comptes Rendus des Séances de l’Aca- démie des Sciences.’ Tome lxxiv. 9. C. Weil—* Beitrage zur Kenntnis der Entwicklung der Knochenfische,” * P.S.—After this paper was in print I received the full monograph of Van Bambeke’s research on Teleostean fishes. His observations on the earlier stages of development have not been noticed in this paper, since they are pot contained in his earlier communication. oo NUCLEI OF ANIMAL AND VEGETABLE CELLS, 131 ‘Sitzungsber. der Wiener Kais. Academie der Wissenschaften.’ Bd. Ixvi, iii. April, 1872. 10. 2. Oellacher.—* Beitrage zur Entwicklungsgeschichte der Knochen- flsche nach Beobachtungen am Bachforellenei,” ‘ Zeitschrift fiir Wiss. Zoologie.’ Bd. xxii, 4. ll. Idem.—Bad. xxiii, 1. 12, Goette.—“ Beitrage zur Entwicklungsgeschichte der Wirbelthiere,” ‘Max Schultze’s Archiv.’ Bad. ix, 4. 13. Romiti.— Studi di Embriologia. _Rivista Clinica di Bologna, Decem- ber, 1873,” quoted in ‘ Jahresber. iiber d. Leistungen und Fortschritte in d. Anatomie u. Physiologie.’ Fur d. Jahr, 1873, p. 95. 14, F. M. Balfour— A Preliminary Account of the Development of the Elasmobranch Fishes,” ‘ Quarterly Journal of Microscopical Science,’ October, 1874. 15. #. H. Haeckel.—‘ Jenaische Zeitschrift, vol. ix, die Gastrula und die Hifurchung der Thiere.’ Recent ReEsrearcuEs on the Nucixt of ANIMAL and VEGE- TABLE CrELis, and especially of Ova. By Joun PrizsTLEy, Assistant Lecturer on Physiology, The Owens College, Manchester. (With Plates XI & XII.) WHILE much important knowledge of the protoplasmic bodies of cells had rewarded the labours of histological workers, only a few ascertained facts, together with various ill-supported theories, existed respecting the intimate nature and the functions of the nuclei. During the present decade however, much of the energy of research has been directed to repair this deficiency ; and the body of fact which we possess concerning nuclei is rapidly gaining in size and solidity. Some of the publications which have assisted in the impulse are those of which an account is now to be attempted. Notwithstanding some marked diversities of theory, the general agreement in the statements of the various authors seemed to suggest that it would be of advantage to the English reader, and certainly fairer to the authors them- selves, to give firstly a separate, brief résumé of the work of each, and afterwards to collate the various statements and indicate their points of divergence. The historical notices, and all the references, are given on the responsibility of the several writers, since it has been impossible from many causes to check them. They are for the most part taken from the admirable critical article of Hertwig. Among the first of the works referred to is that of Pro- fessor Auerbach, of Breslau, published in 1873-4.! In it there is recorded a very extensive series of observations 1 *Qrganologische Studien.’ Breslau. 1382 JOHN PRIESTLEY, upon nuclei, their nature, microchemical reactions, and growth. Nuclei in a perfectly recent and normal state, are de- scribed as flexible and elastic vesicles with a somewhat thick, highly refractile, doubly contoured membrane which appears to be less distinctly separated from the cell proto- plasm than from the interior of the nucleus. This membrane is regarded as having been formed about the original drop- like nucleus by the differentiation of the inmost layer of protoplasm into a species of interior cell membrane. The main body of the nucleus is bright, and is called by Auerbach the ground substance. It includes one or more nu- cleoli,smooth bodies of irregular outline, as well as numbers of extremely fine intermediary granules. ‘These are either impartially scattered throughout the nucleus, or arranged so as to leave a clear zone about the nucleoli, and sometimes also a similar zone beneath the nuclear membrane (Plate XI, fig. 1, a—d). When nuclei liberated from the cell-body were submitted to the action of certain reagents, characteristic changes of appearance occurred. Water, added gradually, by irrigation, to nuclei'so prepared causes, as a first effect, a shrinking, associated with the expression of droplets of a hyaline body which is supposed to exist in the interstices of the nuclear ground substance. These droplets cling to the irregular cir- cumference of the nucleus and fill the hollows due to shrink- ing (Plate XI, fig. 1,e—g,m). The intermediary granules swell up and most of them become invisible ; the interior of the nucleus assumes a darker, shining, almost homogeneous appearance, the distinction of wall and contents becoming less marked; while the nucleoli at this stage are very slightly affected. If irrigation with water be continued the nucleolus begins to swell up, filling the whole of the nucleus and fusing with the membranous wall (Plate XI, fig. 1,1). If several nucleoli coexist in the same nucleus they may present different degrees of resistance to the action of the water. In the mean time the nucleus has attained to its original size ; but continued exposure soon results in increased dimensions. When this point has been reached, the nucleus successfully resists any further destructive action of the water, not, as is generally thought, becoming completely disintegrated. Some of the later steps in the above process may be retraced by treating the nucleus with a 1 per cent. solution of NaCl, when nucleoli and the nuclear wall become again visible, showing that no true solution has occurred. ~~) NUCLEI OF ANIMAL AND VEGETABLE CELLS, i3a The first contraction of the nucleus is called by Auerbach water-shrinking,' while the subsequent tumescence of nucleoli is styled an internal swelling of the nucleus. Solutions of salts and of acetic acid act upon nuclei in a manner which varies according to their percentage strength. For example, so-called indifferent solutions of Na Cl (‘S—1 per cent.) produce a greater distinctness of nucleoli, intermediary granules and nuclear wall, which is in reality a hardening of the nucleus. Solutions which are more dilute produce a shrinking like that above described ; below this, again, shrinking gives place to internal swelling as the characteristic alteration (Plate XI, fig. 2 B); while solutions containing very little salt indeed produce the final result of the action of water, viz. the general swelling of the nucleus. Above the point of indifference the action of solutions resembles that of solutions below it, the stronger solutions producing an internal swelling of the nucleus like the weaker solutions; while the strongest solutions have an action like that of the indifferent strengths themselves (Plate XI, fig. 1,h,i, k). These facts suggested a method of classification of the various strengths of solution which is illustrated in the following table :* NaCl. | K,Cr,0,.{ A. Upper region of hard- § 35 p.c.| 10 p.c. |60 p.c. nies iit 2 12 ening Upper region of in- § 14 2 ternal swelling 15 15 Lower region of hard- ¢ 1°5 15 1:2 ening 0°08 | 12 0:05 L : fj 0°08 | 1:2 0°05 Shrinking wv alas I evelli or M2 0-008] 0°03 | 0-01 ofnucleus. Seattin pene SEO 0:001| 0:006 |0-001 ae Region of general ¢ 0°001| 0:006 | 0-001 | nucleoli. swelling 0000} 0°000 | 0-000 Sugar in all solutions, from the weakest to those which contain 60 per cent., causes the nuclei to swell up; and ' Cf. Heat-rigor. 2 In this table the figures represent the percentage strengths of the solutions used. The whole range of strengths is divided into regions, each characterised by a mode of action upon nuclei. Thus, of solutions of common salt, all those whose percentage strength ranges from 35 per cent, to 14 per cent. exert that action on nuclei which is termed hardening. 134 JOHN PRIESTLEY. it may be used to counteract the shrinking effect of dilute solutions of acetic acid. The above statements, although made primarily concern- ing the nuclei of carp’s liver, may be understood as applying to nuclei in general, whether still within their cells or not. With respect to nucleoli Auerbach has many new facts. Instead of accepting 1—5 as the limits of numbers in which nucleoli may occur, he regards 0—16 as being more correct ; but he adds that even 100 may be seen in some nuclei. The enucleolar condition, besides occurring as a sign of de- generation in the shrunken and thickened nuclei of horny and some connective tissues, is seen in the early develop- mental stages of various ova (of mammalia, articulata, and vermes), as has been known since the time of Bergmann.! Auerbach has shown that this condition obtains in the batrachian ovum. until cleavage has reduced the surface to a finely granular mass. After a few days, however, certain nuclei may be seen to have an ill-defined central cloudiness, which, growing darker and darker, becomes at last the nucleolus. In some cases the central haziness seems to radiate towards the periphery: hence it is supposed that the nucleolus is formed by the aggregation about the centre of nucleolar substance which is derived either from the periphery of the nucleus itself or from the inmost layer of cell protoplasm. Besides increasing in the above manner, nucleoli may multiply by fission. Of this there can be little doubt, for Auerbach noticed a constant inverse relationship between the size and the number of nucleoli within the nuclei. If two nucleoli were present they were constantly smaller than the nucleolus in uni-nucleolar cases; while if three, five, &c., were to be seen, some were distinctly larger than the rest, as if the unevenness of number were due to the fission of some only of the nucleoli. This division is associated with a move- ment of the new nucleoli through the nuclear ground-sub- stance, the cause of which is not understood (Pl. XI, fig. 2 A). In addition to an increase in the number of nucleoli, the converse operation was observed in certain cases, viz. the fusion of several nucleoli into one.” The functional significance of the increase of nucleoli is not apparent. Inthe epithelium of the stomach and intestine of the frog, Rana temporaria, the multinucleolar condition is constant only during the height of summer and in the au- 1 Miiller’s ‘ Archiv,’ 1841. ? This occurs in the fat-bodies and salivary glands of M. vomitoria. NUCLEI OF ANIMAL AND VEGETABLE CELLS. 185 tumn, giving place to a paucinucleolar state during spring and winter. Again, it cannot be merely a phenomenon of the fission of nuclei, since it is seen in nuclei which are not about to divide (e.g. of red blood-corpuscles), or in those which are about to disappear (eg. of the upper layer of epidermic cells). In the larva of Musca vomitoria the layers of cells which, in the pupa stage, undergo histolysis are just those which are characterised by an extraordinary growth of nu- cleoli. Hence it is, perhaps, not impossible that in many cases the multinucleolar condition may be a vain effort of nature in obedience to tendencies which have long since ceased to be of use, and the aims of which are only here and there fully realised, as in the case of Musca vomitoria. We are now in a position to understand Auerbach’s views respecting the origin of nuclei. According to him they first appear as clear spaces—vacuoles filled with a tenacious fluid mass—possessing no distinct wall. Hach droplet then ac- quires a membrane by differentiation of the inmost layer of the cell-protoplasm, and nucleoli and intermediary granules afterwards make their appearance. Once differentiated the nuclear membrane is an integral part of the nucleus, con- stituting the latter a true vesicle, isolable as a whole by mechanical means. Many facts speak for the identity of nucleolar substance and cell-protoplasm. In optical appearance nucleoli and the substance of young cells agree together. Vacuolation may occur in either, large nucleoli being seldom free from clear spaces. Both are ameboid, the movements in nucleoli having been described by many observers.’ Lastly, nucleoli have, as Auerbach himself shows, that characteristic of vital protoplasm, the power of multiplication by division. In other words, nucleoli have all the capabilities of elementary organisms, and are in truth cytodes. Regarded in this light, they are real daughter cells which have arisen by an endogenous process; and the nucleus is the chamber in which they develope. It is now merely necessary for them to find a way out through the body of the mother-cell in order to begin life as independent beings. That this method of increase has become obsolete in the cases of a majority of cells of higher adult animals, or perhaps only occurs in pathological processes, may be quite true with- out offering any obstacle in the way of such a hypothesis. In the specialisation of function which appears as we ascend 1 Metschnikoff (Virchow, ‘ Archiv,’ Bd. xli). Balbiani (Kefersteio, Jah- resber. f. 1865, in ‘ Zeitsch. f. rat. Med.,’ xxvii. La Valette St. George (Max Sehultze’s ‘ Arch.,’ Bd ii). 136 JOHN PRIESTLEY. the animal series new means of gaining the same end present themselves, while the old vanish or are made use of for other purposes. ‘The phases in the act of propagation of a unicellular organism may be less significant and have a far different outcome in a unit-cell of a more complex creature. In examining the origin and growth of nuclei, Auerbach chose two objects which had already been studied by various previous observers, viz. the ova of Strongylus auricularis and Ascaris nigrovenosa, two species of parasitic nematodes. The protoplasm of these elliptical or elongated ova contains yolk- granules ; and in the specimens which have already acquired a yolk-membrane, a clear fluid—liquor ovi—appears to have been expressed into the space immediately subjacent to it, collecting in larger quantities at the extremities. For the purposes of examination the ova were subjected to pressure caused by the capillary adhesion of the cover-slip and glass slide, a pressure which can be easily regulated by varying the size of the cover-slip and the quantity of fluid beneath it. By this means the obstruction to clear vision due to the granules was to a large extent removed ; while the clear peripheral layer of liquor ovi became less or entirely dis- appeared. The specimens were removed from the body of the nematode and mounted in iodized serum. If observations are commenced a short time after fertilisation the ova are seen to consist merely of protoplasm and of yolk- granules, the germinal vesicle having entirely disappeared. Soon the yolk-granules seem to have withdrawn from the periphery, leaving the latter; quite clear. Thereupon the differentiation of the future yolk-membrane begins, either as a true secretory process, or perhaps, merely as a hardening of the outer layer of protoplasm. ‘The completion of the yolk- membrane is a signal for the redistribution of the yolk-gran- ules in the clear zone,—an act, which is, however, imme- diately followed by a contraction of the whole protoplasmic mass and the expression of the before-mentioned liquor ovi. This terminates the formation of the primary cleavage mass (Pl. XI, fig. 3). At this point commences what Auerbach aptly calls the prelude to the cleavage-drama. At two opposite points of the periphery of such a primary cleavage mass—generally at the poles, and immediately below the yolk-membrane, clear, star-like, ill-defined spaces are seen to develope (Plate XI, fig. 4). These quickly grow larger and rounder, attaining a diameter of 12—15 yp, and their borders become sharper. Meanwhile certain pale, round, sharply defined nucleolar structures appear in their interior, giving them the NUCLEI OF ANIMAL AND VEGETABLE NUCLEI, 137 characters of nuclei which are not as yet provided with a wall. From their places of origin these peripheral pro- nuclei (to use Van Beneden’s expression) move towards the centre with slowly increasing velocity, leaving behind them a clear track as of protoplasm destitute of granules (Plate XI, fig. 5). Duirng this migration, which is probably rather the result of contraction or movements of the surrounding protoplasm than of forces driving the bodies through a resistant mass, the nucleoli exhibit a marked activity, changing their relative positions in various directions and with varying velocities. At the centre the pronuclei touch, at first at a point, but afterwards, owing to mutual com- pression, along a straight line which runs in a direction across the long axis of the egg. The mass thus formed then rotates about its centre, so as to bring the previously trans- verse line of contact longitudinal (Plate XI, fig. 6), while the nucleoli one by one dissolve and vanish. The double structure begins to elongate in the direction of the greater axis (Plate XI, fig. 7), in the course of which elongation the dividing line of contact suddenly disappears along its whole length—a phenomenon which affords a strong proof of the absence of any definite pronuclear membrane. This completes the coalescence of the pronuclei, and the resulting rhomb is the true nucleus of the primary cleavage mass. Cleavage proper now begins. Elongation of the nucleus continues in its former direction and a spindle is produced. Soon the tips of the nucleus exhibit clearer spaces, which radiate out in all directions into the granular protoplasm and are united one with the other by two clear lines, one on each side of the main body of the nucleus (Plate XI, fig. 8). In the mean time the latter has become smaller and more fissure-like, and it finally disappears altogether, leaving the twin-stars with an intermediate band (Plate XI, fig. 9). The granules of the proper cell substance now begin to recede from a portion of the wall and a lateral inversion of the margin occurs (Plate XI, fig. 9). This rapidly runs into a furrow which extends around the circumference of the ovum—an operation in which the yolk-membrane is not involved. The furrow deepens inwards and finally constricts the protoplasmic mass into two. While the furrow is still shallow two vacuoles appear in the band uniting the stars, one on each side of, and close to, the plane of the equator, and commence a movement towards the centres of their respective halves. Meanwhile the stel- late figures in each half shrink, and, as they do so, the newly formed yacuoles increase in size. On reaching their re- 138 JOHN PRIESTLEY. spective destinations the nuclei, which are about 15-18 wu in diameter, acquire nucleoli ; but as yet they remain quite naked, Division into two may now be said to be complete. What remains -of the bistellate figure gradually disappears, the nuclei alone remaining ; and activity is succeeded by repose. The following stages of cleavage are but a repetition of the above-described steps. Division into four, into eight, and into sixteen masses can be traced in detail with com- parative ease ; but beyond this the size of the objects renders observation difficult. The theory by which Auerbach explains these appearances is one of a distribution and recollection of nuclear substance, After the disappearance of the original germinal vesicle and the occurrence of fertilisation, two nuclei appear, formed, as, in Auerbach’s view, all nuclei are formed, viz. by a vacuolation of the cell mass. These slowly move together, touch, fuse into one, and pass into an enucleolar state. The resulting body, whose method of formation did not fail to suggest to Auerbach the resemblance to conjuga- tion, elongates and becomes smaller, expelling its contents from both ends into the surrounding granular protoplasm ; and the disturbance in the arrangement of the granules gives rise to the appearance of two stars. This act is what Auerbach describes as faryolytic (kapvov = nucleus.) The formation of the new nuclei is but a reversal of the above operation. The scattered nuclear substance in each half of the ovum aggregates about a point situated in what will shortly become its proper segment. It is, in reality, a return of the nuclear mass to its former state of aggregation after a temporary sojourn in the interstices of the cell; and Auerbach has therefore designated the process of growth of the young nuclei palingenetic. Another work of importance bearing out the life-history of nuclei is one by Prof. Strasburger, of Jena,’ published in 1875. In it three methods of the increase of cells in the animal and vegetable kingdoms are investigated, viz. free cell- formation, cell-division, and the renewal or rejuvenescence of cells (Vollzellbildung). | Of the first method several observations are recorded and figured, of which that of the embryo-sac of Phaseolus multi- florus is typical. There, in the formation of endosperm- cells, the nuclei first appear as punctiform thickenings, surrounded by a clear zone with tolerably defined borders. 1 «Ueber Zellbildung und Zelltheilung.’ Jena, NUCLEI OF ANIMAL AND VEGETABLE NUCLEI. 139 (Plate XII, fig. 1). Both zone and nucleus increase in mass, but the zones in the denser portions are smaller in relation to the nuclei than in those which are less dense. The protoplasm of the sphere enclosing the nucleus often presents an undoubted radial arrangement (Plate XII, fig. 4). After a time the nucleus becomes vacuolated, the hollows being filled with fluid of a rosy tinge. The outer contour of the sphere soon differentiates into a skin (Hautschicht), while the remaining protoplasm of the sphere becomes reticular as thecell grows (Plate XII, fig.5). Not until neighbouring cells have increased so as to allow of the direct contact of their skins is it possible to demonstrate the presence of a sepa- rating cellulose wall. After this stage has been reached and the cells form a complete tissue, all further increase takes place by cell-division. Of cell-division in the vegetable kingdom the case of certain cells in the embryonal vesicle of Picea vulgaris may be taken in illustration (Plate XII, figs. 7—12). On fertili- sation, the original nucleus of the embryonal vesicle dis- appears and four nuclei simultaneously present themselves at the upper extremity (the lower in the diagrams) of the vesicle. These are separated from one another and from the rest of the vesicle by series or rows of granules (Plate XII, fig. 7). Hereupon a process of true division begins. The border of each nucleus becomes circular and ill-defined; and in the equatorial plane a disc or plate appears composed of a single layer of upright, parallel, rod-like granules. On either side of this nuclear disc (Kernplatte) the substance of the nucleus is marked by striz which converge towards the poles of their respective halves (Plate XII, fig. 9), and terminate there in a somewhat circular spot. The nuclear disc commences to thicken by the longitudinal extension of its rods, the latter being drawn out into thin fibres at their centres, while their ends travel in a direction along the converging strize above mentioned, to meet together at the circular polar spots. Here they form clear ellipsoid structures of small size, which are in reality the nuclei of the future segments (Plate XII, fig. 9). They do not long remain clear, but soon exhibit a striation of granules in the direction of their shorter axis (Plate XII, fig.11). They surround themselves with zones of proto- plasm and increase in size. Meanwhile the nuclear fibres (Kernfaden) present central swellings which afterwards fuse into a second equatorial disc or plate (Plate XII, fig. 10). This plate grows in area, 140 JOHN PRIESTLEY. thus causing a greater divergence of the nuclear fibres con- nected with it, and then splits into two laminee—the skins (Hautschichten) of the coming cells, between which cellulose is developed. Hence this plate is called the cell-plate ~ (Zellplatte). Division is completed beyond the circumference of the cell-plate by a, like differentiation of the protoplasm existing there. The above-mentioned phenomena of division, which constitute a favorable illustration for comparison with division in animal cells, were only studied in alcoholic preparation in the case of Picea. No doubt of their continuity can, however, exist, since in the large-celled freshwater alga, Spirogyra orthospira, Neg., with its suspended nucleus and cavernous or tunnelled cell-masses, the similar phenomena may be followed with great ease in the living plant under the microscope. In the animal kingdom, where division seems to be the only well-authenticated mode of increase of cells, Strasburger examined the ova of Phallusia mamillata. On the disappearance of the original nucleus a peripheral lenticular clear body developes. Of this the central part dips into the substance of the ovum and divides off as the new nucleus, which soon presents a vacuole containing nu- cleoli. The nucleus of the first cleavage mass, therefore, is not here formed by the union of two or more pronuclei (Pl. XII, figs. 13 and 14). With the appearance of the clear disc the cell-protoplasm had become radiate, the rays proceeding from the disc ; now, the radiation centres about the nucleus. The latter soon becomes homogeneous and elongates ; and finally exhibits a striation of a few converging lines with equa- torial enlargements (Pl. XII, fig. 15). Strasburger did not himself succeed in observing an actual splitting of a nuclear disc; but Biitschli, in the cases of Cucullanus elegans and Blatta germanica, has figured this stage unmistakeably (Pl. XII, figs. 17 and18)!. The new nuclei are formed much as in plants. The stellation of the cell-mass is now twofold, proceeding from two centres ; and certain of the rays from both centres intercross about the middle of the ovum. The protoplasm gradually clears up in the equatorial plane, whereupon division commences as an inversion of the edge of the ovum at the periphery of that plane, and is often completed as a suddenact. The rejuvenescence of cells takes up but a few pages of 1 Stasbuger had Biitschli’s permission to insert a few figures from an un- published work by the latter. NUCLEI OF ANIMAL AND VEGETABLE NUCLEI, 141 Strasburger’s work, and consists mainly of observations con- firmatory of previous knowledge. The similarity in detail of the process of cell-division in animals and in plants which his researches so fully display were to Strasburger suggestive of a community of descent of animal and vegetable cells. This suggestion cannot be re- garded as being weakened by the existence in plants of other methods of cell-development ; for, as Strasburger repeatedly insists, on the ground of the occurrence of various inter- mediate conditions, as well as from the functional analogy of similar parts, free cell-formation is not an original mode of increase, but has probably been derived from the method of division by the suppression of certain stages. As to the nature of the nucleus itself, Strasburger differs entirely from Auerbach, in that he regards that organ as a solid structure closely allied to the skin (Hautschicht). The diversity of forms exhibited by nuclei, and the complexity of their functional processes, seem to the former observer quite to exclude an hypothesis according to which they are merely fluid-filled vacuoles. wa In his views of the forces concerned in cell-formation Strasburger is at one with those! who consider them as exert- ing an attraction on the surrounding protoplasm, by means of which the latter is collected about the nucleus. Mere physical surface-tension, such as determines the shape of a portion of liberated protoplasm existing in a fluid medium with which it is hardly miscible, is, therefore, not concerned in the formation of cells. A third recent publication, concerned chiefly with the phenomena of cleavage, is by Dr. Oscar Hertwig.? The cells examined were the ova of the sea-urchin, Toxopneustes lividus, fertilisation being effected by artificially mixing in a watchglass the male and female generative elements. The granulous unripe ovum is enclosed in a porous capsule, and contains a germinal vesicle with definite walls, which in its turn includes a germinal spot of grey, compact substance capable of staining deeply in carmine. In addi- tion the germinal vesicle possesses an interior reticular arrangement of protoplasmic threads. About the time of fertilisation the capsule becomes con- verted into a doubly contoured membrane which has a clear substance immediately subjacent to it. The germinal 1 Pringsheim, Sachs, Fol. ? Beitriige zur Kenntniss der Bildung, Befruchtung und Theilung des thierischen Hies. Morpholog. Jahrbuch 1. 