^, ^, IMAGE EVALUATION TEST TARGET (MT-3) 1.0 1.1 ■^ loB IIIII2 2 2.0 1^ 1.8 L25 1 ,.4 p.6 ^ 6" — , ^ Hiotographic Sciences Corporation 23 WEST MA (meaning "CON- TINUED "), or the symbol y (meaning "END"), whichever applies. Un des symboles sulvants apparattra sur la derniira image de chaque microfiche, salon le cas: le symbols — ► signifie "A SUIVRE". le symbols ▼ signifie "FIN". re Maps, plates, charts, etc., may be filmed at different reduction ratios. Those too large to be entirely included in one exposure are filmed beginning in ti^e upper left hand corner, left to right and top to bottom, as many frames as required. The following diagrams illustrate the method: Les cartes, planches, tableaux, etc., peuvent Atre fiimis d des taux de reduction diffirents. Lorsque le document est trop grand pour Atre reproduit en un seul ciichi, il est fiimi A partir de I'angle supirieur gauche, de gauche i droite, et de haut en bas, en prenant le nombre d'images nicessaire. Les diagrammes suivants iilustrent la mithode. y errata >d to nt ne pelure, 1900 d n 1 2 3 32X 1 2 3 4 5 6 ^m ^ii^ ^'^ ¥ % TORONTO UBRAR^ 789 yONGE TORONTO TUHONTO PUBLIC LIBRARIES REFERENCE LIBRARY =>-TO. U 5^3 No. f^: ■1* . . / PUBLICATIONS FROM THE BIOLOGICAL LABORATORY OK THE UNIVERSITY OF TORONTO. No. I.— Contributions to the Morphology and Physiology of the CelJ By A. B. MACALLUM, M.B., Ph.D. (Heprintedfrom the Transactions of the CaiMcliati Imtitute, Vol. I., Pt. S.) = TORONTO: Thk Copp, Clark Company, Limited, Printers. 1891. ' • ' '■ f * i.'k: .•ill "«' ■Ai:- IN 8(>07 9 m 3 1 1951 ' 1 V' ,>.,,' ,1.;, -r ■:;:■: '.-^[i. ■ [Extract from Transactions of the Cana.■•■, TRAN8ACTIO.NH OK THE CANADIAN INSTITITE, [Vol. I. with division 3, the treatment of which I postpone until I have finished my experiments on the methods of the resorption of chromatins ( Nu- deins). To illustrate the parasitic nature of some of these forms, I will now describe undoubted examples of intracellular parasites from the intestines of the spotted newt and the lake lizard (Necturm). I. A Cellular Parasite fko.m the Intestinal Epithelium of DlEMYCTYLUS ViRIUESCENS. In April of this year I obtained from the neighborhood of Toronto a number of spotted newts for the purpose of studying the phenomena of secretion in the pancreas and in making preparations of this organ I found it frequently convenient, on account of the small size of the animal and its organs, to include the anterior portion of the intestine. In the in- testinal epithelium of one of the newts was found a large number of forms like those shown in Figures 3 and 4, and I immediately endeavored to work out their history. Before detailing the results of this work it may be well to state that the particular object