142 JOHN PRIESTLEY. vesicle undergoes a regressive metamorphosis, appearing first at the periphery of the ovum as a lenticular body of punctate mass, and embedding, in addition to the germinal spot, certain larger irregular structures, which do notstain readily in carmine, After a time the germinal spot is no longer visible in the degenerate vesicle ; but, instead, there is seen in the cell- body a clear round structure with no membrane, but with the capability of deeply tinging when treated with carmine. This body, which Hertwig calls the nucleus of the ovum (Hikern), never appears while the germinal spot is yet within the vesicle, from which circumstance, in addition to its deportment towards staining fluids, it is supposed to be the escaped germinal spot. Finally, the vesicle becomes entirely lost to view. After fertilisation has taken place a small clear spot appears at the periphery of the ovum below the yolk-mem- brane, about which as a centre the yolk-granules commence to arrange themselves in radiate lines. Within the lght space a small dark spot is seen having a like reaction to carmine with the nucleus of the ovum. From subsequent phenomena, and from the fact that he occasionally saw a delicate streak extending from the clear spot towards the periphery of the ovum, Hertwig believes the spot to be the head or nucleus of the fertilising spermatozoid, and accord- ingly he has named it the nucleus of the sperm (Spermakern). .The two nuclei (the pronuclei of other observers) approach and meet near the centre of the ovum, where, after the nucleus of the ovum has exhibited certain variations of outline, due possibly to ameboid movement, they fuse together, while the dark central spot of the sperm-nucleus disappears from view. ‘The resulting structure is called the nucleus of the first cleavage mass or first cleavage nucleus. About it a clear substance gradually collects, giving its outlines a faintness or indecision, while itself elongates into a spindle. The collecting clear substance aggregates chiefly about the tips of the spindle, towards which also the radiate strie of the cell-body converge in two sets. There is, in fact, seen the bistellate figure characterised by Auerbach as karyolytic. The stars increase in size, the rays extending to the circumference of the ovum ; and the nucleus between them, suddenly becoming indistinct, seems finally to disappear. Division of the cell-substance now follows in the manner already described, being preceded by ameeboid movement, a circumstance which Auerbach has also mentioned. The young nuclei seem to originate as described by the latter NUCLEI OF ANIMAL AND VEGETABLE CELLS. 148 observer, viz. as vacuoles in the intermediate band of the bistellate figure ; and it may be ascertained that the total mass of the nuclei after division is larger than before. Nothing morecould be made out by examination of freshova. After hardening in ;!; p. ce. solution of perosmic acid for two to five minutes and staining in carmine a further differentiation may be seen, The elongating nucleus is marked by longitu- dinal rods with thickened centres, which together form a me- dian thickened zone. The nucleus, moreover, continues long after the time of its seeming disappearance in the fresh con- dition, extending as a band from the centre of one star to that of the other, and exhibiting at this stage two lateral thickened zones, one midway between the centre and each end. The clear portion of the band situated between them gets longer as cleavage proceeds, and the lateral thickened zones at last become the young nuclei. As the result of his observations, Hertwig concludes that the nucleus is an automatic centre, situated within the cell-body and equipped with active forces. These forces de- termine the changes of form which occur immediately after fertilisation, viz. the amceboid movement and the elongation of the nucleus as well as the formation of the various thickened zones, which must be regargded as due to an aggregation of nuclear substance (Hernsubstanz as dis- tinct from nuclear fluid, Kernsaft) similar to that observed in the growth of nucleoli. By means of other internal forces the nuclei are capable of assimilating to themselves material derived from the general cell-mass—a conclusion rendered necessary by the constant increase in the mass of nuclear matter after division. Other forces, again, acting on the sur- rounding protoplasm, cause a density in the neighbourhood of the nucleus which occasions the radiate arrangement of granules so characteristic of cell-division. With regard to the final act in the latter process, viz. the segmentation of the cell-mass, Hertwig considers the forces at work to be proper to the protoplasm and quite indepen- dent of those acting on the nucleus. In addition to the above complete researches we have before us a preliminary communication by Van Beneden! published in the year 1875. As no extended research in mammalian embryology had appeared since the time of Bischoff, it seemed to Beneden that 1 *Ta Maturation de l’ceuf, la Fécondation et les Premiéres Phases du Développement Embryonnaire des Mammiféres, d’aprés des Recherches faites chez le Lapin.’ Bruxelles. 144 JOHN PRIESTLEY. such a work, to be taken up under the stimulus of present developmental questions, could not fail to be of great use; and he accordingly commenced observations on the ova of rabbits. ‘The present small publication is the forerunner of a larger memoir which is to contain numerous plates. According to Van Beneden’s observations the germinal vesicle of the rabbit’s ovum includes a clear liquid which is traversed by a granulous substance (nucleoplasma) often in the form of a reticulum. In addition to this there exists a nucleolus and a few round bodies called pseudo-nucleoli. As maturity approaches the germinal vesicle moves towards the periphery of the ovum, which has become distinguishable into cortical and medullary portions, and takes on an ellipsoid form applying itself to the pellucid zone. A clear, non- granular portion of the cortex then accumulates about the germinal vesicle, forming what Van Beneden calls la lentille cicatriculaire. Meanwhile the nucleolus fuses with that part of the vesicular membrane which is nearest the edge of the ovum, and the membrane itself, becoming thinner, gradually disappears. While this is taking place the nucleoplasma together with the pseudo-nucleoli melt into a grauular nucleoplasmatic body, and the fluid contents of the germinal vesicle are ab- sorbed into the cicatricular protoplasm.. Simultaneously the changed nucleolus, which, on fusion, had extended into a plate, gathers itself together into an ellipsoidal or lenticular nucleolar body. By the time the vesicular membrane has disappeared the cicatricular protoplasm, after infiltration by the vesicular fluid contents, has become again granular and indistinguishable from the surrounding cortical mass; and the vitellus commences to withdraw from contact with the pellucid zone and to express into the space thus left a clear perivitelline fluid. Into this perivitelline space the nucleo- plasmatic and nucleolar bodies are expelled, forming the Richtungsblaschen (¥ritz Miller) or globules polaires (Robin) of other observers. All distinction of cortex and medulla has by this time vanished, and the yolk, at this stage, truly merits the name Monerula (Haeckel). The ovum is now ready for fertilisation; all the steps hitherto recorded taking place while the ovum is still oyarial. The act of fertilisation is the passage of spermatozoids . through the zona pellucida, which Beneden is now convinced presents no micropyle; once in the perivitelline space, they apply themselves by their heads firmly to the vitelline sphere, without, however, in any case penetrating into its substance, NUCLEL OF ANIMAL AND VEGETABLE CELLS. 145 Fertilisation is, therefore, the fusion of the spermatic sub- stance with the superficial layer of the yolk. After its occurrence the yolk is divisible into three zones, a central finely and regularly granular, an intermediate coarsely and irregularly granular, and a superficial of highly refractile, punctate appearance. In the last-mentioned layer a small, round, homogeneous spot—the peripheral pronucleus —developes, resembling somewhat a vacuole, and not tinting in perosmic acid like the surrounding substance. It increases in size and obtains interior nucleolar structures. At the same time two or three small clear irregular bodies appear in the central zone and fuse into a central pronucleus of somewhat indefinite contour and knobbed or knotted surface (un corps bosselé a sa surface). The pronuclei approach and meet near the centre of the ovum, the protoplasm of ‘which exhibits a radiate arrangement. One of them—the peripheral—continues to grow, while the other diminishes in size ; until finally but one body can be seen, of irregular and indistinct contour and con- taining no nucleoli. The exact mode of formation of this nucleus—whether by fusion of both pronuclei, or by absorp- tion of one by the other—Beneden could not clearly determine. The origin of the peripheral pronucleus from that layer which was immediately touched by the spermatozoids gives colour to an hypothesis of a fusion of male and female elements in the union of the pronuclei; but van Beneden leaves it for the present among the number of mere possibilities. The changes which occur immediately after this stage have not yet been studied by van Beneden as closely as they deserve to be. Nevertheless, he saw the primary cleavage nucleus elongate and a figure develop which he compares with Auerbach’s karyolytic figure ; and he believes, more- over, that he has sufficient ground for stating that the vacuoles which the latter observer describes as occurring in the intermediate band are merely remains of the primary embryonal nucleus, 7. e. the nucleus of the primary cleavage mass, and, as such, are capable of becoming tinted by treat- ment with picrocarminate of ammonia. Immediately after completed cleavage, each segment is of regular spherical shape, and presents a clear spot within, which, under a high power of microscope, is seen to be composed of two distinct parts—a smaller, round part, con- sidered by van Beneden to be derived from the primary embryonal nucleus and called by him the derived pronucleus, and a larger part, with knobbed surface, incompletely enveloping the former, called the produced pronucleus NEW SER,——VOL, XVI. K 146 JOHN PRIESTLEY. (pronucleus engendré). The latter is the remnant of the clear substance which had accumulated at the poles of the spindle-shaped nucleus in the primary cleavage mass, and, being merely differentiated protoplasm of the cell-body, it has no genetic connection with that nucleus. The derived pronucleus increases at the expense of the produced pro- nucleus until the latter is all absorbed, when the former ac- quires nucleoli and constitutes the nucleus of the segment. At this point van Beneden’s research ceases to have special interest for us now; a detailed account of what follows need not, therefore, be given. To be brief, certain differences in size, microchemical reaction, and subsequent development presented by the cleavage segments of the second order, led him to apply the terms ectodermic and endodermic to the larger and smaller masses respectively. Of the masses of subsequent orders, those derived from the ectodermic segment divide more rapidly, and in such a manner as to envelope as a cortex those derived from the endodermic segment. ‘The cortex is provided with an aper- ture, called by Ray Lankester a blastopore, through which is extruded an endodermic stopper ; and the whole structure is designated Metagastrula. In conclusion, van Beneden, after describing the formation of the blastodermic vesicle (germ-vesicle), gives a brief résumé of the results of his researches on the multiplication of the cells of the ectoderm in rabbits—results which are almost identical with those of Strasburger. The elongating nucleus becomes indistinct and irregular in margin. The nucleoli disappear; the contents separate into a clear swe nu- cléaire, indifferent to staining fluids, which collects at the extremities, and a homogeneous essence nuciéaire, tinging deeply in carmine, which collects in the middle as an equatorial plate or disc of highly refringent, ovoid granules. At this stage, it is to be remarked, Beneden never saw the longitudinal striation remarked by Strasburger and others. A finely granular mass now collects about the poles of the nucleus—a mass which is possibly the same as that of the pronucleus engendré spoken of before; and the protoplasm of the cell presents the appearance of stars, indicating clearly an attraction exerted by the nuclear poles. The equatorial plate splits into two discs which move apart, being merely connected by a few threads stretched between their opposing faces. After a time, however, the threads are retracted into the discs. The discs reach the poles and finally become the young nuclei. The intermediate clear tract differentiates at its NUCLEI OF ANIMAL AND VEGETABLE CELLS. 147 centre into a substance which originates a partition between the coming segments, and thus corresponds to Strasburger’s Zellplatie. With the preceding résumés before us, it is not difficult to frame a general account of the earliest stages of cleavage in which all the observers may agree. As the ovum approaches maturity, shortly before or about the period of fertilisation, certain changes of regressive character overtake the germinal vesicle, which result in its total or partial destruction. Under the influence of the fertilising act the ovum again becomes nucleated, the nucleus arising in a fusion of pro- nuclear bodies, one of which may possibly be derived from the fertilismg element. The nucleus at once begins a cycle of changes about the facts of which the unanimity of the vgrious observers leaves us no doubt. It first looses its nuclei and elongates into a spindle, which becomes indistinct and irregular, due probably to the collection about it of a clear or finely granular substance. In the plane of its equator a granular disc or zone of matter more receptive of coloration by staining fluids appears and splits, after a short time, into two parts, each of which begins a movement towards that pole of the spindle which is nearer to it. At the same time, or a little while before, the granular cell-body exhibits in its substance a distinct radiation as of two stellate figures which centre about the tips of the nucleus. The two disc-segments gain at length the extremities of the spindle, and a striation of meridian lines is seen to extend between them. Again, in the equatorial plane, a differentiation of structure occurs, and a second granular plate is formed The disc-segments are the nuclei of the new cleavage-spheres, and the second equatorial plate marks out, and assists in, division of the cell-mass, which has meanwhile been pro- gressing inwards from the equator. With regard to the germinal vesicle, both Auerbach and Strasburger agree with the numerous observers who describe it as disappearing entirely either before or during fertilisation; but neither gives in detail an account of its destruction. Hertwig and van Beneden, on the other hand, have entered into the question fully, and have arrived at very different conclusions. Their descrip- tions of the germinal vesicle in the unripe ova of Toxopneustes lividus and the rabbit agree in all particulars—an agreement which is noteworthy in regard to the granulous reticular threads of protoplasm described as stretching across the vesicle. . 148 JOHN PRIESTLEY, In their accounts of the final dissolution, however, they differ. In Hertwig’s view the whole structure, after having become peripheral, disappears, leaving behind it of its con- tents merely the germinal spot, which remains as the nucleus of the ovum (Eikern). Van Beneden thinks that the walls of the peripherally-seated vesicle fuse with the nucleolus or ger- minal spot, and the body thus formed, together with the re- mains of the reticular threads and pseudo-nucleoli, which constitute another body, are rejected as so-called Richiungs- blischen or corps directeurs. In this connection mention must be made of the state- ments of Balfour'in a monograph on the developmental history of Elasmobranch Fishes, which he is now publishing. He believes'that the vesicle atrophies as the ovum ripens, and that its contents become indistinguishable from the surround- ing protoplasm. The membrane, in the cases where it is thick and resistant, may escape complete absorption and be extruded bodily, as in the instances of osseous and elasmo- branch fishes, birds, &c.; but in the majority of ova it is quite absorbed, although it may appear again in the guise of the Richtungs-Korper. The fate of the germinal vesicle has always excited great interest among embryologists, and three different views exist concerning it. According to the first, which, as we have seen, is shared by Auerbach and Strasburger, the vesicle dis- appears entirely. Purkinje? and von Baer*® both describe it as moving towards the periphery of the ovum while still in the oviduct, and as finally becoming ruptured and dis- appearing ; and in this description they have been followed by many observers in more recent times. According to the second view, the germinal vesicle con- tinues as such, and undergoes division prior to that of the whole cell. This, which is supported in special cases by statements of Joh. Miller, Leydig, Gegenbauer, and Fol,* was maintained by van Beneden? for the whole animal king- 1 Tn the ‘Journal of Anatomy and Physiology,’ January, 1876. 2 Purkinje: ‘Symbol ad ovi avium historiam ante incubationem.’ 3 ©. E. v. Baer: ‘ Untersuch. ii. die Entwicklungsges. der Fische,’ 1835. C. E. v. Baer: ‘ Untersuch. ii. die Entwicklungsges. der Thiere,’ Bd. i. 4 Joh. Miiller: “‘ Ueber die Erzeungng von Schnecken u. Holothurien,” ‘ Archiv f. Anat. ii. Phy.,’? 1852. Leydig: “ Ueber den Bau u. die Sys- tematische Stellung der Raderthiere,” ‘ Zeitsch. f. Wiss. Zool.,’ Bd. vi. Gegenbauer: “Zur Lehre vom Generationswechsel u. der Fortpflanzung bei Medusen u. Polypen,” ‘ Untersuch. iiber Pteropoden u. Heteropoden.’ Fol: “Die Erste Entwicklung des Geryonideneies,’ Jena. ‘ Zeitsch. f. Med. u. Naturwiss.,’ Bd. vil. 5 Van Beneden: ‘ Recherches sur la Composition et la Signification de Peeuf,’ Bruxelles, 1870. NUCLEI OF ANIMAL AND VEGETABLE CELLS. 149 dom. ‘The last-mentioned writer supposed that the germinal vesicle is in no case abolished, but merely rendered for a time invisible by reason of certain changes in the surrounding yolk, and that it reappears before cleavage. According to the third view, of which Hertwig, among the authors whose views we have been examining, is the repre- sentative, the germinal spot is saved in the degeneration of the germinal vesicle and appears as the nucleus of the ovum ready for the changes which fertilisation will bring on. This view of the continuation of the germinal spot is not new. Derbés,! in 1847, described the ovarial ovum as con- sisting of three zones, the germinal spot, the germinal vesicle, and the yolk ; of which the middle one afterwards disappears. Von Baer,” again, in the case of echinoderms, states that the germinal spot remains as a nucleus to the ovum when the germinal vesicle is no longer to be seen. Similar state- ments are also made by Leydig® concerning Piscicola, and by Bischoff‘ in the case of mammals. | With regard to these views it seems impossible, as Hert- wig points out, to doubt the correctness of those who assert a disappearance of the germinal vesicle. ‘To suppose that such a conspicuous object as the germinal vesicle was over- looked by them, becomes extremely difficult when the num- ber and trustworthiness of the observers is considered. Hence it must be concluded that the error lies with those who have believed in its continuance; for it is scarcely possible that both sets of statements can be correct in the particular cases to which they refer; or, in other words, that such a marked difference should exist between, let us say, Hydra on the one hand (where the germinal vesicle disappears) and Meduse (where the vesicle divides according to Gegenbauer) on the other. The possibility of such an error is by no means remote; for the descriptions of the vesicle as a homogeneous non- nucleolated vacuole, which are given by the majority of the observers, are such as to suggest strongly a confusion with the nucleus of the primary cleavage mass, which is acknow- ledged by all to undergo division. The view of a continuance of the germinal spot alone admits of no such objection. Those who, like Auerbach and 1 Derbés: “ Observ. sur le Mécanisme et les Phén. qui accompagnent la Formation de |’Embryon chez l’Oursin comestible,” ‘Ann. des sc. nat. Zoologie,’ 1847, tome viii. 7 C. E. v. Baer: ‘“ Neue Untersuch. ii. die Entwicklung der Thiere,” ‘Frorieps’ Neue Notizen,’ Bd. 39. 3 Leydig: ‘‘ Zur Anatomie von Piscicola Geometrica,” ‘ Zeitsch. f. wiss. Zool., Bd. i. * Bischoff: “‘ Entwicklungsgesch. des Kanincheneies,” 1842. 150 JOHN PRIESTLEY. Strasburger, believe that the germinal vesicle vanishes en- tirely, necessarily suppose the existence of an enuclear stage in the development of the first cleavage mass. But on this point Hertwig thinks there is room for doubt. The difficulty of distinguishing in the fresh condition a small body like the original germinal spot, with refractive powers but slightly different from those of the protoplasmic mass which surrounds it, has been much underrated. Until, therefore, further attempts to differentiate a nucleus or in- terior body by means of staining fluids have led to similar negative results, this criticism must be allowed to have weight. ‘Lhe statements as to the origin of the primary cleavage nucleus are those which present the points of greatest differ- ence among the authors who have recently investigated the matter. Strasburger’s view is, however, singular ; for he con- nects the nucleus in question with the skin or cortical layer of protoplasm of the ovum, by direct descent. This he does without putting forward any strong reasons for such a relationship, and in spite of the fact that the nucleus stains much more deeply than any other part of the ovum when treated with colouring fluids—a circumstance which Hertwig suggests as indicating a distinction of sub- stance. The rest describe a fusion of pronuclei, generally of two, but sometimes of more than two.! ‘To each of these pro- nuclei Auerbach ascribes a similar origin, viz. the vacuola- tion of the cell-body and the collection into the vacuoles of a fluid nuclear matter. Both Hertwig and van Beneden speak of the pronuclei as bodies staining in colouring fluids, and having other nuclear characteristics; and both regard one of the pronuclei (Hertwig’s Spermakern, van Beneden’s pro- nucleus periphérique) as possibly in some way connected with the fertilising element,—indeed, Hertwig believes it to be the head or nucleus of the spermatozoid. In the subsequent history of the nucleus, as has already been said, there is greater agreement. ‘The discrepancies between the account of Auerbach and those of Strasburger, Hertwig, and Van Beneden, or, more correctly speaking, the incompleteness of the former as compared with the latter, is entirely explained by the respective methods of observation adopted; since Hertwig, when he examined recent specimens only, overlooked the interior nuclear changes so fully verified by Strasburger—an oversight which he repaired by a study of 1 Biitschli. NUCLEI OF ANIMAL AND VEGETABLE CELLS. 151 the same objects hardened and carminized. Differences of hypothesis could hardly be avoided. Although it required the perfection of modern instruments and technical methods to furnish a complete history of cell- division, yet certain stages of it had already been dimly or partially seen by earlier observers. Thus Bagge,! Gabriel,’ and Kolliker? have figured appearances similar to those shown in Plate)! (figs. 8—7), which were thought to be due to the division of the central nucleus into two. Moreover, the first-mentioned author describes an elongation of nucleus which resulted in the formation of a finger-biscuit-shaped mass, and ultimately in its division into two parts. Again, the radiate arrangement of the granules in the protoplasmic cell-mass has been from time to time noticed as accompany- ing cleavage, viz. by Derbés* in the case of Echinoderms, and by Krohn®, Kowalewsky,® and Kupffer’ in Ascidians, Of the changes affecting the nucleus during its elongation Auerbach saw nothing but the collection of a clear, stellate mass at its tips, which he thought to be due to the expulsion of its contents, and an appearance as of two movable vacuoles in the intermediate band. Among the rest the chief point of distinction is that Hertwig describes no structure equiva- lent to the cell-plate of Strasburger and Van Beneden, which assists in the partition of the whole cell. It is important to add, however, that Balfour,’ in the fusiform nuclear struc- tures which he describes as taking part in cell-division under certain circumstances, represents an equatorial double row of granules, between which the line of division of the cell undoubtedly passed. The difference of opinion as to the origin of the nuclei of the new segments is very well marked. Auerbach, as we have already seen, regards their origin as palingenetic: the nuclear substance, dispersed for a time throughout the cell- mass, is re-collected into the vacuoles, which will become the young nuclei. Strasburger and van Beneden are equally explicit. According to them, the two halves of the nuclear disc or essence nucléaire move to the extremes of the elon- 1 Bagge, ‘ Diss. inaug. de Evolutione Strongyli, &c.,’ Erlangen, 1841. ? Gabriel, ‘ De Cucullani Elegantis Evolutione,’ Berolini, 1853. 3 Kolliker, ‘ Miller’s Archiv, 1843. 4 Derbés, loc. cit. 5 Krohn, ‘‘ Ueber die Entwick. der Ascidien,’ ‘Archiv f. Anat. u. Phys.,’ 1852. © Kowalewsky, ‘Mém. de l’Acad. imp. de St. Petersburg,’ tome x. 7 Kupffer, “ Die Stammesverwandtschaft zwichen Ascidien u. Wirbel- thieren,” ‘ Archiv f. Mikro. Anat.,’ Bd. vi. 8 FF. M. Balfour, loc. cit.= Jou. Gy Phas, Tan’ 152 NUCLEI OF ANIMAL AND VEGETABLE CELLS. gated parent-structure and become at length the daughter- nuclei, increasing, as van Beneden asserts, at the expense of the substance which has already collected about the tips of the original nucleus. Although Hertwig in his hardened and stained specimens does not certainly speak of the derivation of the young nuclei from the first median thickened zones, there can hardly be a doubt that the lateral thickened zone (which afterwards became the nuclei) correspond entirely to the segments of the nuclear disc described by Biitschli, Strasburger, and Beneden, and resulted from division of the former zone. In a paper in which the names of so many histologists have been mentioned as in this, it would be unfair to omit a reference to the researches of two observers who were among the first to note certain of the new phenomena. Fol! and Flemming” have described the appearance of two stellate figures, which the former calls ‘ centres of attraction,’ in cells which are about to divide. Between these for a time certain structures, supposed by them to be the remnants of the old nucleus, are demonstrable by means of reagents (Fol used acetic acid, Flemming carmine). During and after division of the whole cell the new nuclei develope either in the fusion of several vacuoles in the centre of the star-like figures (Fol), or as new carminizable structures which appear in the same situation (Flemming). It is hard to resist the conclusion that the intermediate structures seen by Fol and Flemming were the same with those described by others as portions of the dividing primary nucleus. The researches which have been considered represent an important step in the progress of histological inquiry —a step which now awaits the confirmation of a more numerous series of observations. 1 Fol: “ Die erste Entwicklung des Geryonideneies,” ‘ Jenaische Zeitsch. f. Med. u. Naturwiss,’ Bd. vii, 1873. ? Flemming : “ Ueber die ersten Entwicklungserscheinungen am Hi der Teichmuschel,” ‘ Archiv. f. Mikros. Anat.,’ Bd. x. ‘Studien in der Ent- wicklungsgesch. der Najaden,” ‘ Sitzb. der K. Acad. d.. Wissensch,’ iii, Abth., Jahrg, 1874, Bd. lxxi. GERMINAL VESICLE AND FIRST EMBRYONIC NUCLEUS. 153 ConrrisuTions to the History of the GeRMINAL VESICLE, and of the First Empryonic Nucteus. By Epovarp VAN BrEneEDEN, Professor in the University of Liége. (With Plate XIII.) I nave had the honour of presenting to the Academy a summary exposition of the results of my researches on the maturation of the ovule, the fecundation and earliest phe- nomena in the embryonic development of the rabbit, up to the moment of the appearance of the primitive streak more especially. I have made known those of my researches which establish the disappearance of the germinal vesicle, the formation of the directive bodies (Richtungsblaschen of Fritz Miller), and the appearance of the first embryonic nucleus. The recent publication of the. researches of Auerbach,} Bitschli,? and Strasburger,? on the formation and division of the nuclei of cells, led me, in the course of my researches on the development of mammalia, to investigate very specially how the first nucleus of the embryo appears and how cells multiply in the embryonic lamine. Long before I became acquainted with these researches, I had observed that the germinal vesicle of the rabbit disappears, independent of fecundation, and that the disappearance of this element is the indication of the complete maturity of the ovum. At the very moment when I was completing the drawing up of my preliminary communication‘ on the maturation of the egg, its fecundation and development in the rabbit, there appeared in Germany an important memoir by Oscar Hertwig® on the formation, fecundation, and division of the egg of an echinoderm belonging to the order Echinida, Toxopneustes lividus. M. Hertwig and I, endeavouring to solve the same problems, but having chosen for our researches animals belonging to different groups, have arrived at 1 Leopold Auerbach: ‘Organologische Studien,’ Breslau, 1874. ? O. Biitschli: “ Vorlaufige Mittheilung iber Untersuchungen betreffend die ersten Entwickelungsvorgange im Befruchteten Ei von Nematoden und Schnecken,” ‘ Zeitsch. fiir Wiss. Zoologie,’ Bd. xxv. O. Biitschli: ‘ Vor- laufige Mittheilung einiger Resultate von Studien iiber die Conjugation der Infusorien und die Zelltheilung,” ‘ Zeitsch. fiir Wiss, Zoologie,’ Bd. xxv. 3 Eduard Strasburger : ‘ Ueber Zellbildung und Zelltheilung,’ Jena, 1875. 4 Edouard van Beneden, “ Le maturation de l’ceuf, la fécondation et les Premiéres Phases du Developement Embryonnaire des Mammiféres d’apres des recherches faites chez le Lapin,” (Communication preliminaire), ‘ Bull. de Acad. Roy. de Belgique,’ 2e sérié, t. xl, 1875. ° Oscar Hertwig, ‘“ Beitrage zur Kenntniss der Bildung, Befruchtung, und Theilung des Thierischen Kies,’ ‘Morphologisches Jahrbuch von C. Gegenbauer.’ 154 EDOUARD VAN BENEDEN. identical conclusions on certain questions of capital im- portance, and at very different results with regard to other, equally fundamental points. Among the problems which he and I have solved very differently are, in the first place, the history of the germinal vesicle ; and secondly, the question of the formation of the first embryonic nucleus. My researches on the ovum of the rabbit have proved to me that no morphological part of the germinal vesicle is found in the yolk at the moment of fecundation. The nucleolus united with the substance which constituted the membrane of the vesicle is eliminated to form one of the “ directive bodies ;” the nucleoplast with the pseudo-nucleoli are thrown off into the perivitelline liquid, to form there the second polar globule. The liquid of the vesicle remains in the yolk, and becomes confounded with the cortical substance of the ovum, which from this moment is no longer distinguishable from the medullary substance. There cannot then be, in the rabbit, any genetic connection between the germinal vesicle or one of its parts, and the embryonic nucleus which appears in the egg after fecundation. I have moreover been able to observe all the phases in the formation of the latter. The first nucleus is developed at the expense of a body formed in the cortical layer of the ovum, which I have called the peripheral pronucleus, and of another body which appears in the centre of the yolk, and which I have called the central pronucleus. It is probable that the first embryonic nucleus is not formed by the fusion of the two pronuclei :—the peripheral pronucleus, at first smaller than the other, en- larges at the expense of the central. The latter becomes attached to the former, and then its substance becomes absorbed by it, as, I think, by a process of endosmosis. According to.the observations of M. Hertwig on the Towxo- pneustes lividus, things take place in a different way. When the germinal vesicle has quitted the centre of the ovum to place itself under the membrane, and has, so to speak, left the yolk, the germinal spot in its turn forsakes the germinal vesicle, penetrates into the yolk, and becomes what the author calls the nucleus of the ovum (Ev/ern); the germinal vesicle then undergoes retrograde metamorphosis ; its membrane becomes dissolved, and the rest is finally absorbed by the yolk. As to the formation of the first nucleus of the embryo, which he calls nucleus of the first cleavage-sphere, or more simply, cleavage nucleus, M. Hertwig has ascertained that it is the product of the copulation of two nuclei. In from five to ten minutes after the sperm has been mixed with the eggs, GERMINAL VESICLE AND FIRST EMBRYONIC NUCLEUS. 155 there appears near the surface of them a small homogeneous body. This element, which appears like a spot or a clear space, is formed by a mass of protoplasm, devoid of granula- tions. This space enlarges slightly and becomes the centre of a radiated figure, a little sun, from which rays, the length of which gradually increases, spread out in every direction. ‘These rays are nothing but lines along which the yolk-granules are arranged. If the clear space is carefully examined, it is seen to contain a small homogeneous cor- puscle. This has nearly the same refractive power as the sur- rounding protoplasm which makes it a little difficult to see. Sometimes Hertwig has seen a delicate line start from this corpuscle, pass to the periphery of the egg, and become con- tinued into a little filament floating in the perivitelline liquid. The clear spot changes its place; it gradually approaches the centre of the yolk and goes to meet the nucleus of the egg, which also reaches the centre of the vitelline sphere. The two bodies finally meet near the middle of the egg. The homogeneous corpuscle contained in the clear space which comes from the periphery appears to be formed of the same substance as the nucleus of the egg, and is, like that, coloured by carmine. Hertwig calls it a small nucleus—measuring 4u, while the nucleus of the egg is not less than 15u. The nucleus of the egg changes its shape, executes amoeboid move- meuts, grows, and is soon surrounded by the clear protoplas- mic substance which comes from the periphery; finally, it becomes fused with the little nucleus and from the fusion of these two nuclei arises the first cleavage-nucleus. While these last modifications are being accomplished, the radiated figure remains ; it even extends, and becomes still more clearly marked ; it involves all the yolk. The nucleus of the egg, and the little peripheral nucleus which becomes attached to it, both surrounded by a layer of transparent protoplasm without granulations, occupy the centre of the stellate figure. Since the clear space near the periphery of the egg appears constantly five or ten minutes after the eggs have been mixed with the spermatic fluid, Hertwig does not hesitate to con- sider the formation of this space as the result of fecundation. The small homogeneous body which he has found to exist there is the head of a spermatozoon; the filament which starts from it is the tail of the same The head of the sperma- tozoon is one of the two nuclei which meet and conjugate ; for this reason Hertwig calls it the spermatic nucleus (Sperma- kern). The first cleavage-nucleus is then the product of the fusion of the nucleus of the egg (Hikern), which is only the original germinal spot, with the spermatic nucleus (Sperma- 156 EDOUARD VAN BENEDEN. kern) which is the head of a spermatozoon. It is the result of the conjugation of two nucler. It is plain that there is in certain respects a remarkable agreement between Hertwig’s observations on Tozopneustes and my researches on the rabbit. We have both ascertained (1.) that there appears near the surface of the egg a clear space, which I have called, in order not to prejudge in any way its significance, the peripheral pronucleus. This peripheral nucleus I have regarded, as at least in part, formed of spermatic substance. (2.) This superficial nucleus penetrates into the yolk and goes to meet another transparent body, of which the character and significance are different from those of the peripheral nucleus; and which I have: called the | central pronucieus in contradistinction to the peripheral pro- nucleus. Hertwig calls it nucleus of the egg, in opposition to his spermatic nucleus. (3.) We have both ascertained that the nucleus of the first cleavage-sphere, called by me first embryonic nucleus, by Hertwig cleavage-nucleus, is developed at the expense of the two nuclear elements after they have joined and become united in the centre of the yolk. We have both supposed that the formation of the first embryonic nucleus results from the union of a male element and a female element; and although I have not applied the term conjugation to the fact which essentially characterises the formation of the first nucleus, still the idea was not the less present in my mind, at least in a hypothetical form. The facts as to which we are in complete disagreement are two in number. (1.) According to Hertwig, the germinal spot does not disappear—it becomes the nucleus of the egg (Eikern). According to my observations there exists no genetic bond between the central pronucleus (xucleus of the egg of Hert- wig) and the germinal vesicle or any of its parts; the central pronucleus which appears after fecundation is an element of new formation. (2.) According to Hertwig, the peripheral nu- cleus—the Spermakern—is the head of a spermatozoon, and the transparent material surrounding it is protoplasm with- out granulations. In my opinion the clear space which appears in the cortical layer of the egg is a nuclear body (the peripheral pronucleus) ; the refractive corpuscles which appear on the spot are nucleolar elements. I propose in the following pages to criticise the opinions expressed by Hert- wig, and to make known the observations which I have had the opportunity of making on the germinal vesicle of the egg of an echinoderm belonging to the order Asterida ; Asteracanthion rubens. GERMINAL VESICLE AND FIRST EMBRYONIC NUCLEUS. 157 1. Does the germinal spot disappear or does it remain so as to become the central pronucleus (EKikern of Hertwig) ? Hertwig expresses the opinion that the germinal vesicle disappears in Toxopneustes lividus ; but he believes that the germinal spot (body or spot of Wagner) remains so as to become his (nucleus of the egg). He has not been able to arrive at a direct proof of this proposition ; his opinion rests upon indirect proofs, of which I will give a summary. (1.) The nucleus of the egg -has the same dimensions (13 yw) as Wagner’s germinal spot; both are corpuscles without any membrane and formed of tolerably firm and homogeneous substance. (2.) Like the substance of Wagner’s spot, the nucleus of the egg is coagulated by osmic acid and coloured more deeply black than the yolk. Both are stained red by carmine. When acted on by acetic acid and chromic acid the two elements undergo a sort of superficial coagulation which produces a finely granular cortical layer, and some spots also granular, in the interior. (3.) We never observe the nucleus of the egg and the germinal spot at the same moment in the same egg. So long as the germinal spot is seen in the germinal vesicle, when the latter has become superficial and lenticular, it is impossible to discover a nucleus in the yolk. From the time when this nucleus exists the germinal vesicle is destitute of its spot. The twoelements are never wanting at the same time. (4.) Hertwig has never succeeded in tracing any alteration in Wagner’s spot, even in those eggs where the germinal vesicle was undergoing retrograde metamorphosis. “ Fur die Annahme, dass der Keimfleck, wie das Keimblaschen zerfallt, lasst sich daher keine directe Beobachtung anfiihren.” He was equally unable to observe a new formation of the nucleus of the egg. (5.) The nucleus of the egg at the moment when it appears is situated near the germinal vesicle; the Wagner’s spot at the moment of its disappearance is adjacent to the olk. ‘ Hertwig has not then observed directly the transformation of Wagner’s spot into the body which he calls the nucleus of the egg ; he has never seen Wagner's body leave the germinal vesicle in order to penetrate into the yolk. Hence some doubt must remain as to the identity of these two elements, what- ever may be the arguments by which he seeks to establish this conclusion. Hertwig himself acknowledges this fact when he writes: ‘‘ Bei Abwagung aller dieser Verhaltnisse 158 EDOUARD VAN BENEDEN. kann zwar die Moglichkeit dass der Keimfleck sich auflést und der Eikern neu entsteht, solange nicht der directe Uebergang des ersteren in den letzteren beobachtet ist, nicht ganz von der Hand gewiesen werden.” We may, moreover, make the following remarks on the arguments adduced by Hertwig: 1. Proofs drawn from the physical and microchemical characters of the nucleus of the egg can have only a second- ary value ; but the characters common to this structure and the spot of Wagner belong to all young nuclei. The only conclusion from these facts is then that the nucleus of the egg is a young nucleus. 2. It does not necessarily follow from our never finding the germinal spot and the nucleus of the egg existing at the same moment that one is a transformation of the other; the dis- appearance of the nucleolus coincides with the appearance of the nucleus of the egg; but does it follow that the one fact is caused by the other? Nevertheless, the coincidence is surprising ; according to my observations on the Mammalia, the formation of the central pronucleus is subsequent to fecundation, and happens, consequently, long after the disappearance of the germinal vesicle. 3. I was much surprised, after reading Hertwig’s memoir, to find no mention there of the “‘ directive bodies ” (Rich- tungs-blaschen) which have been seen in many Echinoderms, and which cannot fail to occur also in Towxopneustes. It would have been interesting to know how these elements are formed in Echinodermata, since, according to my observa- tions on the Rabbit, one of these bodies is nothing else than the germinal spot thrown off into the perivitelline liquid after its previous fusion with the membrane of the germinal vesicle. 4. I cannot pass over so easily as Hertwig does the differences between the appearance presented by the nucleus and the characters of the germinal spot. For my own part, I do not believe that the nature of the media in which these elements are respectively observed can account for the marked differences which, according to Hertwig’s drawings, exist between Wagner’s spot and the nucleus of the egg. This opinion is founded upon observations which will be related further on. 5. Hertwig has never seen any modifications produced in the characters of Wagner’s body during the retrograde meta- morphosis of the germinal vesicle. In this I have been more fortunate, not that I have studied the eggs of Toxo- pneustes, but I made some observations eighteen months ago GERMINAL VESICLE AND FIRST EMBRYONIC NUCLEUS. 159 on the germinal vesicle in an echinoderm of our coasts, Asteracanthion rubens. The result of my researches is that, in this animal, Wagner’s spot disappears in the germinal vesicle before the characters of the latter are altered. It cannot be supposed that in the starfish Wagner’s spot under- goes progressive modifications which bring about its com- plete dissolution in the germinal vesicle, and that this element becomes in Toxopneustes lividus one of the first constituent parts of the first cleavage nucleus. If M. Hertwig has not seen the germinal spot undergo any modifications, we need not conclude, I think, that such modifications do not occur. His own figures, indeed, will I think show that in Tozo- pneustes Wagner’s spot undergoes the same modifications as in the starfish. Before relating my observations on the disappearance of the germinal vesicle in Asteracanthion rubens, I must state shortly in what circumstances, for what reasons, and with what ideas I undertook these researches. When I presented to the Academy my memoir on the composition and signifi- cance of the egg, I was convinced that the germinal vesicle does not disappear, but that it divides, after fecundation, so as to produce the nuclei of the two first cleavage-spheres.! Up to the time when Jobann Miller published his researches on Entoconcha mirabilis* no one had any doubts as to the disappearance of the germinal vesicle. Leuckart, in his article “‘Zeugung,” published in Wagner’s Hand- worterbuch, expressed as follows the conclusion which he felt authorised to draw from a comparison of all the re- searches made on this subject.® “ Fassen wir alle diese Thatsachen zusammen, dann kann es wirklich kaum noch zweifelhaft bleiben dass das Ver- schwinden des Keimblaschens einen Vorgang bezeichnet, der mehr der Bildungsgeschichte des Eies, als der En- twickelungsgeschichte des spateren Embryo zugehért. Das Einzige, was der Aufbau eines neuen Thieres voraussetzt, ist die Anwesenheit eines entwickelungsfahigen Materiales.” “If we put all these facts together, there can indeed hardly remain any doubt that the disappearance of the germinal vesicle indicates a process belonging more to the history of the formation of the egg than to the later history of develop- 1 “ Recherches sur la Composition et la Signification de l’ceuf basées sur Etude de son mode de Formation et des Premiers Phénoménes Embryon- naires,” ‘ Mém. Couronné de l’Acad. Roy. de Belgique,’ T. xxxiv. 2 Joh. Miiller, “Ueber Synapta Digitata und uber die Erzeugung von Schnecken in Holothurien,” Berlin, 1852, p. 17. 3 “Handworterbuch der Physiologie von R, Wagner,” ‘Art. Zeugung von R. Leuckart,’ p. 922. 160 EDOUARD VAN BENEDEN. ment. The only essential condition of the construction of a new animal is the presence of a material capable of de- velopment.” This was at that time the opinion of most, if not of all naturalists. Butso great was the authority of Johann Miller, so complete the confidence which was placed in his observa- tions and conclusions, that the opinion he promulgated on the permanence and division of the germinal vesicle in Ento- concha caused all the affirmative statements made in previous works to be called in question. Shortly afterwards Leydig! announced that he had con- firmed in the Rotifera the results obtained by Miller in his researches on the development of Entoconcha. Mecznikow? observed the division of the germinal vesicle in the Coeci- domyz and Aphides. “ It is hardly possible,” he writes, * to call in question the generality of this fact in insects.” Pagenstecher® made the same observation in the Trichine ; Leuckart* in the Oxyurides; Keferstein® in Leptoplana tre- mellaris ; Gegenbauer in the Medusze, Siphonophora (Corynide, Calycophoride, Physophoride), Pteropoda, Heteropoda, and finally in Sagitta. Hackel’ and Kolliker® confirmed as regards the Siphonophora the statements of Gegenbauer. Finally, I myself observed the division of the germinal vesiclein the transparent and easily observed egg .of Distoma Cignordes.9 Resting as much on my personal observations as on those of the eminent naturalists just quoted, I expressed in a hesitating form the opinion already announced in a manner equally general by Leydig,’° that the disappearance of the germinal vesicle is only apparent, and that the embryonic development begins with the division of 1 Fr. Leydig, “Ueber den Bau und die Systematische Stellung der Raderthiere,” ‘ Zeit. fiir Wiss. Zoologie,’ Bd. vi, p. 102. 2 Mecznikow, “ Embryologische Studien an Insecten,” ‘Zeitsch. fiir Wiss. Zool.,’ Bd. xvi, p. 484. ? Pagenstecher, ‘ Die Trichinen,’ Leipzig, 1865. 4 Leuckart, ‘ Die Menschlichen Parasiten,’ Bd. ii, 2e Lief., p. 322. 5 Keferstein, ‘Beitrage zur Anatomie und Entwickelungsgeschichte einiger Seeplanarien,’ Gottingen, 1868. 6 Gegenbauer, “ Beitrage zur Naheren Kenntniss der Siphonophoren,” «Zeit. fir Wiss. Zool., Bd. v. ‘Zur Lehre vom Generationswechsel bei Medusen und Polypen,’ p. 24. ‘Untersuchungen iiber Pteropoden und Heteropoden,’ Leipzig, 1855. ‘Ueber die Entwickelung der Sagitta,’ Halle, 1856. 7 E. Hackel, ‘Zur Entwickelungsgeschichte der Siphonophoren,’ Utrecht, 869. 8 KOlliker, ‘Die Schwimmpolypen von Messina,’ Leipzig, 1853. 9 Edouard van Beneden, loc cit., p. 30. 0 Leydig, ‘Lehrbuch der Histologie,’ p. 10. GERMINAL VESICLE AND FIRST EMBRYONIC NUCLEUs, 16l this element. I expressed myself in the following words: «En résumé, je considére non comme demontré, mais comme trés-probable, que la vésicule germinative se divise au lieu de disparaitre ” (loc. cit., p. 244). I supported this proposition by various more or less plau- stble considerations, and in particular, I pointed out that the disappearance of the vesicle had never been observed, no direct proof of this disappearance having been given. I should add that the contrary proposition did not, any more than.this, rest upon direct observation; no one of the authors who maintained the permanence of the vesicle rested his opinion upon any other considerations than upon a nega- tive fact. The vesicle is asserted not to disappear because no egg has ever been observed entirely devoid of a nucleus. Some observers have been inclined to trace a genetic bond between the nucleus of the impregnated egg and the ‘ spot’ of Wagner. Leydig! has expressed this opinion so far as regards the eggs of Piscicola; von Baer? with respect to the development of a sea urchin. Bischoff* derives from the germinal spot of the rabbit, not only the directive bodies but also the nucleus which is found in the egg after impregna- tion. But Bischoff confesses that it is in his eyes, a pure hypothesis; he moreover, abandoned it shortly afterwards,* and in his later works, has declared it to be untenable. Quite recently, Fol? has found in the ripe egg of the Meduse a nucleus, with respect to which he feels some doubt, not knowing whether it is the modified germinal vesicle or the spot of Wagner. Finally, Hertwig has just given new credit to this tottering opinion by the publication of his recent memoir, which cannot fail to produce a great effect. A short time after the publication of my memoir on the composition and significance of the egg, two publications appeared in Germany, which are of the highest importance with respect to the question now occupying our attention. Oellacher® established the fact that in the matured ovum of the salmon the germinal vesicle reaches the surface of the . germ and opeus itself into the space which exists at that 1 Leydig, “Zur Anatomie von Piscicola geometrica,” ‘ Zeit. fiir Wiss. Zool.,’ Bd. i. 2 K. E. von Baer, ‘Neue Untersuchungen iiber die Entwickelung der Thiere,” ‘ Froriep’s Neue Notizen,’ Bd. xxxix, p. 38. 3 Bischoff, ‘ Entwickelungsgeschichte des Kanincheneies,’ 1842. 4 Bischoff, ‘ Entwickelungsgeschichte des Hundeies,’ 1845. ® Fol, “Die erste Entwickelung des Geryouideneies,” ‘Jenaische Zeit- schrift,’ Bd. vii, p. 474. ® Oellacher, “ Beitraige zur Geschichte des Keimblaschens im Wirbel- thierei,” ‘ Archiv. fiir Mikrosk. Anat.,’ Bd. viii. VOL. XVI.——NEW SER. L 162 : EDOUARD VAN BENEDEN. period between the yolk and the ovular membrane. The opening enlarges, and the membrane of the vesicle becomes gradually detached from its contents. Finally, the latter are evacuated and the membrane spreads itself out on the surface of the germ. Some observation of the same author on “ The Germinal Vesicle of the Fowl ” furnish a remark- able and complete confirmation of the conclusions at which the illustrious von Baer arrived fifty years ago. Soon afterwards there appeared the beautiful researches of Kleinenberg “On the Anatomy and Development of the Fresh Water Hydra.” Kleinenberg! thus describes the mode of disappearance of the germinal vesicle :—‘‘ About the time when the production of pseudocells is completed the germinal spot undergoes a retrograde metamorphosis. At first it loses its circular outline and becomes irregular and angular; its substance appears coagulated; then it breaks up into little fragments, and these, unless I am mistaken, finally dissolve. So long as the egg was an ameebiform body, the germinal vesicle was situated at the centre of the yolk ; but from the time that the egg begins to become rounded it takes an excentric position, and approaches that pole which is turned towards the surface. It takes its situation near to the surface, and is now covered only with a thin layer of plastic material. Here it also begins to undergo a retrograde metamorphosis which ends in its complete disappearance. Its granular contents become more and more liquefied; a part of these contents escape from the membrane with the result that the latter, which had previously remained uni- formly stretched, becomes collapsed ‘so as to form a tube of generally ovoid shape, the wall of which is thickened and folded at certain points. The part of the contents which has remained in the interior breaks up into isolated shining bodies of rounded or angular form, and of very different dimensions ; amongst them are scattered some drops of a fatty liquid.” Kleinenberg thinks that these bodies are composed of a fatty material, or at least that they consist of the material which results from the transformation of albuminoid sub- stances, and which we observe in many pathological tissues, where this appearance is a sign of fatty degeneration. According to his view the germinal vesicle disappears by fatty degeneration. On one occasion Kleinenberg believed he observed an actual hole in the germinal vesicle. ‘‘ If this is a normal phenomenon,” says Kleinenberg, *‘ it is possible that the contents of the vesicle escape and mix with the sur- ? Kleinenberg, ‘ Hydra,’ Leipzig, 1872, p. 42. GERMINAL VESICLE AND FIRST EMBRYONIC NUCLEUS. 163 rounding plasma.” The question as to what becomes of the membrane has not as yet been resolved; but what is certain, according to Kleinenberg, is that every trace of the germinal vesicle has already disappeared long before the moment when fecundation takes place. Thanks to the observations of Oellacher and Kleinenberg, the disappearance of the germinal vesicle was directly demonstrated ; from that time it was no longer possible to maintain the persistence of the germinal vesicle in all animals, without denying the facts observed. The question then entered into a new phase. Two observers, who must certainly be reckoned amongst the most eminent of the age, had just established the mode in which the germinal vesicle disappears. From that time only two alternatives were further possible; either it must be admitted that the germinal vesicle does not play the same part in all animals; that it disappears in some, and that it persists and under- goes division in others; or else it must be allowed that all the observations made by Miller, Leydig, Gegenbauer, Leuckart, Pagenstecher, Mecznikow, Kolliker, Haeckel, and myself were erroneous, or, at any rate, that the conclusions drawn from the facts observed were but little in conformity with the principles of logic. I believe that the latter of these hypotheses is of the two more probable; the opinion which affirmed the permanence of the germinal vesicle rested, in fact,on negative grounds. It was affirmed that this element does not disappear, because no egg had ever been found which was entirely devoid of all central nucleus; but it does not strictly follow that because no egg had been found entirely deprived of a central nucleus, that therefore the germinal vesicle persists. ‘The doubts which the researches of Oellacher and Kleinenberg had aroused in my mind led me to make fresh researches. In order to make fresh obser- vations on this point it was of importance to choose eggs in which the yolk possessed in the greatest possible degree the properties of transparency and homogeneity, and it was requisite, moreover, that they should be distinguished by the dimensions of the germinal vesicle and the (germinal) spot of Wagner. The ova of the Echinodermata, and in particular those of the Asteracanthion rubens realise these conditions in the highest degree. At the end of April, 1874, I betook myself to Ostend with the object of carrying out the artificial fecundation of these ova. It is not very long ago that we were still in ignorance as to whether the sea starfish are of different sexes. Tiedemann declares that that he has never found the male organs of these 164 EDOUARD VAN BENEDEN, animals.! Nothing, however, is easier than to distinguish the ovaries from the testicles. It is true that the sexual organs have the same form, the same position, and the same volume in the two sexes; when they have reached their complete development the five pairs of sexual glands extend along the entire length of the arms, and they cause a marked elevation of the skin of the side of the back, so that one can recegnise, even from the external appearance, the individuals in which the sexual products have attained maturity. The most superficial microscopic examination suffices to distin- guish the contents of the testicle from those of the ovaries, and one soon learns to recognise the sex, even with the naked eye; the ovaries have a yellowish or very pale brownish tint; the testicles are of a pure milk-white colour. Moreover, the lobules of the ovarian clusters are more rounded and shorter, whilst those of the male sexual gland are elongated and rather of tubular shape. I will first describe the ovarian ovum, such: as it appears when, already free in the cavity of the ovary, it has attained the dimensions of the ripe ovum, but while its germinal vesicle is still lodged in the centre of the yolk. These ova may have either an ellipsoid form, or else they may be pyriform. Their dimensions vary between ‘16 by °13 and °19 by °17 milli- métres. They are composed of a thick and entirely homo- geneous envelope, of a finely granular yolk, and of a germinal vesicle which is situated in the neighbourhood of the centre of the yolk. Membrane.—It is still a question whether there exists around the ovum of the Asteridea a single one or two mem- branes ; nor is anything more certain known as to the nature and signification of these envelopes. If fresh ova which have reached the dimensions of the ripe ovum, and which still have the germinal vesicle central, are examined, we can distin- guish a clear zone around the yolk which has a thickness of from °003 to 004 millimétres. This is quite clear, trans- parent, and homogeneous. It is limited where in contact with the yolk by a sharply defined contour; on the outer aspect, on the contrary, its contour is pale and so slightly marked, that it requires great attention to discern it. The index of refraction of the substance which constitutes this membrane must be very similar to that of water. This substance is very soft; it appears to be a gelatinous, muci- laginous, or albuminoid body; whence the names of ** Gallerthiille, Eiweisschicht, and mucilaginous layer” which ‘Tiedemann, ‘ Anatomie der Rohren-Holothurie, des Pomeranzfarb. Sees- terns,’ &c., 1816, p. 42. GERMINAL VESICLE AND FIRST EMBRYONIC NUCLEUs. 165 have been given to it. More recently Hoffmann! has called it the ‘ vitelline membrane.” 'To see well the characters of this membrane, examination in water must be avoided. The moment it comes in contact with water it swells con- siderably ; one may study it conveniently by placing the eggs directly in 1 per cent. solution of osmic acid, or in a concentrated solution of picric acid. It is not coloured red by the colouring matter of carmine fluids; it is partially dissolved or contracts strongly by a prolonged stay in absolute alcohol. After remaining for a certain length of time in absolute alcohol it is only a very thin membrane which immediately envelops the vitellus, and which at certain points is in immediate contact with it, whilst at other points it is separated from it, thus forming more or less regular undulations. If one examines the transparent layer in ova which have not attained their complete development, it exhibits a radiated striation which is due to the presence of pores in the form of canaliculi of extreme tenuity; it has been long known that the ova of Holothurians present the same character. I have never observed in the ova of the sea starfish the eanal which J. Miller has discovered in several Holothuria, and which has been thought to fulfil the function of a micro- pyle. However, in the pearshaped ova the transparent layer is oftentimes a little thinner at the tail end of the pear. Derbés has found in the ovum of the Echinida, indepen- dent of the mucilaginous zone, which I have just described, a thin membrane which is immediately applied to the sur- face of the vitellus, and which he calls a vitelline membrane. Several authors have affirmed since Derbés, the existence of this second membrane, not only in the Echinida, but also in the Holothurida and Asterida. I have not been able to convince myself of the existence of this membrane in the egg of the Asteracanthion rubens. What is the signification of the transparent layer which exists around the ovum in all the Echinodermata? What name must be given to it? It is not possible to answer these questions in the present state of our knowledge on the forma- tion of the egg of these animals. I have reasons for believ- ing that the membrane is not produced by the egg itself, and that its mode of formation is the same as that of the zona pellucida of mammals. If my opinion is correct, there would be grounds for designating it by the name of Chorion. As its microscopical characters have some resemblance to 1 C. K. Hoffman, “Sur Anatomie des Astérides,” ‘Extrait des Ar- chives Néerlandaises,’ vol. ix. 166 EDOUARD VAN BENEDEN. those of the zona pellucida of Mammalia, we may, at least provisionally, call it by that name which has the advantage of recalling its physical characters without in any way prejudging its eguivalence from a morphological point of view. Vitellus.—The yolk is formed of a clear and transparent fundamental substance (protoplasm) and of feebly refracting vitelline granules held in suspension in the protoplasm. These granules are formed of a substance, the refractive index of which is but little greater than that of the vitelline protoplasm. Hence it results that the transparence of the ovum is scarcely altered by their presence. The absence from the yolk of all vesicular or globular elements and of all highly refractive substances, causes the body of the ovum to be far from the appearance of an emulsion. It is a clear, transparent and finely granular mass. This circumstance renders the eggs eminently favorable to the study of the modifications which the germinal vesicle undergoes in the egg which has reached maturity. It is possible to distinguish in the yolk of the ova of the sea starfish two layers, or if the expression be preferred, two substances ; a cortical layer, the thickness of which is nearly equal to one third of the radius of the yolk, and a medullary mass. The cortical layer is clearer and less granular than the medullary mass; it presents, moreover, a slight radiated striation which appears to me to be wanting in the medullary mass. ‘The limit between the two constituent parts of the vitellus is not marked by a very clear line, the cortical sub- stance of the ovum passes insensibly into the medullary sub- stance. Yet the zone of transition is very narrow. This distinction between the two constituent substances of the ovum of the Asterida has escaped the observation of all those who have studied the sexual products of these Echinoderms. It has not been mentioned up to the present time in any animal of this division, and I am surprised that Hertwig, who has studied the ova of the Toxopneustes with so much care, should not have observed it. Germinal vesicle.—The germinal vesicle is perfectly spheri- cal; it is lodged in the centre of the medullary mass of the egg. It is marked out by a very sharply defined and deep line. It encloses a transparent and perfectly homogeneous liquid. If we examine the germinal vesicle whilst it is in the yolk, we perceive in this liquid a voluminous and very conspicuous germinal spot, and around it a cer- tain number of much smaller globules which are pseudo- nucleoli. The germinal spot, or spot of Wagner, is of large GERMINAL VESICLE AND FIRST EMBRYONIC NUCLEUS, 167 size. It is circular, and its contour is quite regular. Some- times it appears nodulated on its surface ; and lastly, at other times it is angular and altogether irregular. It is formed of a highly refractive aud very brilliant sub- stance which encloses clear vacuoles, whose number and volume vary in different eggs. The vacuoles have already been noticed by Leydig, in the eggs of Holothuria tubulosa: ‘ Der Keimfleck des fertigen Eies ist bedeutend scharfer con- tourirt als das Keimblischen, fast fettartig und zeigt ein oder mehrere Cavitaten.” Hertwig has observed that in the ova of the Toxopneustes Wagner’s spot contains sometimes one, sometimes several vacuoles. In the ova which I am describing, I have not observed amoeboid movements performed by the nucleolus. Long ago de la Valette’ had noted the fact that in the ovum of a Libellula the germinal spot might be seen to change its form and place. Mecznikow’ has seen, not only in the spot of Wagner of several of the lower animals, but also in the salivary cells of the larva of insects, spontaneous movemeuts of the nucleoli. Balbiani? has made a similar observation with regard to the ova of spiders. Alexander Brandt* has observed that in the eggs of Periplaneta orientalis the ger- minal spot affects all sorts of forms, and that these changes, which are really active, must be ascribed to the contractility of the substance of the spot or nucleolus. He has remarked that under the influence of heat, these movements become so active, that it is difficult to draw the successive changes of form which are produced. Auerbach' has recognised changes of form, presenting all the characters of amoeboid movements, in the large nuclei of the embryonic cells of the Muscida and in the germinal spots of the ovum of the pike; Hertwig has seen the same phenomenon occur in the nuclei of the eggs of the frog and also in the germinal spot of Wagner, in the ovum of Pterotrachea. More than four years ago, I observed changes in form, and enlargement and diminution of size, whilst watching Wagner’s spot in the germinal cells of the Polystomum imtegerrimum, and I had remarked that the diminution in volume of these nucleoli corresponded with ' De la Valette : “Uber den Keimfleck und die Deutung der Hitheile,” ‘ Archiv. fiir Mikrosk. Anat.,’ bd. ii, 1866. 2 Mecznikow, ‘ Virchow’s Archiv.,’ bd. xii. se Balbiani (quoted by Auerbach), see Keferstein. ‘Jahresbericht,’ fir 1865. 4 Alexander Brandt, “ Uber retive Formveranderungen des Kernkorper- chens;,” ‘ Archiv. fiir Mikrosk. Anat.,’ bd. ix. ® L. Auerbach, ‘Organologsche Studien,’ heft i, pp. 167 and 168. 168 EDOUARD VAN BENEDEN, the disappearance of the vacuole which is seen in these at certain moments. I observed, also, that the very numerous nucleoli in the young ova of the frog likewise execute move- ments which consist in changes of form. I made these observations a little while after having established the alternate disappearance and reappearance of the nucleoli in the nucleus of the Gregarina gigantea.’ Since then I have several times observed the same fact in the Monocysiis lumbricorum. In the latter species, however, there exists one nucleolus of larger size, which is more voluminous than the rest, and which never disappears, but which changes its form. and in which one sees vacuoles appear and dis- appear: sometimes the nucleolus encloses only a single very extensive vacuole ; a few seconds later it may show a crowd of little ones of all dimensions; at other times there are no longer so many. Other researches and numerous occupa- tions have, however, hindered me from publishing these observatious before now. I have not seen changes of form take place under my eyes in the ova of the Starfish; but I have no doubt whatever that the differences shown to exist in the form of the germinal spot must be attributed to the contractility of the substance of the nucleoli. This con- clusion results from observations made on mature ova of changes which essentially consist in the reduction of the nucleolus into fragments, a reduction which immediately precedes the disappearance of Wagner’s spot. The pseudo-nucleoli, eight to fifteen in number, are cor- puscles of very variable size, composed of a substance which is much less refractive than the nucleolar matter. Some- times they are disseminated throughout the whole extent of the germinal vesicle ; more frequently they are situated in the neighbourhood of the true nucleolus. ‘They have an entirely different composition, and different properties from the latter. It is incorrect, therefore, to say with Hoffmann that there are from one to ten nucleoli in the ovum of the Asteracanthion rubens. Hertwig, in his work, declares himself a partisan of the opinion of Auerbach, who holds that the membrane of the germinal vesicle, and of nuclei in general is produced by the differentiation of a thin layer of protoplasm around a vacuole which is filled with the nuclear substance. Although I do not wish here to go fully into my views on the subject of the 1 Edouard van Beneden, “Sur une nouvelle espece de Grégarine, Désigneé sous le nom de Gregarina gigantea” (On a New Species of Gre- garina, named the Gregarina gigantea), ‘ Bull. Acad. Roy. de Belg.,’ 2nd series, vol. xxviii. See also translation of same in this Journal. GERMINAL VESICLE AND FIRST EMBRYONIC NUCLEUS. 169 constitution of the cell-nucleus in general, and the germinal vesicle in particular, I think it right to say that I do not in the least coincide in this opinion of Auerbach and Hertwig. A young nucleus is formed by a homogeneous substance which I have called the nuclear essence.1_ When this young nucleus enlarges, the nuclear essence becomes united with a substance which is taken up from the protoplasm of the young cell. The nuclear substance which results from this union forms the body of the nucleus. The membrane of the nucleus, as well as the nucleoli, are the unmodified remains of the primitive young nucleus; they are formed exclusively by the nuclear essence. At the moment when a nucleus is about to divide the nucleoli as well as the nuclear membrane dissolve in the nuclear sub- stance. Hence it results that the contour of the nucleus becomes scarcely distinguishable, and that the nucleoli dis- appear. All who have studied the multiplication of cells know how little the nucleus is apparent at the moment when the division of the cell is about to take place. It is this fact which has given rise to the theory according to which all cell- multiplication is preceded by the disappearance of the nuclei. The momentary disappearance and reappearance of the nucleoli in the nucleus of the Gregarina were mentioned by me in 1869. I was at that time unable to give any inter- pretation of these facts ; still, they bear witness to the facility with which the nucleolar substance dissolves in the nuclear substance. The observations of which I shall give an account later on, and which show that the spot of Wagner dissolves in the germinal vesicle with the disappearance of the latter element, form an argument which may be turned to account in support of my opinion. Immediately after the dissolution of the nucleoli and of the membrane in the nuclear substance, a separation is effected between the nuclear essence which goes to form the equatorial zone and the nuclear liquid which is driven back to the poles of the nucleus. The latter, after the division of the zone into two nuclear discs which are to become new nuclei, loses itself in the body of the cell. The vacuoles which appear in so large a number of the nucleoli are, I think, nothing but the result of the momentary union of certain parts of the nucleolar substance with the nuclear liquid. I believe that this way of looking at the constitution of the nucleus is the only one which can explain the physical and 1 Edouard van Beneden, “De la Maturation de Pceuf, de la Féconda- tion et des Premiéres Phénomeénes du Developpement Embryonnaire des Mammiféres,” p. 50. 170 EDOUARP VAN BENEDEN. microcliemical characters of the element and of its various parts—to wit, the nuclear membrane, the body of the nucleus, and the nucleoli. It depends entirely on the phenomena which one knows in relation to the vital manifestations, the de- velopment and the multiplication of nuclei. If one breaks the membrane of the egg of a Starfish so as to allow the contents of the egg to escape into weak osmic acid or picric acid, the germinal vesicle presents a peculiarity which I have been able also to make out in the living egg, after having first observed it in the germinal vesicle isolated and treated with these reagents. In the nucleus of the egg there exists a network with large meshes, formed by a very finely granular substance. It is in this reticulum that the pseudo-nucleoli are found; the germinal spot appears to be the centre from which the reticulated filaments start. The characters of this network vary, moreover, in different ova ; sometimes one even sees in place of the network a small granular collection formed by the substance of the reticulum and the pseudo-nucleoli. ‘This reticulum I have also found in the rabbit, and I have proposed to designate the substance which constitutes it by the name of mnucleo-plasma. The first to describe a network similar to that of the Starfish was W. Flemming.! He found that in the Anodons and the Unios the transparent liquid of the germinal vesicle is traversed by numerous anastomosing filaments. Kleinenberg has mentioned something similar in the germinal vesicle of the fresh-water Hydra; and lastly Hertwig has observed it in the Toxopneustes lividus and in the ova of the mouse. I do not know that anything of a similar nature has been shown to exist in other nuclei of cells. I think, therefore, it will not be without interest to mention here my observations on the constitution of the nucleus of an enormous cell which con- stitutes by itself alone the whole central part of the body (Leibeshéhle of Kolliker) of the Dicyema. The nucleus, which is more or less regularly ellipsoid in shape, presents a thick membrane, beneath which there exists a very close network composed of a finely granular material, whilst the contents of the nucleus (nuclear substance) are perfectly homogeneous and transparent. The body of the nucleus is traversed in certain individuals by a reticulum which makes it resemble a spongy tissue; in other individuals there exists only a bundle of filaments like pseudopodia. I have re- presented one of these nuclei (figs. 20 and 21). By the application of the picrocarminate after previous treat- 1 W. Flemming: “ Studien in der Entwickelungsgeschichte der Naja- den,” ‘ Sitzb. der K. Acad. der Wissensch. zu. Wien.,’ vol. Ixxi. GERMINAL VESICLE AND FIRST EMBRYONIC NUCLEUS. 171 ment with osmic acid, the nuclear substance is stained of a rose colour; the nucleolus and the membrane bright red ; the reticular substance does not stain. This is also the case with the nucleo-plasmic meshwork of the germinal vesicle of the rabbit. But if one opens the ovary of the starfish at the moment of sexual maturity one finds in it not only ova similar to those which I have just now described, and which are essen- tially distinguished by the fact that although they have attained the dimensions of the ripe ovum they still have their germinal vesicle in the centre of the vitellus ; together with these ova one sees others which have not yet attained maturity; others in every respect similar to those of which I have before spoken, but differing from them in the fact that the germinal vesicle has become superficial ; and others which show no longer any trace of the vesicle of Purkinje. These latter, however, are ova which are only exceptionally observed ; generally speaking, all the ovarian ova still possess their germinal vesicle. The ova in which the germinal vesicle has reached the surface of the yolk scarcely differ from those above described ; they have an ellipsoidal or spheroidal form; their zona pellucida, swollen by the seawater, is very thick, and its surface quite irregular. The yolk invariably presents the same characters; the germinal vesicle has retained its spherical form, and all the sharp definition of its contour. It is difficult to make out whether it is in immediate contact with the zona pellucida, or whether it is separated from that membrane by a thin layer of vitelline protoplasm. Inside the vesicle are seen the nucletis and pseudo-nucleoli in the midst of a little cluster of granules. I have never found the nucleo-plasmic network in the germinal vesicle when it has become superficial, whatever the method to which I have had recourse to convince myself of its presence. From the time when the germinal vesicle has taken its peripheral and superficial position, the nucleo-plasma, together with the pseudo-nucleoli, forms a small nucleo-plasmic mass by the side uf the nucleolus. If the ovarian ova of a completely developed ovary are received into a small vesicle containing sea-water, and if a fragment of testicle which has reached maturity be shaken in it for a moment, a certain number of ova are fecundated, and two or two and a half hours after having performed the artificial fecundation, divided ova are found at the bottom of the vessel. If one has taken care to leave only a small number of ova in the vessel, and to renew the water from 172 EDOUARD VAN BENEDEN. time to time the embryonic development advances rapidly, and at the end of from two to three days the ciliated em- bryos swim freely in the water. Artificial fecundation was performed for the first time in an Echinoderm by K. E. Von Baer ; after him several embryologists have had recourse to the same procedure in order to study the development of Echinida, Asterida, and Holothurida. I will mention only Derbés, Krohn, Busch, J. Miller, A. Agassiz, and Selenka. If, some few seconds after having performed artificial fecundation, one places a certain number of ova on an object glass, having taken them with a pipette from the bottom of the vessel in which the sexual products have been mixed, one observes that a crowd of spermatozoa have collected to- gether at the surface of the zona pellucida. They move their tails with such force, that they even succeed in making the ova move. If, in order to study the successive phenomena which take place in it, one chooses an ovum which presents a superficially situated germinal vesicle, and if one watches it continuously, one finds that three quarters of an hour or an hour after the fecundation, the germinal vesicle which was so distinct at the time of the commencement of the obser- vation, has completely disappeared. Subsequently one sees the vitellus undergo in succession the phenomena of retrac- tion; distinctive bodies and polar globules) appear in the peri-vitelline fluid; then the primary vitelline globe breaks into two parts. The ova, therefore, which are provided with a superficial germinal vesicle, are fit and liable, as well as those which no longer showed any traces of it in the ovary, to be fecundated. In order to be sure whether the disappearance of the germinal vesicle is the consequence of fecundation, or whether it occurs independently of the action of the semen, it is sufficient to turn some ovarian eggs into another vessel, taking care to avoid ail mixture with the spermatic fluid. Hf one observes under the conditions above described ova taken from the bottom of the vessel, one can follow and observe the successive phases of the disappear- ance of the vesicle, just as when one follows them in fecun- dated ova. This disappearance then is independent of the action of the spermatozoa. This conclusion might, moreover, be drawn from this fact, that some of the ova lose their germinal vesicle, whilst still in the ovary, and that neverthe- less these ova are perfectly fertile. It was of the highest importance to study the successive phases of the disappearance of the vesicle, in order that we might be able to determine with certainty how it GERMINAL VESICLE AND FIRST EMBRYONIC NUCLEUS. 173 disappeared. Nothing is easier than to make this observa- tion on the ova of the starfish, since the eggs continue to develop on the object-slide, and the changes which they undergo take place under the very eye of the observer. Hight or ten times have I seen the series of modifications which lead to the disappearance of the vesicle of Purkinje evolve themselves under my very eyes. ‘The succession of these phenomena is the same, whether one observes a fecun- dated ovum or follows them in a non-fecundated one. The whole series of these changes is completed in the same time in either case. I believe, however, that fecundation is often the immediate cause of the disappearance of the germinal vesicle, in this sense, that in a ripe ovum the germinal vesicle disappears from the time that the ovum is placed in the presence of the sperm, whilst that element might have still remained for some time if the ovum had not been fecun- dated. ‘his appears to me to be shown by the following observation: if we place in two glasses ova from the same ovary, and fecundate those in the one, carefully preventing the sperm from becoming mixed with the contents of the other, an hour after the fecundation all the mature ova of the first glass will have lost their vesicle, whilst most of those in the second still show it perfectly distinct. If amongst the fecundated ova one be chosen which shows a quite super- ficial germinal vesicle, we may be almost certain to see the germinal vesicle disappear in less than an hour. But it is not so if we select from amongst the non-fecundated ova one which presents similar conditions. Let us now see the series of modifications that are observed. 1. At first the little granular mass which is situated by the side of the nucleolus, and which is composed of nucleoplasma and pseudo-nucleoli, becomes less and less apparent ; soon it becomes impossible to distinguish it; the germinal vesicle now contains only an entirely homogeneous and transparent liquid; with no other granule than the spot of Wagner or nucleolus. 2. The contour of the germinal vesicle becomes paler ; the same is the case with the nucleolus, the substance of which appears to become less and less refractile. At the same time the nucleolar vacuoles become united with a single central vacuole, which appears asa clear spot, circumscribed by an irregular ring formed of a highly refractile substance. The nucleolus becomes very irregular, its surface is now knobbed, and the projections are separated from each other by fissures. The nucleolus resembles a small raspberry-like mass, 174 EDOUARD VAN BENEDEN. 3. The nucleolus (germinal spot) breaks up abruptly into a large number of fragments which continue to diverge from one another and to spread themselves out into the whole mass of the germinal vesicle. ‘These fragments are of unequal size. There is one of them which is notably of larger size than all the others, and which contains the central vacuole of the old germinal spot. This vacuole is now only surrounded by a thin layer of nucleolar substance, which as looked at appears only as a narrow and irregular ring. The formerly homogeneous contents of the germinal vesicle are now granular, and hold in suspension small bodies of variable form and dimensions which are only the fragments of nucleoli. 4, All the nucleolar fragments increase a little in volume and become less and less refractive. Soon they appear as only little clouds with ill-defined contours, forming spots on the uniformly homogeneous ground of the germinal vesicle. They at last completely disappear from view. The frag- ment of the nucleolus which encloses the central vacuole is still visible when all the others have already disappeared. Soon afterwards the last traces of this body hkewise dis- appear. The germinal vesicle, which is still perfectly spherical, is now quite clear and transparent. We can no longer see in it any trace of nucleolus, nor any granule of whatever kind. The contour of the vesicle has become less and less marked, as if the substance of the membrane were dissolved at the same time with the nucléus in the nuclear substance. ‘The progressive diminution of. refrac- tiveness of the nucleolar substance proceeds side by side with the vanishing of the contour of the germinal vesicle. 5. Some seconds after the last traces of Wagner’s spot have disappeared, the membrane of the germinal vesicle be- comes torn, or rather a hole is formed in it. This solution of continuity always appears in that part uf the vesicle which is turned towards the centre of the ovum. ‘The contents of the vesicle immediately flow out through the hole, forming a clear drop outside the vesicle. This little drop has the appearance of a bud or of a hernia. It enlarges very rapidly. At the same time the membrane of the vesicle withers and becomes wrinkled. The germinal vesicle has now withdrawn itself slightly from the surface and has got nearer the centre of the vitellus. It is enveloped on every side by the vitelline pro- toplasm. At one moment it appears as if formed of two clear masses adjacent to each other, which on account of their homogeneous appearance encroach upon the granular ground of the vitellus. One of these is formed by that part of GERMINAL VESICLE AND FIRST EMBRYONIC NUCLEUS. 175 the nuclear substance which is still enclosed in the wrinkled membrane of the germinal vesicle, the other is formed by a drop of nuclear fluid expressed and projecting into the vitellus. 6. The extravasated drop is flattened against the vesicle, in the neighbourhood of the hole by which it made its exit. The nuclear mass then reassumes a more or less rounded form. But in this clear mass one distinguishes an irregular line, which separates the intravesicular from the extravesi- cular portion of the nuclear substance. This line is produced by the membrane which separates the two parts of the nuclear substance which has become very thin. ‘This line at last completely disappears, proving that the membrane is entirely disolved in the nuclear sub- stance. There then no longer remains any portion of the germinal vesicle, except a clear spot, whose ill-defined contours become more and more irregular. The spot becomes smaller and smaller, and ends by disappearing completely. It seems as if the clear and homogeneous matter of the germinal vesicle became granular from the periphery to the centre. This appearance is probably the result of the progressive dissolution of the nuclear sub- tance by the vitelline protoplasm. The successive phenomena which precede the complete disappearance of the germinal vesicle are these: 1. The solution of the nucleoplasmic mass and of the pseudo- nucleoli in the nuclear juice; 2. The breaking up of the germinal spot into fragments, and the progressive solution of these fragments in the nuclear substance; 3. The per- foration of the membrane followed by the partial expulsion of the contents of the nucleus; 4. The complete solution of the membrane in the juice of the germinal vesicle; 5, lastly, the solution of the nuclear substance in the vitelline protoplasm. The modifications which I have shown to exist in the nucleolus, the reduction of the vacuoles into a single vesicle, the changes in the form of that element, and its breaking into fragments cannot be explained unless we admit the contractility of the nucleolar substance. This view is more- over in conformity with the conclusion which one has been able to draw from the ameboid movements which have been seen to be executed by the nucleoli of other cells. The facts which I have just related have not been observed by M. Hertwig in his Towopneustes lividus. M. Hertwig thinks, on the contrary, without however, being able to affirm it from direct observations, that in that 176 EDOUARD VAN BENEDEN. Echinoderm the germinal spot passes out of the germinal vesicle, and becomes free in the vitellus, and there forms the nucleus of the yolk. But if 1 may judge from the figures which he gives of the germinal vesicle in process of retrogressive metamorphosis, I am convinced that the spot of Wagner in that Echinoderm undergoes the same fragmen- tation which I have ‘mentioned in the starfish. I believe that the germinal bodies which M. Hertwig figures in the germinal vesicle (figs. 8, 4,5, and 6) are nothing but the fragments of the nucleolar substance. It may be remarked that in his work M. Hertwig gives no information on the subject of these granules: he does not describe the pheno- mena which relate to the progressive metamorphosis of the vesicle; he confines himself to saying: ‘‘ At the time of maturity of the egg, the germinal vesicle undergoes retro- grade metamorphosis, and is driven by contraction of the protoplasm to the surface of the yolk. Its membrane is dissolved, its contents liquefy, and are finally absorbed by the yolk; the germinal spot, however, appears to remain unchanged, to penetrate into the yolk-mass, and to become the permanent nucleus of the ripe fertilizable egg.’”! Of all the observations published up to the present time relating to the history of the germinal vesicle, the only ones which present any analogy with those which I have made on the starfish are those of Kleinenberg, on the fresh-water Hydra’ Kleinenberg, in fact, recognised that in that animal the germinal spot of the mature ovum undergoes a retrogres- sive metamorphosis ; it presents an irregular and angular contour ; then it breaks up into little fragments, and these at last dissolve. So far as concerns the spot of Wagner, the description of Kleinenberg is as applicable to the Star- fish as to the Hydra. As to the description which he gives of the mode of disappearance of the germinal vesicle, it differs very notably from what I have seen in the Star- fish. But at the end of his description Kleinenberg says: ‘“Once I thought I saw an actual hole in the membrane of the germinal vesicle. If this is a normal phenomenon, it would be possible for the contents of the vesicle to escape and mingle with the surrounding plasma.” I believe that the formation of the hole which Kleinenberg believed he saw is anormal phenomenon, and that it is by the formation of this hole that the contents of the vesicle are partially eliminated, both in the Hydra and in the Starfish; and it is as a result of the rupture of the membrane that the 1 O. Hertwig, loc cit., pages 11 and 12. * Kleinenberg, Hydra, page 24. GERMINAL VESICLE AND FIRST EMBRYONIC NUCLEUS. 177 contents of the germinal vesicle became dissolved in the vitelline protoplasm. This identity of the phenomena which lead to the disappearance of the germinal vesicle in the star- fish on the one hand, and the hydra on the other, is so sig- nificant that it is needless to insist upon its importance. The fact of the dissolution of the nucleolus in the nuclear substance is not an isolated fact. I have long ago observed the alternate disappearance and reappearance of nucleoli in the nucleus of the Gregarinida. Strasburger states that the fusion of the nucleoli in the body of the nucleus constantly precedes the division of the nucleus; and I have noticed the same fact in my researches on the division of the cells in the embryonic layers of the rabbit. I believe that what is true of the nucleoli is true also of the nuclear membrane: the substance which constitutes that membrane may be dissolved in the nuclear substance. When, eighteen months ago, I made the observations of which I have just given an account, I had the intention also of studying the origin of the polar globules and the mode of formation of the first embryonic nucleus. But I was interrupted in my study of the earliest phenomena of development by the opportunity which presented itself one day when I had not any Asterida at my disposal, for inves- tigating the origin of the sexual organs in Zoophytes, in some organisms which came into my hands accidentally. From the time when I entered upon the study of this question in thelHydractinia, I thought I saw the possibility of arriving at a positive solution of it. 1 abandoned for the moment the study of the development of the Starfish ; reckoning on being able to resume it when I wished ; but since that time I have not again had the opportunity of completing my re- searches. I have seen the directive bodies (Richtungsblaschen) formed under my eyes, and I am in a position to affirm that a fresh nucleus appears in the vitellus before the first segmen- tation; but I am not in a position to say either how the directive bodies appear, nor how the first nucleus is formed in the embryo. If I compare the results of my study of the germinal vesicle of the starfish with my observations on the rabbit, I find a complete analogy as to the essential facts; but also differences the importance of which I do not wish to diminish. In the rabbit as in the starfish the germinal vesicle dis- appears in so far as it is a morphological element ; no part formed by the germinal vesicle exists any longer in the ovum at the moment when the first embryonic nucleus is seen to appear; no genetic bond then exist between the germinal VOL. XVI—NEW SER. M 178 EDOUARD VAN BENEDEN. vesicle or one of its parts and the first nucleus of the embryo. But whilst in the Starfish the collection of the nucleo- plasma, the germinal spot and the membrane of the germinal vesicle are dissolved in the nuclear fluid and secondarily in the protoplasm of the yolk—in the mammalia these elements are thrown off into the perivitelline liquid to form directive bodies, and only the contents of the germinal vesicle remain in the yolk. Since in the starfish directive bodies are elimi- nated by the yolk, it is probable that in the Echinodermata, as in Mammalia, these bodies are formed by the nucleoplastic substance on the one hand, and by the nucleolar matter, joined to the substance of the membrane, on the other hand. It must be admitted that this is a pure hypothesis. But, however this may be, it follows from my observations that in the starfish as well as in the rabbit, there is no filiation whatever between the germinal spot and the first embryonic nucleus. II. There is a second point in the observations and views of M. Hertwig, which appears to me irreconcilable with the results of my researches on the rabbit. Is the clear spot which appears im the cortical layer of the yolk protoplasm without granulations, and is the corpuscle which as found there, and which Hertwig regards as the head of a sperma- tozoon a cell-nucleus ? Or is the clear spot a nuclear body and the corpuscle contained in it a nucleolar element having no morpho- logical connection with a spermatozoon ? The spermakern of Hertwig is the head of a spermatozoon enclosed in a clearspot. This is composed of protoplasm without granulations. My peripheral pronucleus, which is certainly homologous with Hertwig’s clear spot and with the peripheral nuclei of Auerbach, Bitschli, and Strasburger is, according to the terms employed in my preliminary commu- nication, ‘‘a small, round homogeneous body, without granu- lations; it has, in fact, the appearance of a vacuole. But when treated by osmic acid, the clear substance of the so- called vacuole becomes darker and of a grey colour, while the substance of the yolk is coloured brown.” It is only later when the pronucleus is already buried in the yolk, that several very highly refractive corpuscles, which would be taken for so many nucleoli, if seen in an ordinary nucleus, appear in its interior. If then our observations agree so far, that we have both seen the body which appears in the yolk, near the surface of the egg with (according to Hertwig) one, or (according to me) several refractive corpuscles, we differ (a) as regards the moment of appearance of the nucleoli-form GERMINAL VESICLE AND FIRST EMBRYONIC NUCLEUS. 179 elements, (0) as regards the number of the elements. More- over the interpretation of the observed facts is different. Hertwig regards the refractive corpuscle as a nucleus (Spermakern), and this nucleus is, according to him, only the head of a spermatozoon; the clear space in which it is seen 1s occupied by protoplasma belonging to the yolk. In my opinion the clear spot is the nucleus; the transparent corpuscles which afterwards appear are nucleolar elements, the mode of formation of which I have never been able to observe, but which, in my opinion, based upon the time of their appearance, their number, and even their characters cannot be heads of spermatozoa. I will make in the first place two observations which are entirely in favour of M. Hertwig. (1.) M. Hertwig has seen the successive phases in the formation of the first cleavage- nucleus produced under his eyes, observing the same egg continue its development on the stage, while my conclu- sions rest on the comparison of a large number of eggs at different stages of development. (2.) M. Hertwig’s view, with respect to the significance of his spermatic nucleus, rests on a positive fact; he has seen a line start from this body and become continued beyond the yolk into a spermatic filament. For my own part I have not been able to account for the formation of my nucleolar elements, and my opinion as to their significance rests upon negative facts. But all those who have made observations of the kind of which I am now speaking, will acknowledge how easy it is to deceive oneself about the meaning of a delicate line ob- served in the yolk of a large egg. It is easy to deceive one- self with respect to the continuity of such a line with a spermatic filament contained in a narrow space between the yolk and the ovular membrane. Hertwig, on the other hand, says himself that he has never seen a spermatozoon bury its head in the yolk, and this head become his spermatic nu- cleus. Hertwig’s direct proof is then still wanting. As to the opinion which Hertwig has expressed with respect to the nuclear nature of his spermatozoon-head, it appears to me that little can be said for it. I see no reason for calling the little body which he regards as the head of a sperma- tozoon, a cell-nucleus. The clear spot in which is contained the homogeneous corpuscle seems to me to present rather the character of a nuclear element. Still the name of cell- nucleus cannot be given either to the clear spot or to the corpuscle contained in it; for the element looking like a nucleus which is formed near the surface of the yolk does 180 EDOUARD VAN BENEDEN. not become the nucleus of the first cleavage-sphere till it has become united to another element having also the ap- pearance of acell-nucleus. It is for this reason that I have spoken of the peripheral body as the peripheral pronucleus, and of the element which is formed in the centre of the yolk as the central pronucleus. If we grant what appears to me hardly to admit of doubt that the small highly refractive bodies of my peripheral pronucleus are homologous with the spermakern of Hertwig, and that in the same way as there are nuclei with a single nucleolus and nuclei with many nucleoli, so also there are pronuclei with a single corpuscle, and others with many corpuscles, then it seems to me that Hertwig’s view of his spermatic nucleus being the head of a spermatozoon has little probability. In Mammalia the nucleolar elements of the peripheral pronucleus have certainly not this significance. (1.) The pronucleus at the moment of its appearance is in the rabbit without any granulations. Now this would be inconceivable on Hertwig’s hypothesis ; the formation of the clear spot is, according to him the consequence of fecunda- tion ; now, fecundation begins with the presence of the sperma- tozoon. The formation of the spot must then always be consecutive to the penetration of the spermatozoon. The spot must accordingly be formed around the head of the spermatozoon, for the head can never penetrate into a pre- formed spot. We ought then never to see a peripheral pro- nucleus without at least one nucleolar corpuscle. (2.) The pronucleus in Mammalia contains several granu- lations. These granulations have neither the appearance nor the dimensions of the heads of spermatozoa; they are spherical or oval globules, the dimensions of which show much variation; the smallest are almost points, and even the largest have not half the size of the heads of spermatozoa. (3.) Auerbach, Biitschli, and Strasburger, observed before Hertwig and before myself, in the Nematodes, the Mollusca, and the Ascidians, sometimes one, sometimes several clear elements appear near the surface of the egg. All three describe these bodies as globules which are at first homo- geneous, and are, at the moment they appear, quite without granulations. According to the observations. of Auerbach, which on this point quite agree with all that I have observed in Mammalia, corpuscles, three to six in number, which Auerbach calls nucleoli, afterwards appear in the homo- geneous liquid of these globules. There is not the slightest analogy between these corpuscles and the Nematode sper- matozoa. GERMINAL VESICLE AND FIRST EMBRYONIC NUCLEUS. 181] (4.) Refractive corpuscles, perfectly identical with those which arise in the peripheral pronucleus were seen also in the central pronucleus. (5.) Corpuscles which perfectly resemble those observed in the peripheral pronucleus are found also in the completely developed nuclei of the cleavage-spheres, and these corpuscles are certainly nucleoli. The opinion expressed by Hertwig that the transparent body which is formed near the surface of the egg is nota nuclear element, but protoplasm without granulations, appears to me untenable, so far as regards Mammalia, for the following reasons: (1.) The transparent substance of the peripheral pro- nucleus does not behave with re-agents like the protoplasm of the yolk, but like the substance which forms the, cleavage- spheres. If the pronucleus be treated with a one-per-cent. solution of osmic acid, it is not coloured brown like the pro- toplasm of the yolk ; on the contrary, it appears on the brown ground of the yolk as a clear well defined spot. The pro- nucleus is faintly coloured pink by picrocarminate applied after osmic acid. By hematoxylin it is coloured violet-blue. (2.) When the peripheral pronucleus has become attached to the central pronucleus, it presents after a time the same appearance as the latter, enlarges, and its outlines become more and more distinct. The nucleolar elements remain for a certain time; finally they disappear and the enlarged peri- pheral nucleus, not one of the refractive corpuscles, becomes the first embryonic nucleus. (3.) My view is in harmony with the opinion expressed by Auerbach, Bitschli, and Strasburger, who have all through regarded the transparent bodies formed at the periphery of the egg as nuclear elements. From all the foregoing considerations I conclude that Hertwig’s view respecting the mode of formation, constitu- tion, and morphological significance of the transparent body which appears near the surface of the egg in Toxopneustes lividus 1s very improbable. I regard this body, which is homologous with the peripheral pronucleus of the rabbit, as being a nuclear element, and I regard the corpuscle con- tained in it which Hertwig calls spermatic nucleus not as the head of a spermatozoon, but on the contrary, as a nucleolar element homologous with those which exist in large numbers in the peripheral pronucleus of Mammalia, Nematodes and Ascidians. I have found the number of these nucleolar elements to be very variable in Mammalia. In the rabbit the number 182 H. R. OCTAVIUS SANKEY. varies from one egg to another. In all the eggs of Cheirop- tera containing two pronuclei which I have examined, each of these elements contained a single nucleolar element.! These variations in the number of the nucleoli have also been pointed out by Auerbach in Nematodes, and by Strasburger in Ascidians. A New Procnss for Examintne the Structure of the Brain. With a review of some points in the HistoLocy of the CureBELLUM. By H. R. Ocravius Sankey. (With Plate XIV.) Tue methods usually adopted in the microscopical exami- nation of the brain have all proved in my hands more or less unsatisfactory. I find that when thin sections of hardened brain are cut and stained, the dye does not sufficiently dif- ferentiate the various structures so as to render their form and arrangement obvious, while in teased preparations the shape of the cells, the connection of their processes, and the fibres of the brain are just as likely to be torn to pieces as to be separated from the substance which surrounds them. The plan which I am about to describe will, I think, be found to overcome, to a certain degree, several of these de- fects. The dye which I employ causes the nuclei to appear black ; the cells and their processes are rendered dark purple, while the rest of the section is of a faint purplish-blue colour, so that the processes and fibres are rendered by these means extremely distinct, and may often be readily traced to dis- tances of a quarter or half an inch, and in some cases even to greater length. For the sake of clearness of description I will divide my process into several stages :— I. The first stage consists in making slices of brain, which should be made from the organ as it is obtained from the post-mortem room, neither hardened nor altered in any way by reagents. The sections should be cut as thin as practi- cable, but slices of one eighth of an inch in thickness will not be found too thick for the subsequent treatment. I find the following a convenient mode of making such sections. A large brush is to be fixed to the back of the left ring-finger by means of two elastic bands; the operator then holding a piece of brain in the left hand, slices it with a large knife kept constantly wetted with spirit by means of the brush. 1 Edouard Van Beneden, ‘ De la Maturation de l’Ciuf.,’ &c., p. 18, NEW PROCESS FOR EXAMINING THE BRAIN STRUCTURE. 183 A large amputating knife answers well for this purpose. The sections should be cut in the direction from the operator. As they are made they should be wiped off the blade with the brush, and allowed to fall into a vessel containing about half a pint of water. The fixing of the brush in the position described will be found to give very important assistance. The piece of brain, as freshly taken from the subject, is difficult to seize and to hold in one position while making the slices as described, and the attempt to hold the brush and the brain at the same time will be found to be a work of difficulty; and as it is desirable to take several sections from the same place, the piece must not be allowed to slip from the operator’s grasp, as it would be very difficult to readjust it. II. The next stage is to subject the sections to the action of the dye. The water in which the sections were placed is to be poured off, until there remains only just enough to cover them. To this there is to be added an equal quantity of a one per cent. solution of the dye, so that in fact the sec- tions will be now in a solution containing half per cent. of the dyeing material. The Dye.—The material that I have found most satisfac- tory in its effects is called aniline blue black. It can be obtained from Messrs. Hopkins and Williams, Cross Street, Hatton Garden. In the dry state this material is a blackish powder, not unlike gunpowder in appearance. It is very soluble in water, to which it imparts an intense purple colour, Its chemical composition is, I believe, not exactly known. I regard the use of this dye as an essential part of the process. I have experimented with some thirty or forty other dyes, but have had no results at all equal to those obtained by aniline black. It is also useful for sections made after the usual process of hardening, &c. The sections should be allowed to remain in the dye for about twelve hours; twenty-four or even thirty-six hours’ immersion does not, however, often injure them. ‘The next step is to pour off the dye, and add clear water until all the colouring fluid has been washed away. ‘The stained sections may be poured, with the water they are in, into a large shal- low basin, and each slice floated into the middle of a glass slide, and removed carefully from the water and allowed to drain. As each section in this stage should be marked, I can recommend for the purpose a very conyenient slide, invented by Mr. Hicks, and described by him in ‘ Quart. Journ. Microsc. Science.’ In this slide the portion on which the label is usually gummed is of ground-glass, and will 184 H. R. OCTAVIUS SANKEY. receive a pencil-mark without danger of its being oblite- rated. III. The next step in the process is to dry the stained slices upon the glasses. ‘They should be placed in an airy and dry situation, but not,as a rule, subjected to heat, but to a current of air. In certain states of the atmosphere, however, they may require the assistance of a very moderate degree of artificial heat. Or should the sections be very thick, a slight amount of dry heat is required so as to antici- pate any liability to putrefaction before desiccation has had time to occur. When the sections are dry it will be found that they are firmly adherent to the glass, and that they, as a rule, show no tendency to crack, except just at the edges. Should it be desirable to obtain a good view of the edge of any section, a slice of unstained brain may be placed over the stained piece so as to overlap the edge, which virtually removes the edge it is desired to see from its external posi- tion, and renders it far less liable to crack. _ IV. When the sections are properly dried they will be found to be of the consistence of somewhat dry cheese, and rather uneven in thickness. The next step is to bring them to a condition suitable for microscopical examination, by re- ducing them to the necessary thickness. The sections, before they are reduced, may be described as consisting of three layers—an upper, middle, and lower, the latter being in con- tact with the glass. It is obvious that the upper and the lower, which were the parts in immediate contact with the dye, will be more deeply stained than the central layer, which in fact remains white. In reducing the thickness of the sec- tions by paring, it is clear that if the upper layer is removed but one of the deeply stained layers will remain. The object to be gained by the paring is to reduce the section to the lower stained layer only. The unstained or intermediate layer may be removed as completely as practicable without endangering the deeper blue layer; but if any unstained substance be left it is of not much consequence, as the pro- cess of clearing renders it invisible. The operation of paring can readily be performed with a razor, but it can be much more efficiently and quickly accomplished by means of an instrument that I have devised for the purpose. This in- strument is a kind of plane, and is a modification of an ordinary carpenter’s two-inch skew rabbet-plane, but with which I operate in the reverse position, that is, with the cutting edge uppermost. The modification consists in screw- ing to the sides of the plane two slips of iron, having per- ectly true and level edges, which are also adjustable by set NEW PROCESS FOR EXAMINING THE BRAIN STRUCTURE. 185 screws, and can be raised just above the level of the cutting blade of the plane. They thus forms guides, one on either side of the knife, by means of which the thickness of the section is regulated. In using the plane, each end of the glass side is pressed tightly down on the iron on either side, so that the slide bridges over the interval between the guides. The dried slice of brain being on its under surface, by passing the.slide along the guides from front to back all the project- ing portions of the tissue come in contact with the knife, and are pared off until the whole is reduced to the requisite thickness, this thickness being regulated at will by raising or depressing the side guides. V. The preparation is now to be cleared by means of dammar varnish or Canada balsam. ‘The intervention of oil of cloves is not required. ‘The section may be examined at once with a low power, and, if found satisfactory, a cover glass should be placed over it. Sections prepared as above may be made of almost any size. I have some which cover an extent of glass equal to three or four square inches. By employing a very long knife sec- tions extending from the medulla oblongata through the pons varolii into the crus cerebri, for instance, can be readily prepared ; and in many of my finished preparations fibres can be traced to extraordinary distances. I have now treated the brains of a considerable number of animals by this method, and have also made a few prepara- tions of the spinal cord and sympathetic and spinal ganglia of the larger domestic animals, and always with more or less satisfactory results. I find that the larger and older the animal is, the better are the preparations which are obtained. In the smaller animals the brain is softer and much more difficult to cut, and it is apt to break down in the dye into a ropy tenacious mass, showing no trace of the outline it pre- viously presented. The brains of young and small animals become also more brittle when dried, and are liable to crack or contract in the process of drying. In spite of these diffi- culties, however, with care sections of even fetal brains may be prepared. Of all brains, that of the human adult is the one for which my process is most suited, and especially, it has seemed to me, in cases in which the temperature has been elevated for some time previous to death. The process is inapplicable to brains which have been hardened by any reagent, as they will crack or become brittle in the drying ; nor is it suitable for brains which have been in any fluid cap- able of crystallization ; for crystals form during the desicca- tion and spoil the preparations. If sections cannot be made 186 H. RK. OCTAVIUS SANKEY. as soon as the brain is removed from the skull, or at latest on the day following, the best fluid to place it in to preserve it is a strong solution of ammonium acetate of a sp. gr. of about 1040. This solution does not harden the brain to any marked degree, nor does it crystallize out during desiccation, nor by its deliquescent properties does it prevent the brain from drying. ‘The brain, indeed, may be preserved in this solution for many weeks in a suitable state for the process. It should, however, be cut into pieces not larger than wal- nuts before being placed in the fluid, and should be soaked in water for several hours before the sections are made, other- wise it will be found that the brain has for some reason become much more adhesive, and sections when made cannot be removed from the knife without laceration. Part II. By means of the process of which I have now given the principal details, I have carefully examined many brains, with the view of testing various views which have been published at different times in connection with the Histology of the Brain and Nervous Centres. I propose here to give some of the results which my study has afforded me, and in the present communication I intend to confine my observa- tions to the structure of the cerebellum ; since my mode of investigation enables me to confirm some observations made originally by Dr. Obersteiner' concerning it, which, so far as I am aware, have not yet met with substantiation, and which do not even appear to be accepted, if I may judge from the fact, that though Dr. Meynert, writing in ‘Stricker’s Com- parative and Human Histology,’ alludes to several points mentioned in the paper quoted, yet omits all mention of those points to which I am about to allude. Dr. Obersteiner, in speaking of the pure grey or outer layer of the cerebellum, says :—“ The neuroglia of this layer is scattered over with round and elongated nuclei of a diameter of 0°007 mil. The latter scarcely present any cell around them, and probably belong to the connective tissue. A clear border surrounds the round nuclei, which is either round or angularly drawn out. It is these nerve-cells with processes which unite themselves to the end branches of Purkinje’s cells.” I find that if in a section made perpen- dicularly to the surface of the cerebellum, and at right angles to the lamella, one of Purkinje’s cells be brought into view under a magnifying power of about 600 diameters and 1 “ Beitrage zur Kenntniss vom Bau der Kleinhirnrinde,” ‘Sitzungsbericht d. K. K. Acad. der Wissenschaft,’ Band. lx, Heft i. Wien. NEW PROCESS FOR EXAMINING THE BRAIN STRUCTURE. 187 its peripheral process traced out, the following appearances will be observed. ‘There is a slight variation in the detail, according as the cell is situated at the top or side of the lamelle or at the bottom of the sulci, but the general arrange- ment is as follows:—(Fig. 1.) Each cell gives off in a direction toward the surface one primary trunk. ‘This soon divides into two secondary trunks which pass to the right and left in a direction parallel to the surface, and in their progress send off at right angles numerous branches, which also are directed towards the surface or towards the pia mater ; and, finally, the secondary branches, having gradu- ally become smaller and smaller in their course, terminate themselves by turning, like the smaller processes, towards the surface. At each point of division there is to be observed a small triangular swelling, which might aptly be compared to a small blot, such as might occur when a line of ink is drawn across another which is still wet. Various opinions have been broached as to the nature of this swelling. Dr. Mey- nert seems to have considered it due to a loose hyaline sheath which he describes as investing the cells, and as prolonged a short distance on the larger processes. Dr. Obersteiner, on the other hand, appears to think it due to the passage from one branch to another of fibres which do not pass back to the cells of Purkinje, but appear, as it were, to form a direct outer communication between the branches. I have been able to see distinctly this triangular enlargement at the union of some of the finest branches, such as are described below as connected with small cells in the pure grey layer, and which cannot, I think, be bundles of fibres, but must be single fibres. If such is the case, the enlargement cannot be produced in the mode supposed by Dr. Obersteiner. On the other hand, if this appearance is due to a sheath, then such hyaline investment must extend to the very finest fibres, and cannot cease at the point men- tioned by Dr. Meynert. The fibres running outwards may be seen to divide and subdivide into very fine branches, dividing, not three or four times only, as might be supposed from the drawings given in the text-books or by Dr. Obersteiner, but much more fre- quently. Ihave observed one branch to divide more than twenty-five times. The more distant branches are given off at very acute angles, and the fibres, after a very short distance, run a nearly or quite parallel course. The division is not strictly dichotomous ; for in many instances small branches are given 188 H, R, OCTAVIUS SANKEY. off from the sides of larger ones, though more usually two branches of equal size result from the division of one. Dichotomous division prevails more as the fibres become smaller. The branches, having thus become finer and finer, are ultimately lost to view, or terminate in the remarkable manner described by Dr. Obersteiner (op. cit.). In my preparations the union of the fibres with the protoplasm of the cells, in the pure grey layer, is plain and unmistakable, and resembles very closely the appearance described by Dr. Obersteiner, though some differences may be noted. I think it is probable that these are due to the mode of preparation of the object, and I believe that my method shows the structure to greater advantage than did the process at his command. Certainly I am able to see the structure, and the artist has been able to figure it much more decisively than Dr. Obersteiner has ventured to show it. (Fig. 2.) The fine fibres, on approaching the cells in which they are about to end, are seen in my preparation to enlarge and assume the character of the protoplasm of the cell, into which they pass without line of demarcation. Dr. Ober- steiner, however, in his drawing, depicts the cell as a round ball, into which, in one sketch, a fine filament is seen to pass, and only into its proximity in his other sketch. Probably this appearance, as given by him, was due to the shrinking of the tissue in the process of hardening, for most of the cells seen in my preparations are not round, but, as Dr. Meynert describes them, triangular or pastille-shaped. Again, there is no clear space around the cell in my preparations, which in Dr. Obersteiner’s drawing appears so wide as to be nearly equal to half of the diameter of the cell itself. These small cells are not only seen to be provided with a process of union connecting them to the fibres in the manner described, but also to give off others, three or four in number, which are short, but which divide and subdivide until they are finally lost in the reticulo-molecular ground substance of the part. The “processes of union,’’ which are connected with Purkinje’s cells are usually directed toward that layer of cells, but this is not an invariable arrangement. My preparations corroborate Dr. Obersteiner’s opinion that the terminal branches of the processes are directly united to cells, and that also side twigs are given off in a more or less rectangular direction, which also join cells, each fibre making for its cell akindof stalk. Ihave, however, more frequently observed the former kind of connection than the latter. With regard to the questions—are the processes invariably connected with cells? and are all the cells of similar character NEW PROCESS FOR EXAMINING THE BRAIN STRUCTURE. 189 and appearance connected with fibres? I can give no definite answer. I have observed that the cells in any given space that are unconnected with these processes are about equal in number to the terminal fibres, which appear to end in- definitely or appear unconnected with the cells, though there may be a slight excess in number on the side of the fibres. The more intensely the preparation is stained, the greater the number of those connections is there to be seen. I am there- fore, inclined to believe that it is owing to the defective method of examination that we are still unable to assert that the fibres uniformly terminate in cells. With regard to the statement of Dr. Obersteiner that there are nuclei of two kinds in the pure grey layer of Kolliker, I may state that I believe such to be thecase. Firstly, there are those cells which are surrounded by a protoplasmic substance, and which are the most numerous; these are the cells which are connected with the fibres from the cells of Purkinje ; and, secondly, there are undoubtedly others around which no protoplasm is to be seen; these are either round or more frequently slightly elongated, and both Dr. Obersteiner and Dr. Meynert regard them as free nuclei belonging to the neuroglia. In my preparation small arteries and veins may be detected, but no capillaries, or, if any, very few, and these but faintly indicated ; but by taking a freshly stained portion of cerebellum and teasing it, the nuclei of the capillaries may be readily detected, and will be found to resemble in appear- ance, shape, and size the second kind of nuclei referred to above. Iam therefore of opinion that this kind of nucleus belongs to the capillaries, and is not an element of the neuroglia. That such is the case, I think, derives support from the fact that their numbers are about what such an origin would readily account for. The annexed chromo-lithographs represent with great accuracy the appearances seen in the preparations, both with regard to form and to colour. Fig 1 is magnified 140 dia- meters. It includes a part of every layer of the cortex of the human cerebellum. Fig 2 is a highly magnified view (950 diam.) of a part of the pure grey layer of the cerebellum taken at a spot rather nearer to the pia mater than to the layer of Purkinje’s cells. This figure is, to a certain degree, diagrammatic. ‘The most central process, with its attached cell, which really occupied a position below and to the left of the structures represented, has been inserted in place of a fibre which did not show any- thing noteworthy. There was in the preparation, in the left upper corner, a small rent; this has not been represented, a 190 : DR. JAMES FOULIS. group of six cells and nuclei taken from another part of the preparation having been inserted instead. Of the four nuclei in this group, three are elongated and appear to belong to the vessels ; the most internal one, on the contrary, is round, and resembles those contained in the cells. Some clue to the existence of these free rounded nuclei may perhaps be afforded by the appearance seen at another part of the plate. In the right upper corner is a cell of which the protoplasm is so pale as scarcely to be visible, while nearer the middle of the lithograph is a nucleus nearly extruded from its cell. In the lower part of the plate are to be seen three or four unequi- vocal instances of union of fibres derived from Purkinje’s cells with the protoplasm of small cells in the outermost layer of the adult cerebellar cortex. Such connections are, of course, faithfully copied from the preparation. On the Devetopment of the Ova and Structure of the Ovary ix Man and other Mammatta. By James Fovutis, M.D, Ed. (With Plates XVI, XVII, XVIII.) Contents.—Introduction. The Ovary of the Kitten. The Human Ovary: (a) Nature of the germ epithelium; (4) The relation of the germ epithelium to the peritoneal epithelium; (c) The manner of inclusion of the primordial ova and germ epithelial corpuscles in the stroma of the ovary ; (d) Development of the egg clusters; (e) The development of the membrana granulosa. General observations on the development of the membrana granulosa in adult ovaries. General conclusions. In the month of August, 1872, Professor Turner suggested as a subject of investigation, the structure of the ovary and the development of the ova, more especially with reference to the recently published observations of Waldeyer. On the 21st December, 1874, Professsor Turner read before the Royal Society of Edinburgh a paper setting forth the result of my observations ;! but in the month of April of the same year, in my graduation thesis for the degree of M.D.,? I had already demonstrated that the eggs and follicular epithelial cells have a different origin. In the year 1870, W. Waldeyer published his observations on the development of the ovary and ova.® In this beautiful 1 «On the Development of the Ova and Structure of the Ovary in Man and other Mammalia,” ‘ Transactions of the Royal Society of Edinburgh,’ vol. xxvii, 1875. * Contributions to the Normal and Pathological Anatomy of the Ovary and Parovarium,’ April, 1874. 3 ‘Hierstock und Hi,’ Leipzig, 1870. THE OVA AND OVARY IN MAN AND OTHER MAMMaLIA, 191 memoir we find an interesting history of the views of earlier observers on the origin of the ova and Graafian follicles. Waldeyer refers to the work of Valentin,! who, in 1838, first demonstrated the branched and tubular glandular structure of the ovary, and he makes special mention of the observations of Pfliiger,? who first described the ova in mammals as arising out of the epithelium lining tubular glands in the ovary, and the formation of Graafian follicles from such tubular structures. Formerly the ova and follicles were considered to be derived from the cells of the stroma of the ovary. The observations of Pfliger led to the publication of numerous works on the origin and develop- ment of the ova, and the tubular formations were soon dis- covered in the ovary of the human subject, first by Spiegel- berg. In Waldeyer’s observations, however, we have the most recent addition to our knowledge of the development of the ova and the formation of the Graafian follicles, and the following quotations from his work will place before us, briefly, the conclusions he has drawn from them: In summing up, Waldeyer thus remarks : “ As the chief result of my investigations, it must be stated that both the egg and the follicular epithelial cells are _ derived directly from the germ epithelium. There is a reciprocal growth of vascular connective tissue and germ epithelial cells, in consequence of which large and small masses of the latter become imbedded more and more in the stroma of the ovary. The imbedded cells present a variety. Some of them, by simple increase in size, grow into ova, viz. primordial ova, while others keep to their original size, and by numerous divisions, at least as it appears to me, produce still smaller cells, viz. the follicular epithelial cells. A genetical distinction between primordial ova and follicular epithelial cells has consequently no existence. The germ epithelium is the common source of both.” In describing the ovary of a newly born child, Waldeyer thus states, in reference to its tubular structure: ** One sees long branching formations in the form of tubes, anastomosing with each other, as Valentin first described, and lying separate from each other at considerable distances. They pass upwards opening with narrow mouths into the epithelium, and appear as direct tubular gland-like processes of it. * At the time at which the tubes described by Pfliger 1“ QUeber die Entwickelung der Follikel in dem Hierstock der Saiige- thiere,” ‘J. Miiller’s Archiv. fiir Anat. u. Physiol.,’ 1838. 2 «Die Hierstécke der Saiigethiere und des Menschen,’ Leipzig, 1863, 192 DR, JAMES FOULIS. exist, that is, as far as I can find, from the ninth month till a short time after birth, they present the structure ascribed to them by Pfliiger, with the exception already mentioned, that there is as little of membrana propria in them as there is in the primary follicles. In the tubes, and mostly in the middle of them, as Pfluger described, we meet with egg cells distinguished by their size and form, often immediately con- catenated one behind the other. Whether in the tubes new egg cells are formed, I cannot decide; but I think it likely, because here, as well as on the surface epithelium, some epithelial cells may develop into egg cells. Division of the egg cells in the tubes Pfliiger seems to have observed, but I have not seen it in fresh specimens. “‘ Follicles are formed from the tubes as well as from the egg compartments, directly through the growth of interstitial issue. At the lower end of the tube, as may be well explained from the want of a membrana propria, interstitial tissue grows into the tubes and incloses the individual egg cells along with a portion of the not fully developed epithelial cells which surround them, and in this way primary follicles are produced. ‘“‘' The tubes of Valentin and Pfluger can lay claim only to a secondary importance, and are not essential for the egg and follicle formation; the greater part of the follicles have undoubtedly an earlier existence, long before these tubes are formed.” My observations have been made on the ovaries of calves, kittens, cats, puppies, rabbits, human foetus, &c., and in the present paper I have described what I have seen in the ovaries of young kittens and of the human foetus with the object in particular of demonstrating that, whereas the eggs are derived from the germ epithelium, the nutrient cells of the ovum, or the follicular epithelial cells, are derived from the cells of the stroma of the ovary. Tuer Ovary OF THE KITTEN. I shall first describe the structure of the ovary and the development of the ova in a kitten of two or three weeks; and I may here remark, that I know of no animal better suited than the kitten to show the relation of the germ epithelium to the stroma of the ovary. In a thin vertical transverse section of a two weeks’ old kitten’s ovary we may distinguish Ist, The germ epithelium. 2nd, The zone of egg clusters. 3rd, The fibrovascular stroma. THE OVA AND OVARY IN MAN AND OTHER MAMMALIA. 193 The germ epithelium consists of distinct copuscles ar- ranged in a layer which passes round the ovary from one lateral border to the other, and becomes continuous with the peritoneal epithelium which covers the stalk or peduncle. The corpuscles of the germ epithelium consist of clearly defined nuclei, all of which have a thin investing film of protoplasm. In some instances this protoplasm is very clearly made out, and is in considerable quantity, but in other cases it can scarcely be seen, even under very high powers of the microscope. As in the ovary of the feetal calf, there is a constant proliferation of the germ corpuscles by a process of fission. They are somewhat granular, and vary considerably in size ; some are oval, but the greater number are spherical. In the ovary of a kitten of four weeks the corpuscles of the germ epithelium appear columnar in form and compressed laterally. In the round or spherical cor- puscles, which are generally larger than the others, the nucleus is extremely well marked, and frequently possesses a bright nucleolus The spherical form of the larger cor- puscles appears to be produced by the swelling out of the nucleus. In the two weeks’ old kitten the corpuscles of the Ovarian germ epithelium are several deep, and the layer itself is of irregular thickness. As we examine the layer of germ epithelium as it passes round the ovary, we are at once struck by the fact that the corpuscles present a great variation in size and degree of development. Here and there we see large spherical nuclei, having round them a thin investing layer of protoplasm, while in other situations certain individual corpuscles stand forth prominently among their neighbours, and are conspicuous by their size and the size of their nuclei. In these latter the protoplasm surrounding the nuclei is in the form of a thick layer. Between these largest corpuscles and the ordinary small ones every variety of size and form is to be met with. The largest corpuscles, which present so much protoplasm round the nucleus, are evidently individuals which have reached an advanced stage of development. These have been termed primordial ova, and there can be no doubt that a great number of the larger or spherical germ epithelial corpuscles are developing into similar bodies. The ordinary size germ epithelial corpuscles measure about th part of an inch in diameter. GENERAL CoNCLUSIONS. The following general conclusions have been arrived at in the course of my investigations : The corpuscles of the germ epithelium are derived by direct 218 DR. JAMES FOULIS. proliferation from those columnar corpuscles which invest the median side or surface of the Wolffian body, and which are continuous with the layer of columnar corpuscles that lines the pleuro-peritoneal cavity of the embryo in the early stages of development. The stroma of the ovary in the early stages of development is produced by a direct growth out from the interstitial tissue of the Wolffian body immediately beneath the germ epithelium on the median side of the Wolffian body. The germ epithelial corpuscles proliferate by fission. In the human foetal ovary of 74 months they measure z,'5,th to =;l5sth of an inch in their longest diameter, and about ss'ssth of an inch in their shortest diameter. Eath germ epithelial corpuscle is a nucleus surrounded by a thin film or investment of clear protopiasm. In the act of becoming primordial ova, the nucleus of each germ epithelial corpuscle swells up into a spherical body, within which is generally seen a nucleolus, and around which is produced clear homo- geneous protoplasm which subsequently forms the yelk of the ovum. Germ epithelial corpuscles are seen on the surface of the ovary in all stages of development into primordial ova. In each primordial ovum the spherical germinal vesicle pre- sent a sharply defined limiting membranous wall. Within the germinal vesicle is the nucleolus or germinal spot. All the ova in the ovary are derived from germ epithelial corpuscles. In all parts of the germ epithelium processes of vascular connective tissue stroma grow in between and around certain of the germ epithelial corpuscles, whereby the latter become more and more imbedded in the stroma of the ovary. Germ epithelial corpuscles are being constantly produced on the surface of the ovary, to take the place of those already imbedded in the stroma. The imbedded corpuscles increase in number by division, and the nucleus of each swells up into a spherical germinal vesicle, around which is gradually produced the yelk.of the ovum. In all parts of the young ovary under the germ epithelium, groups of germ epithelial corpuscles become imbedded in meshes of the stroma. As each individual in the group swells up the nucleus or germinal vesicle becomes very distinct as a round or spherical body. From the swelling out of each germ epithelial corpuscle in the group, the whole group expands and becomes more or less spherical. Such groups of developing corpuscles are called egg clusters. Hach egg cluster is enclosed in a mesh or capsule of vascular stroma of the ovary. Each imbedded germ epithelial corpuscle is potentially an ovum. THE OVA AND OVARY IN MAN AND OTHER MAMMALIA. 219 The stroma of the young ovary consists for the most part of fusiform connective tissue corpuscles, and blood-vessels. The walls of the young blood-vessels in the young stroma consist of connective tissue corpuscles. The connective tissue corpuscles are direct offshoots from the ovarian stroma, and are found in contact with the yelk or protoplasm of each primordial ovum situated among the germ epithelial cor- puscles on the surface of the ovary. Wherever we find primordial ova we see connective tissue corpuscles in contact with the yelk of each. In all parts of the ovary we find the nuclei of connective tissue corpuscles dividing. Sometimes these corpuscles are swollen out into round bodies containing three to four nuclei. In each egg cluster several of the in- cluded germ epithelial corpuscles are in a much farther advanced stage of development than their fellows. From the walls of the meshes inclosing the egg clusters, delicate processes of vascular connective tissue grow in, between, and around individual corpuscles in the egg clusters, and by a continued intergrowth of the young stroma in this manner each individual of the group becomes at last enclosed in a separate mesh or capsule. These last formed meshes are the Graafian follicles. As a rule, each Graafian follicle is occupied by one young ovum. The protoplasm or yelk of each ovum is in close contact with the wall of each Graafian follicle. In contact with the yelk of each young ovum, and indenting it, are connective tissue corpuscles, which form part of the wall of each Graafian follicle. In the formation of the membrana granulosa, these connective tissue corpuscles in the wall of the Graafian follicle, and in contact with the yelk of the contained ovum, increase in number by division, their nuclei swell out into little vesicles, and at last a perfect capsule of such corpuscles is produced round the ovum. This capsule is the membrana granulosa or follicular epithelium of the follicle. At first the membrana granulosa consists of a simple layer of corpuscles lining the follicle.’ The individual cor- puscles of the membrana granulosa in the human ovary measures about ;,5,th inch. As the ovum becomes mature, the corpuscles of the membrana granulosa proliferate, and then many layers of small corpuscles are produced between the ovum and the follicular wall. The cells of the mem- ' The formation of the granulosa from a single layer of connective tissue cells which are in origin independent of the germ-epithelium is paralleled by the mode of development of the inner cellular membrane of the egg- capsule in Cephalopods, as described by Ray Lankester, ‘ Phil. Trans.,’ 1875, “ Contributions to the Developmental History of the Mollusca.” 220 DR. JAMES FOULIS. brana granulosa are thus derived from the corpuscles of the connective tissue stroma, and not, as Waldeyer states, from the germ epithelial corpuscles. The follicular space is formed by a breaking down and probable solution of certain of the corpuscles of the thickened follicular epithelium in the midde parts of the same. The discus proligerus consists of folli- cular epithelial corpuscles, which are in contact with the zona pellucida of the ovum. ‘The zona pellucida or vitelline membrane is formed by a hardening of the outer part of the yelk or protoplasm of the ovum, and is not, as Reichert, Pfliiger, and Waldeyer stated, a product of the follicular epithelium. At birth the human ovary contains not less than 35,000 ova, few of which reach maturity. In the human ovary at birth the germinal vesicles measure rs'coth to sooth of an inch. Most of them are about the same size, and present a sharply-defined membranous wall. In some germinal vesicles two or three germinal spots are seen. The tunica albuginea is the thickened stroma growing round the ovary. At the age of two and a half years all formation of ova from the germ epithelium has ceased. Graafian follicles are not formed from tubular structures in the manner described by Pfliiger, Spiegelberg, and Waldeyer. The appearance of tubular structures passing into the stroma of the ovary are produced by sections through furrows and depressions between irregular prominences on the surface of the foetal ovary. The irregularities of the surface of the foetal ovary are produced at first by the expansion of egg clusters upwards under the germ epithe- lium. When the walls of furrows and depressions come in contact, egg clusters are formed by the imbedding of germ epithelial corpuscles in that situation, just as in other situa- tions. Egg clusters are formed in connection with the germ epithelium lining the furrows and depressions. Among the germ epithelial corpuscles lining the furrows, &c., we find large primordial ova, and corpuscles in all stages of develop- maeait into the same, just as in other situations among the ordinary germ epithelial corpuscles. In the human fcetus, round and oval-shaped groups of germ epithelial corpuscles are found in connection with the germ epithelium all round the ovary. When vertical sections are made through these they present the appearance as if tubular structures filled with developing germ epithelial corpuscles passed from the germ epithelium downwards into the stroma of the ovary. The development of the corpuscles of the membrana granulosa, from connective tissue corpuscles of the stroma, THE GENUS ASTROKHIZA OF SANDAHL. 221 can be well followed out in the ovaries of adult rabbits and cats. At the age of six years the epithelium of the human ovary consists of very small flat hexagonal-shaped corpuscles, measuring ==!,,th to ,.;th of an inch. The corpuscles are undergoing division. ‘This layer can be stripped off without difficulty. At the age of twelve the epithelium has little difference in appearance from the above, the small size of the epithelial corpuscles being remarkable. The epithelium is beautifully seen in old cats, and must be regarded as homologous with the peritoneal epithelium. In old cats the epithelium on the surface of the ovary consists of very small distinct cells, measuring from ;,';5th to z,5;th inch, with granular oval nuclei. Edinburgh. On the GENUS ASTRORHIZA Of SANDAHL, LATELY DESCRIBED as HaEckELIna by Dr. Bessers. By W.B. Carpenter, M.D., C.B., F.R.S. (With Plate XIX.) [In consequence of the publication by Dr. Bessels in the ‘Jenaische Zeitschrift,’ vol. ix, of a description of the animal and test of Astrorhiza as a new genus, the following extract from a paper ‘On the Rhizopodal Fauna of the Deep Sea,” presented to the Royal Society June 17, 1869 (abstracted in ‘ Proceedings of the Royal Society,’ vol. xviii, p- 99), has been forwarded to us for publication by Dr. Carpenter.— Eps. | ALTHOUGH twelve years have elapsed since Dr. Sandahl first characterised the genus Astrorhiza,' on the basis of specimens discovered in muddy shallows on the Scandinavian coast, its existence in British seas had not (so far as I am aware) been recognised previously to the Lightning expe- dition. My own attention was first drawn to this type about four years ago by Professor Lovén, who was good enough to send me some specimens of it, at the same time informing me that it was not at all uncommon, and might probably be found on our own shores if carefully looked for. These specimens fully bear out the description given of the type by Dr. Sandahl, who characterises the genus as follows :— “Corpus discoideum, orbiculatum, testa tectum stellata e materlis diversis composita, sive poris, margine in radios plures tubulosos excurrente.” The species is thus defined :— 1 Ofvers. af. K. Vet.-Akad. Forh. d. 14, Oktober, 1847. 222 DR. W. B, CARPENTER. Color teste stellate obscurus, griseo-brunneus, maculis parvis flavo-brunneis sparsus, ineequalibus, irregularibus, paullum intentibus. Numerus denticulorum varians (10-19). Diameter teste (sine dentic.) 5-6 mm. Longitudo denticu- lorum 1-2 mm. The diameter of the central disk in the specimens sent me by Prof. Lovén commonly;reaches one fifth of an inch, and its radiating prolongations sometimes attain a nearly equal length./ The test is essentially composed of sandy particles ;—of very irregular size and form, large rounded fragments ‘being occasionally interposed among the ordinary angular grains, and the external surface being smoothed over by a kind of plaster, which seems composed of particles of fine mud, whilst the internal surface is roughened by the pro- jection of angular sand-grains into the cavity, as in Sacca- mina (loc. cit.). These materials appear to be held together by an organic cement, which imparts a certain degree of flexibility to the envelope. The walls of the radiating extensions are composed of small sand-grains embedded in agglutinated mud, and the tubes thus formed are quite flexible. \_— A few specimens were collected by Mr. H. Brady about two years ago from the Dogger Bank, at depths of from 12 to 20 fathoms, which seem referable to this genus, though differing from Dr. Sandahl’s type-form in some important particulars. ‘The lenticular disk attains in some specimens a diameter of three tenths of an inch, but it is destitute of radiating prolongations. The sand-grains, which (as in Dr. Sandahl’s specimens) are of very irregular size and shape, seem to be more firmly united together; but it still appears probable, from the flexibility of the envelope they form, that the uniting material is an organic, not a mineral cement. The external surface (so far as can be judged from specimens preserved dry) was not smoothed over by the plaster, which concealed all but the largest sand-grains in Dr. Sandahl’s specimens. The Lightning dredgings at 170 fathoms (temp. 413°) in the “cold area,’ and at 530 fathoms (temp. 48°) in the part of the “warm area” most nearly approaching this, where the Globigerina-mud was mingled with a considerable quantity of the fine sand characteristic of its bottom, yielded a remark- able series of arenaceous tests, presenting exactly the com- position and mode of aggregation of the tests of the Lituole found in the same dredgings (loc. cit.), but exhibiting singular diversities in form, some of the most remarkable of which are represented in Plate XIX, figs. 1—13. ass THE GENUS ASTRORHIZA OF SANDHAL. 22:3 seem referable to two fundamental types, which may be | designated respectively the lenticular and the cervicorn. (Tn - the former the test may be considered as deriving its shape from a spheroid (fig. 5), which, whilst becoming flattened, sends out irregular digitate prolongations (figs. 6, 8, LO 11S) so as very much to resemble the different aspects of an Ameba. In the latter (figs. 1 and 2) there is nothing that can be called a centrum, but a somewhat flattened stem puts forth irregular branches of the like shape, which often again branch, sometimes widening out into palmate expansions before doing so. Between these there are various inter- mediate gradations, the simpler or more elementary examples of the “ cervicorn” type (figs. 4, 7, 18) having an obvious tendency to approach those modifications of the “lenticular” in which-the radiations enlarge at the expense of the central disk. When the cavity is laid open, which is best done in the manner recommended in the case of the Lituole having the like texture (loc. cit.), it is found to be small in pro- portion to the size of the test, the walls of which are extremely thick (figs. 3 and 4). It is in all cases simple and undi- vided, and is occupied by a sarcode-body of a greenish- brown hue. a A very remarkable feature in this organism, which in other respects might be likened to a Rhabdammina (loc. cit.) of gigantic size and coarse construction, is the absence of any definite aperture. 'This I have carefully substantiated by the examination of a considerable number of specimens under various methods of observation; the extremities of the branches or radiations, like every part of the general surface, exhibiting a continuity of the arenaceous layer, save where it has obviously been interrupted by fracture. From the looseness with which the sand-grains are aggregated, how- ever, it seems quite conceivable that the pseudopodial extensions of the sarcode-body may find their way out between them at any points, but more especially at the extremities of the branches or radiations, where the wall is thinner than it is elsewhere. And this deficiency of firmness in the test further suggests that it may undergo enlargement by the extension of its ramifications, the terminal layer of sand-grains being pushed away (so to speak) by the growth | of the sarcode-body. anil Any doubt I might have entertained as to the essential identity of our own types with the Asérorhiza of Sandahl have been removed by the fact that specimens collected by M. Sars, jun., from a depth of 450 fathoms, whilst corre- sponding in every particular with our own gatherings, are 224 DR. W. B. CARPENTER. unhesitatingly referred to that genus by Prof. Sars, who has kindly transmitted to me examples of them. | The variety in the composition of the test would thus seem referable to the nature of the material furnished by the bottom on which these Rhizopods live, and partly to the condition of that bottom as regards: stillness. We have seen that there are some arenaceous Foraminifera whose very symmetrical tests seem only capable of being built up where materials can be found of a precisely suitable kind, whilst there are others which appear able to use for their ruder constructions any material which the bottom may happen to supply; and Astrorhiza obviously belongs to the latter category. But the most curious difference between the original specimens and those more recently collected by M. Sars and ourselves lies in the manner in which these materials are united. For whilst in the latter the sand-grains are so slightly connected together by any intermediate material that the test is very easily disintegrated, the wall in the former has at the same time a considerable amount both of tenacity and flexibility— in this last particular differing from all other arenaceous tests with which I am acquainted. Now the extreme brit- tleness of the Astrorhize brought from great depths, where a profound stillness prevails, would be speedily destructive to them if they lived amidst the agitated waters of the surface ; and it would seem as if the animal had the power, by exuding an organic cement, of so uniting the materials of its test as to give it just that power of resistance which is most suitable to its habztat. NOTES AND MEMORANDA. Relation between the Limit of the Powers of the Microscope and the Ultimate Molecules of Matter.! THE subject which I have selected for my address is the relation between the limit of the powers of the microscope, and the ultimate molecules of organic and inorganic matter. I think I may at all events claim for this question sufficient novelty. Until the last few years the subject could scarcely have been attempted, and even now so many necessary facts are imperfectly known, that nothing more can be done than to fill the gaps with plausible assumptions. This neces- sarily imparts more or less of a speculative character to some of my remarks; but it appears to me that in his annual address the president of a society cannot do better than endeavour to point out the general bearings of what is already known on some great question, even if for no other object than to prove the need of further inquiry. Though fully impressed with the imperfect state of our knowledge, yet, even now, the facts are sufficiently definite to indicate, if not to prove, the existence of as wide a world of structure beyond the limit of the power of the microscope, as what has been revealed to us by it is beyond the powers of the unassisted eye. I propose to divide my subject into three heads— 1. The limits of the power of the microscope. 2. The size of the ultimate molecules of organic and inor- ganic matter. 3. Conclusions to be drawn from the general facts. In considering the limits of the power of the microscope, I shall assume that the instrument itself is perfect. and that the invisibility of the objects examined is in no way depen- dent on the want of the necessary characters. The point ' Abstract of the Anniversary Address of the President of the Royal Microscopical Society, H. C. Sorby, F.R.S., &c. With corrections com- municated by the Author. VOL. XV—NEW SER. P 226 NOTES AND MEMORANDA, to which I particularly wish to direct attention is the limit of visibility depending on the constitution of light, beyond which light itself is of too coarse a nature to allow of our seeing objects distinctly defined. ‘This question has been treated of in an admirable manner by Helmholtz in the jubilee volume of ‘ Poggendorff’s Annalen’ (1874, p. 573). The conclusion to which he arrives is that the limit depends on the confusion in the image due to the bright interference fringes overlapping the dark outlines of the object. This limit varies directly as the wave-length of the light, and in- versely as the sine of half the angle of the aperture of the object-glass when illuminated by means of a condenser of equal aperture. According to this principle the limit for dry object glasses of 60° aperture is, roughly speaking, about equal to the wave-length of the light, and for the largest possible aperture equal to $ the wave-length. In the case of immersion object glasses of the same aperture, the limit is about ? of that for dry. On comparing this theory with the results of observation, the agreement is very striking. It indicates exactly the same law for the increased defining powers of lenses of large aperture, as has been determined by experiment, and gives for the theoretical limit of distinct visibility g>4,,th part of an inch, which is exactly the same as the mean of the experimental determination of a number of the most skilful microscopists. It also shows why in the case of lines at equal intervals, like Nobert’s bands or the markings on Dia- tomacez, it is possible so to manage the illumination that the dark fringes of interference may coincide with the true lines of structure, and give rise to good definition, even be- yond the normal limit, and also agrees with the fact that lines less than +;75scth of an inch apart can be photographed, though seen with extreme difficulty, if indeed truly resolved, except under very peculiar and exceptional conditions ; since the waves of light at the blue end of the spectrum, which are concerned in photography, are short enough to give good definition of lines so near together that the interference fringes due to the longer waves at the red end would give an indistinct image. Taking everything into consideration, the agreement between observation and the theory is so close as to make it extremely probable, and to justify the conclusion that the normal limit of distinct visibility with the most perfect microscope is $ of the wave-length of the light. If so, we must conclude that, even with the very best lenses, except under special conditions, light itself is of too coarse a nature to enable us to define objects less than ,,4, th or NOTES AND MEMORANDA. 227 sodoccth of an inch apart, according as a dry or an immer- sion lens is used. The limit of 3,4,, of an inch deduced on Helmholtz’s principle from the physical characters of light agrees admir- ably with the estimate formed independently by various great authorities on the microscope. The mean of the estimate thus formed by Quekett, Moss, De la Rue, and Carpenter, as quoted by Stodder, is in fact exactly the same (54,5 of an inch), so that we cannot, I think, be far from the truth, if we take that as the base on which to build further con- clusions. With an immersion object-glass of very large aperture it might be possible to define an interval of some- what less than +450 of an inch, but probably the above- named determinations were made with dry lenses. At all events, since the limit of visibility as determined by the use of the best modern microscopes agrees so completely with what appears to be the limit due to the physical constitution of light, we must, I think, conclude that our instruments do now enable us to see intervals so small in relation to the wave-length of light, that we can scarcely hope for improve- ment as far as the mere visibility of minute objects is concerned, whatever may remain to be done to improve their performances in other respects. 2. The Size of the Ultimate Atoms of Matter. Having then come to the conclusion that the limit of distinct and unequivocal definition is somewhere about from Botcs tO saa'se0 Of an inch, it appears to me very desirable to consider what relation such a magnitude bears to the size of the ultimate atoms of organic and inorganic matter. From the very nature of the case the microscope altogether fails to threw any light on this question, and the only course as yet open to us is to draw the best conclusions we can from the various properties of gases. This problem has been attacked by Stoney,! Thomson,” and Clerk-Maxwell,? who, from various data, and by various methods of reasoning, have endeavoured to determine the number of ultimate atoms in a given volume of any permanent and perfect gas. In order to avoid inconveniently long rows of figures, I have reduced all their results to the number of ultimate atoms contained in a space of ,,'5, of an inch cube, that is to say, in qg9900000 1 © Philosophical Magazine,’ 1868, vol. xxxvi, p. 132. 2 * Nature,’ March 31, 1870, vol. i, p. 551. 3 Tbid., August 11, 1873, vol. viii, p. 298. 228 NOTES AND MEMORANDA. y of a cubic inch, at 0° C. and a pressure of one atmosphere These numbers are as fatlews Stoney 5 : 4 . 1,901,000,000,000 Thomson : 3 ; - 98,320,000,000,000 Clerk-Maxwell : : , 311,000,000,000 Mean mn : : - 50,260,000,000,000 As will be seen, there is a very great discrepancy between the numbers given by Thomson and Clerk-Maxwell. This is in part due to the fact that Thomson gives the greatest probable number, whilst Clerk-Maxwell has endeavoured to express the true number indicated by the phenomena of inter- diffusion of gases. ‘The determinations do to a great extent depend on the measurements of length, and any differences are of course greatly increased when the number of atoms in a given volume is calculated, since that varies as the cube of the linear dimensions. Extracting the cube root of each of the above numbers, we obtain the number of atoms that would lie end to end in the space of ;,),, of an inch in length. It also appears desirable to give double weight to the determi- nation by Clerk-Maxwell, since it is founded on the best data. We thus obtain as follows: Stoney : ; : . 12,390 Thomson é : : ‘ se teee 29,275 Clerk-Maxwell Y 5 ‘ : 6,770 Mean . 2 : : m : 18,022 Calculating from this mean, we may perhaps conclude that the number of atoms in a cubic -;,!,,th of an inch is about 6,000,000,000,000. As will be apparent from the wide difference in the determinations, this result can be looked upon in no other light than a very rough approximation ; but still, when we bear in mind that Thomson’s result is given as a limit, it must be admitted that the numbers belong sufficiently to one general order of magnitude to justify our looking upon the mean as a tolerably satisfactory ground on which to form some provisional conclusions. Now, if the gas containing the above-named number of atoms consisted of two volumes of hydrogen to one volume of oxygen, when combined to form vapour of water there would be a condensation of volume from three to two, and on con- densing into a liquid a further contraction to +.;; of the bulk of the vapour. Each molecule of water would however consist of three atoms of gas, and hence in order to determine the number of molecules of liquid water in =,5, of an inch cube, it is necessary to multiply the number in a gas by 3x 1234 xi=617. This gives for the number of molecules NOTES AND MEMORANDA, 229 of water in =, inch cube about 3,700,000,000,000,000. In this and all other cases I give round numbers, since any nearer approximation is impossible. Though living organisms contain much water, yet far more complex substances enter into their composition. As an example of one of these, we may take albumen. Accord- ing to Lieberkiihn its composition is expressed by the formula C,,H,,.N,,50... It therefore contains seventy-one times as many ultimate atoms as water, and its atomic weight is about eighty-two times that of water. In the condition of horn I find that its specific gravity is about 1:31. Calcu- lating from these data, I conclude that when the various con- stituents combine they contract to 5% of the total volume, and not, as water, to2; and that the volume of a single molecule of albumen is about 55°6 that of a molecule of liquid water. If their form be similar, their diameter must therefore be 3°82 times that of a molecule of water. This would lead us to conclude that in a cube of =1,, of an inch of horn there are about 65,000,000,000,000 molecules of albumen. According, then, to these principles there would be in the length of 3745, of an inch of about 2000 molecules of water, or 500 of albumen, and hence, in order to see the ultimate constitution of organic bodies, it would be necessary to use a magnifying power of from 500 to 2000 times greater than those we now possess. ‘These, however, for the reasons already given, would be of no use unless the waves of light wer some 3,55 part of the length they are, and our eyes and instruments correspondingly perfect. It will thus be seen that, even with our highest and best powers, we are about as far from seeing the ultimate constitution of organic matter as the naked eye is from seeing the smallest objects which they now reveal tous. Nor does there appear to be much hope that we ever shall see the ultimate constituents, since light itself is manifestly of too coarse a nature,even if it were possible to still further develop our optical resources. As matters now stand we are about as far from a knowledge of the ultimate struc- ture of organic bodies as we should be of the contents of a newspaper seen with the naked eye at a distance of a third of a mile, under which circumstances the letters of various sizes would correspond to the smaller and larger ultimate molecules. ‘This being the case, we may feel persuaded that particles of organic matter, like the spores of many living organisms scarcely visible with the highest magnifying powers, and, if seen, quite undistinguishable from one another, might yet differ in an almost infinite number of structural characters, just as any number of different news- 230 NOTES AND MEMORANDA. papers in various languages or with varying contents would look alike at the distance of a third of a mile. 3. General Conclusions to be deduced from the above Facts. When we come to the application of these principles to the study of living matter, we are immediately led to feel how very little we know respecting some of the most important questions that could occupy our attention—questions which certainly never presented themselves to me, until I looked upon them from this point of view, and which perhaps have not occurred to any one before. As illustrations of the sub- ject now under consideration I do not think I can select better than the facts bearing on the size and character of minute germs, and on Darwin’s theory of ultimate organized gemmules, as described in Part ii, chapter xxvii, of his work on the variation of animals and plants under domestication. So far as I have been able to learn, he has nowhere given any opinion as to the probable szze of such gemmules, nor discussed the probability of some of his speculations when examined from a numerical point of view, and in connection with the probable size of the ultimate molecules of organized matter. I therefore propose to do so; since, though not actually a microscopical question, it is most intimately con- nected with our studies, and as microscopists I think we have a good claim to investigate objects that are just beyond our magnifying powers. For the sake of simplicity I will take into consideration only the albuminous constituents of animals, using the term albumen in a sort of generic sense, to include many com- pounds, which differ in many particulars, and yet have many incommon. With slight modifications the same principles would apply in the case of other substances. Whatever be the special variety of this constituent, it 1s so associated with water in living tissues that in most, if not in all, cases they would cease to live if thoroughly dried. This is exemplified by the case of hair and horn, which must con- tain much water at the growing end, but are dead where hard and dry. In living tissues much of the water is no doubt present simply as a liquid mechanically mixed with the living particles, but it appears to me that we ought to look upon some portion as being in a state of molecular combination. So little attention has been directed to this kind of weak affinity, that its very existence is almost or quite ignored in many large and important chemical works, and yet probably many of the phenomena of life are mani- NOTES AND MEMORANDA, 231 fested only by such compounds. Very much light is thrown on this question by the study of the spectra and other optical characters of coloured substances. These clearly prove that when dissolved in any liquid the optical proper- ties of the solution depend in part on the nature of the solvent, and are by no means the same as they would be if minute particles of the solid substance were diffused in the liquid. These facts cannot, I think, be explained unless we conclude that the solvent is to some extent in the state of molecular combination with the substance dissolved. This molecular affinity is also in some cases manifested by a swelling up of a solid substance when placed in some liquids, even when perfect solution occurs to a very limited extent. Such a condition appears to be very characteristic of the living tissues of animals, and makes it sufficiently probable that the ultimate living particles are molecular compounds with water, and not molecules of free dry albuminous substances. Unfortunately, nothing definite is known respecting this question, and all that can now be done by way of illustration is to make some sort of a probable supposition. Taking everything into consideration, it appears to me that, as a reasonable example, we may assume that living albuminous tissue contains one-half of its volume of water mechanically mixed, and one-fourth its volume of free albumen united molecularly with an equal volume of water. On_ this sup- position the number of molecules in ;,)5, of an inch cube would be about Albumen : Z ‘ . 17,000,000,000,000 Water in molecular combination . 923,000,000,000,000 94.0,000,000,000,000 Since, however, the form of minute living organisms more nearly approximates to spheres than to cubes, it will be more convenient to give the numbers in a sphere of +5, of an inch in diameter. For this there would be about as follows: Albumen 2 4 : . 10,000,000,000,000 Water in molecular combination . 490,000,000,000,000 500,000,000,000,000 In the present state of our knowledge it is perhaps im- possible to say whether or not the essential characters of living particles are due to the structural arrangement of the molecules of this combined water as well as of those of the albumen, and whether or not in considering the possible variations in structure the total number of molecules should 232 NOTES AND MEMORANDA. be taken into account. The very small relative amount of dry matter in some living animals does, however, make it very probable that molecularly combined water really plays a part in their structure; and on the whole we may, I think, base our provisional calculations on the total number of molecules given above. The Theory of Invisible Germs. The relation between the size of the smallest object that can be seen, and that of the ultimate molecules of living matter, is manifestly a question of great importance in con- nection with the theory of germs. If the ultimate mole- cules were much larger than they appear to be, there would be serious objections to the theory; but, as far as we can judge, they are sufficiently small to make it possible for an almost endless variety of germs to exist, each having a dis- tinct structural character, and yet each so small that there is no probability of our ever being able to see them, even as indefinite points. Thus, according to the principles de- scribed above, a sphere of organized matter one-tenth of the diameter of the smallest particle that could be clearly de- fined with our highest powers, might contain a million molecules of albumen and moleculary combined water. Variations in number, chemical character, and arrangement, would in such a case admit of an almost boundless variety of structural characters. The final velocity with which such extremely minute particles would subside in air must be so slow that they could penetrate into almost every place to which the atmosphere has access. Darwin’s Theory of Pangenesis. Darwin’s theory of pangenesis is an attempt to give some- thing like a reasonable explanation of the phenomena of inheritance, and is not necessarily connected with the ques- tion of the evolution of new species. A full account of the theory will be found in his work on the variation of animals. At p. 374 of vol. ii. he says that. ‘* he assumes that cells before their conversion into completely passive or formed material, throw off minute granules or atoms, which circulate freely throughout the system, and when supplied with proper nutri- ment multiply by self-division, subsequently becoming de- veloped into cells like those from which they were derived. These granules for the sake of distinctness may be called cell-gemmules, or, as the cellular theory is not fully estab- lished, simply gemmules. They are supposed to be trans- mitted from the parents to their offspring, and are generally NOTES AND MEMORANDA. 2393 developed in the generation which immediately succeeds, but are often transmitted in a dormant state during many gener- ations, and are then developed. ‘Their development is sup- posed to depend on their union with other partially developed cells or gemmules which precede them in the regular course of growth.” He nowhere gives any opinion as to the actual size of gemmules, or the number present in particular cases, but it appears to me interesting to consider how far the theory will hold good when examined from this more physical point of view. For the sake of argument, I assume that gemmules on an average contain one million structural molecules of albumen and molecularly combined water. Variations in number, composition, and arrangement would then admit of an almost infinite variety of characters. On this supposition it would require a thousand gemmules to be massed together into a sphere, in order to form a speck just distinctly visible with our highest and best magnifying powers. By calculation I find that a single mammalian spermatozoon might contain so many of such gemmules, that, if one were lost, destroyed, or fully developed in each second, they would not be completely exhausted until after the period of one month. Hence, since probably a number are concerned in producing perfect fertili- sation, we can readily understand why the influence of the male parent may be very marked, even after having been, as regards particular characters, apparently dormant for many years. In a similar manner [ calculate that the germinal vesicle of a mammalian ovum might contain enough gemmules for one to be destroyed, lost, or fully developed in each second, and yet the entire number not be exhausted until after a period of seventeen years, and the entire ovum might contain enough to last at the same rate for no less than 5,600 years. These calculations are made on the supposition that the entire mass is composed of gemmules. Of this there is little probability ; ; but still, even if a considerable portion of the ovum consists of completely formed material and of mere nutritive matter, it might yet contain a sufficient number of gemmules to explain all the facts contemplated by the theory of pangenesis. ‘The presence of any considerable amount of such passive matter in spermatozoa would, however, be a serious difficulty in the way of the theory, unless indeed very many spermatozoa are invariably concerned in producing fertilisation. Taking everything into consideration, it does not appear 234 NOTES AND MEMORANDA. to me that any serious objection can be raised against pan- genesis when examined from a purely physical point of view, as far as relates to the inheritance of a very complex variety of characters by the first generation, though there would have been many serious difficulties to contend with, if the ultimate atoms of matter had been very much larger than is indicated by the properties of gases. When we come to apply similar reasonings to the second or following generations, we are compelled, along with Darwin, to conclude that gemmules have the power of pro- ducing other gemmules more or less closely resembling themselves, and of being collected together in the sexual elements, since otherwise the number that could be trans- mitted in a dormant state for several generations would be far too small to meet the requirements of the case. Conclusion. In my remarks I have made no endeavour to conceal our present ignorance of many very important questions con- nected with my subject. Want of the requisite data neces- sarily imparts a speculative character to many of my conclu- sions; but perhaps there is no more fruitful source of know- ledge than to see and feel how little is accurately known, and how much remains to be learned. PROCEEDINGS OF SOCIETIES. Dustin Microscortcat Crus. 28th October, 1875. Acetta coriacea from Oban Bay, new to the British Seas, in its simplest form.—Dr. A. Macalister presented a specimen of Acetta coriacea from Oban Bay, showing the simplest form of that species. The “persone” were separate, each with a single “mouth.” Hitherto the only recorded forms of this common sponge from the west coast were examples of the Aulophlegma- type, and Dr. Macalister believed this to be the first specimen of the simple form of this species recorded from the British seas. Lejeunia microscopica, Taylor, exhibited—Dr. D. Moore showed. the rare and pretty Lejewnia microscopica, Taylor, which he had just brought from the west of Ireland; although to the naked eye little in the shape of an organized plant makes itself evi- dent, this little gem, probably the very smallest of our higher Cryptogams, forms a very pretty object under a 1” objective. Germinating Seedlings of Drosera.—Dr. Moore likewise drew attention to some germinating seedlings of Drosera; these had two ovate, smooth, cotyledonary leaves, the third leaf in all assum- ing the spathulate form of those of the species, and was clothed with the pretty characteristic insect-capturing glandular hairs. Navicula Stokesiana,n.s.,O’ Meara.—Rev. EK. O’Meara presented what he considered to be a new form of Navicula, of which the following is a description:—Valve large, rhombic-lanceolate, length ‘0045", breadth 0018” ; marginal striate band wide, inner striate band contiguous to the median line, narrow, and appearing to be elevated above the general surface, free space included within the inner margins of the inner striate bands narrow-linear, forming in the middle a very narrow stauriform line ; space inter- mediate between the inner and outer striate bands occupied by lines of strize which seem to be prolongations of the striz of the outer striate band; striz close, punctate, radiate. This form in most respects is similar to Nav. irrorata, Schmidt, ‘ Atlas der Diat.,’ t. ii, f.14; but it seems distinguished by the fact of having the intermediate space striate, whilst in Nav. irrorata this portion of the valve is represented as unstriate. Mr. O’ Meara proposed under the name of Navicula Stokesiana to identify this beautiful and rare form with the name of the present respected President of the Royal Irish Academy. Penium curtum, Bréb., exhibited.—On the part of Mr. Crowe 236 PROCEEDINGS OF SOCIETIES. (who was absent) Mr. Archer showed examples of that somewhat remarkable desmid Peniwm (Cosmariwm, Autt.) curtum, Bréb. These specimens were taken by Mr. Crowe from a shallow acci- dental water-deposit on the railway platform at the Bray station, thus showing the somewhat capricious distribution of this form, seemingly always occurring so far away from its congeners, the Desmidiex at large, delighting, as they do, in boggy and mossy localities, not roadside ruts! Mr. Crowe had taken it on a former occasion on a roadside in Wales; again, scarcely a step from his own halldoor at Bray; and Mr. Archer on the roadside near Dargle Gate. This kind of habitat seems to accord with de Bré- bisson’s description of that of Cosmarium Regelianum, (Nag.) Bréb., seemingly equivalent to P. curtum, Autt. (though de Bré- bisson records both as distinct) ; the former, at least, occurs, he says, ‘‘ tapissant des cavités sablonneuses, recemment inondées par la pluie.” Sporocybe byssoides, Fries, exhibited. — Mr. Pim showed Sporocybe byssoides, Fries, which grew on a decaying bulb of Sophronitis grandiflora; the dense heads of brown, minutely echinulate spores were very characteristic. Structure of petioles in the genus Nymphea.—Mr. Mackintosh laid before the Club the results of some further examinations of the structure of the petioles in Nymphea. Through the kind- ness of Dr. Moore, Director of the Glasnevin Botanic Gardens, he had ebtained specimens of eight species, which he had found could be arranged in two groups. One of the groups, of which Nymphea alba might be taken as the type, was characterised by having more than two primary air-passages of subequal size, and were provided with stellate cells. The other section, repre- sented by Mymphea Devoniana or N. dentata, had two large primary air-passages, several smaller secondary ones, and no stellate cells. Mr. Mackintosh exhibited sections of three species of the latter group—WN. lotus, N. Devoniana, and N. thermalis—in order to draw attention to the only difference which he could detect in the different species: this consisted in the septum between the two primary air-passages being made up of two, three, or four cells. It mightas yet seem doubtful if any depend- ence could be placed on a character of apparently so trivial a nature, but further investigation which he hoped to pursue would doubtless throw light on this interesting subject.—He likewise showed a cross section of the petiole of N. lotus, in which two or three of the cells bounding one of the secondary air- passages had become hypertrophied and grown out into a cel- lular mass which nearly filled up the cavity. A Pseudo-Cosmarium—in other words, a Cosmarium-like excep- tional state of an Arthrodesmus-incus-form—exhibited.—Mr. Archer drew attention to what at first blush looked like anew Cosmarium; and though this, as will be seen, was not so, he had actually labelled the bottle in which it occurred, in order to distinguish it from others, ‘“*Cosmarium inquirendum.” This little form was very minute, of DUBLIN MICROSCOPICAL CLUB. 237 quadratic general figure, but rounded at upper angles, and the constriction forming a rounded sinus at each side, the upper margin continuously with the “angles” broadly and gently con- vex, length and breadth about equal. It was thus different from any (British) Cosmarium. Never wasa more deceptive little form, for it was not a species of Cosmarium at all, though, with- out doubt, desmidian. Some examination of the slide, indeed, soon showed that this quasi-Cosmarium owed its origin to an ex- ceptional state of an Arthrodesmus-incus-form, that minute one, in fact, with simple cuneiform semicells and spines very slightly divergent. As is well known, when the growth of the new semicell, subsequent to division, is but partially advanced, the spines are not yet produced and the angles bluntly rounded; at this point, in several cases, the new semicell, as regards its own growth, had remained stationary, but, that notwithstanding, self-division had once more set in and new semicells were formed, the result of this second generation being these pseudo-Cosmaria. Here was a case, some might be found to advance, of the direct ‘jump ” of one species into another “new’”’ one! But it was notso. Cases were to be seen on the slide where some of these examples, which at the proper epoch had neglected to develop the spines, were now trying, as it were, to make up for lost time, and young short spines were at one end making their appearance. The whole thing was thus a mere “sport ’—still a puzzling case of excep- tional growth, such as Mr. Archer believed had not been before noticed, and therefore worthy of a passing record. Euglypha spinosa, Carter, exhibited.—Mr. Archer, on the part of Mr. Crowe, who had taken the example in North Wales, ex- hibited Huglypha spinosa, Carter, a fine and large species, and most marked ; it appears very rare indeed ; as yet in Ireland it has only been found in Co. Kerry, and at ‘“ Toole’s Rocks,” Co. Wicklow, by Mr. Archer. “Headed” Bacterian exhibited and note thereon.—Mr. Archer showed the active, and the stillgloeogenous “ Zooglcea”’ condition of a bacterian seemingly very close to the “ headed ” form figured by Cohn in his memoir in his ‘ Beitr. z. Biol. d. Pflangzen,’ Heft 2 (1872), t. ii, f. 13, though the actual identity seemed to be at least doubtful. Cobn’s examples occurred in an infusion of dead flies ; on the other hand, the present occurs in quite fresh gather- ings where all around—desmids, diatoms and other alge, infusoria and rotatoria, &c.—are in quite active and healthy condition. Hence Mr. Archer ventured to suppose, at least ad interim, that it could be hardiy correct to relegate such forms as this, of local occurrence (and qguere also Prof. Lankester’s B. rubescens), and which live and flourish in seemingly only healthy environments, to one of the same genus as the common and universal forms of putrescent or decaying, often stinking, infusions. The long and slender little filamentary bodies combined in a manner approxi- mately parallel, in 4’s and 8’s, or sometimes in 16’s, within their common oblong or ovate, definitely bounded mucous matrix, might 238 PROCEEDINGS OF SOCIETIES. in the present form be taken for some possibly undescribed alga, whilst the refringent ‘“ heads,” in the active state, though in as- pect and nature not seemingly materially different, except perhaps in size, from the similar bodies forming the joints or links in a Vibrio, &c., still, from their occupying but one extremity, would more forcibly call to mind the “spores” of certain veritable Nos- tochacean forms; they are sometimes detached and met with separately. This when in the active or moving state is a very agile form, incessantly darting backwards and forwards, and “ poking” its way into and again out from the masses of “dirt” and heterogeneous objects around. 18th November, 1875. Notice of some Desmidian forms allied to Closterium obtusum, Bréb.—Mr. Archer showed specimens of two new doubtlessly Des- midian forms closely allied to, but distinct from, that referred to Closterium under the name of Cl. obtuswm by de Brébisson. Onewas long, very slender, slightly curved, and ends truncate,close to which a colourless space, without granules, beyond the medium longitu- dinal string of green contents. The other was considerably stouter, margin subparallel for a considerable distance, towards the ends somewhat suddenly attenuated ; apices bluntly rounded, close to which a colourless space beyond the median longitudinal string of green contents, containing a single fixed and still granule. In both forms the nucleus was excentric, and placed ina lateral depression of the endochrome-mass. It will be seen that these hardly accord with the characters of Closterium. Mr. Archer thought it possible that, with some twoor three others kindred thereto, they might take a place as a distinct genus close to Closterium and Penium, as well as near Spirotenia and Cylindrocystis. Before arriving at a conclusion some of the other forms in view would have to be refound and submitted toa further study. Aulacodiscus Johnsonii, exhibited.—Rev. EK. O’ Meara showed a fine mounting by the Club’s Corresponding Member, Mr. Kitton, of Norwich, of Aulacodiscus Johnsonii ; in one instance the num- ber of nodules on one valve was greater than on the other. Structure of Petioles in genus Nymphea.—Mr. Mackintosh ex- hibited transverse sections of the petioles of Mymphea odorata and N. cerulea, along with N. alba, all belonging to the series with stellate cells in their air-passages, and which might therefore be termed “ asteridiophorous.”’ So far as he could make out, the only difference discernible in the petioles was in the numberof secondary air-passages, which were generally two in N. odorata, from six to eight in N. cerulea, and from nine to twelve and even more in N. alba. The number of primary air-passages was four instead of ten, as in the “anasteridiophorous”’ series, examples of which were exhibited at last meeting of the Club. Tetraspores in Stennogramme interrupta——Dr. E. Perceval Wright showed examples with tetraspores of Stennogramme inter- rupta, and referred to a notice in ‘ Grevillea’ by E. M. Holmes, in DUBLIN MICROSCOPICAL CLUB. 239 which that writer had stated that the tetrasporic fruit had not been noticed or figured in any work published in Great Britain; Dr. Wright, however, pointed out that this was not so, but that Dr. Harvey and Miss Gifford had long since recorded it, as already fully detailed in last number of this Journal. Another “headed” Bacterian and note thereon——Mr. Archer wished once again to draw attention to another example of the gleogenous, still (‘‘ Zoogloea’”’) condition of the bacterian which he had exhibited at last meeting of the Club, then both in the same and in the active moving state, and this the more especially as he desired to place side by side therewith a specimen or gleogenous group of the very same “species,” “form” or “thing,” which he. had just noticed on a slide of Herr Otto Nordstedt’s mounting, put-up for a single specimen of a Stau- rastrum, the bacterian being there, of course, quite unwittingly and accidentally. This was absolutely, and in its (granted, very few) details precisely, one and the same thing to which Mr. Archer had previously drawn attention. It was very pretty to see how nicely it had “ mounted’’ and maintained its ordinary aspect. If it were met by an observer in the condition of both examples for the first time it would, without doubt, be naturally & priori regarded (as before mentioned) as an alga, and its bac- terian nature would hardly suggest itself. Generally speaking, no heads (or “spores’’?) are then visible on the little threads, but Mr. Archer had seen gloeogenous specimens showing the heads more or less grown, and in different examples of different sizes. Mr. Archer was gladto be able to show the form in ques- tion (in both native and “foreign” examples!) simultaneously with another “ species” to which he now drew the Club’s atten- tion, doubtless closely resembling the preceding, but yet different. ' Here the little threads were, microscopically speaking, consider- ably thinner, of a bluer tint, rigid, likewise “headed,” but the heads considerably more minute. Their movements were far more lazy ; the pin-like examples might be said, so to speak, merely to totter about. Of this form Mr. Archer had not been able to see any gleeogenous state, so far as he could make out, but on the other hand he drew attention to certain slender filaments in the same gathering of great tenuity, bearing along their length little bluish bead-like bodies at even distances, distributed in pairs, suggesting the breaking up of the filament into short pieces between the beads, which would thus stand as the “heads.” At other times filaments could be found in still the same gathering, showing at shorter, but likewise even, distances pairs of beads extremely minute and pairs of the ordinary size alternating with each other. To say the least, the moving forms, slowly wabbling about in their vacillating manner (so different from the vivacious and restless up-and-down fidget of the preceding form, though there were none of those in the free and active state to-night), were extremely like the several portions of the filament, as described, mutually detached and become independent. Their colour, especially the 240 PROCEEDINGS OF SOCIETIES. heads, was of a bluish tint; in fact, they looked very like some minute “nostochaceous” joints, and Mr. Archer thought there could be little doubt but that these beads or heads corresponded to the “spores”’ of characteristic “ Nostochacer,”’ with which, indeed, the “ Bacteriew” should be associated. (It is but right to mention that these notes and views were laid before the Club prior to the appearance, in Dublin at least, of Professor Cobn’s recent paper on Bacterians in the late number of his ‘ Beitrage,’ in which similar views are expressed, and in which he now refers the various forms to their respective places in the system amongst Phycochromaceous Alge.) Mepicat Microscoricat Society. 17th December, 1875. H. Power, Esq., Vice-President, in the Chair. Dr. Prircuarp exhibited in action the new freezing machine for cutting microscopic sections, which has already been described in the ‘ Lancet’ for December 11th, 1875. The principle upon which its action depends is that a block of copper cooled by immersion in ice and salt will retain its low temperature sufficiently long in order to enable sections to be cut from a small piece of any soft tissue placed upon it, and which by contact with it has become frozen and adherent to its surface. Tf first immersed in gum water the specimens solidified better. In the discussion that followed, Mr. Ward suggested using a metal plug in the same way as Dr. Pritchard recommended, only dropping it into an ordinary microtome tube, so as to obtain the additional advantage of a rest for the razor. Mr. Groves thought that if the plug were hollowed so as to contain some ice and salt it would remain cold much longer. Acari in diabetic urine—Mr. Jabez Hogg showed a specimen of urine from a case of incipient diabetes which contained large quantities of the Acarus domesticus, as well as particles of indigo. ‘Twenty-four hours after being voided he observed their presence, and up to the present time they were still alive and breeding (for they were seen in all stages of development), though six weeks had elapsed. The mycelium of the diabetic fungus had appeared and the indigo was increasing. It was possible that the animal fed on these two substances. He had only examined this one specimen, and had kept it in the bottle in which it had been sent to bim, in the window all the time. He had no doubt it was the ordinary sugar acarus, and must have obtained access to the urine in the first instance by accident. MEMOIRS. On the Formation of BLoop-vEssELs, as observed in the Omentum of Younec Rassits. By G. Tun, M.D. (With Plate XV, figs. 1—5.) WHEN new capillary blood-vessels are formed from pre- viously existing vessels in a tissue sufficiently transparent to be examined under the microscope, there is an appearance constantly observed, regarding the cause of which there is much difference of opinion. From the wall of the capillary a conical mass projects into the surrounding non-vascular tissue, and it is frequently continued into a similar mass that meets it from a neighbouring or from another part of the same vessel. Nuclei are visible at various parts of the tract thus indicated, and later a fully formed capillary is found to have been formed in it. Golobew! believes that the first stage in this projecting mass is an outgrowth from a spindle-cell in the wall of the vessel, and that the new formation extends by a hollowing out of the process and a multiplication of spindle-cells, which bend over towards each other. The dark lines seen ina capillary treated by silver correspond to the line of junction of the borders of two spindle-cells. KGlliker? at one time taught that a “ bud” (sprossen) grew out from the wall, and either joined a similar bud from another or the same vessel, or became continuous with the spindle or stellate cells of the tissue. In the last edition of his work he acknowledges that with the present better knowledge of the structure of capillaries these views are no longer tenable. Stricker considers that the projecting process proceeds from a stellate cell in the wall of the capillary, which anastomoses with other stellate cells in the tissue. The widening of the lumen of these processes constitutes the new vessel. This, of ‘ » ¢ Arch, f. Mic. Anat.,’ 1869, p. 49. 2_* Handbuch der Gewebelehre,’ VOL. XVI——NEW SER, ; Q 242 DR, G. THIN. course, presupposes a communication between the lumen of the processes of stellate cells and the lumen of the capillary. My information regarding Stricker’s views is obtained from Klein’s treatise on the omentum.! The last- named author adopts Stricker’s view, with the addition that this excavation of branched cells “goes on both from the already formed vessel, and also isolated in the branched lymph-canalicular cells, which are in direct continuity with the endothelium of the blood-vessels.” Schafer? regards the capillaries as being formed in spindle cells, absorption of part of the cell walls where the one over- laps the other being the mechanism by which a continuous tube is formed. Rouget® regards the projecting process as a development of the protoplasm of the wall of the vessel, which “‘ vacuolates”’ and then forms a tube continuous with that of the parent vessel. Ranvier*regards this process as a cellular bud (bourgeon cellulaire), which gradually hollows out as it forms. Thus the same appearance is regarded by Stricker and Klein as an enlarging process of a stellate cell ; by Golobew as an outgrowth of aspindle-cell; by Rouget as an outgrowth of the protoplasm of the wall of the vessel; and by Ranvier as acellular bud. Kdlliker used a similar term (sprossen) as applied to the same object, without defining it further. Kolliker was the first to describe this projecting mass as being continuous with the stellate cells of the tissue, in which Stricker and Klein follow him. Golobew, Rouget, aud Ranvier, on the other hand, state that they have never seen it become continuous with a stellate cell. Ranvier draws a sharp distinction between the growth of a capillary from a pre-existing vessel and the independent development of capillaries in the connective tissue which, after they are fully formed, join the other blood-vessels. Klein describes, as we have seen, an independent forma- tion in stellate cells which “ vacuolate,” and Schafer by the hollowing out of spindle-cells. To return to the projecting mass from an already formed capillary. This has been most frequently observed and figured, as seen in the tail of the tadpole. It is easy of ob- servation, and there can be little doubt that the different observers saw exactly the same appearance, as is besides clear enough from their figures. Since the same appearance has 1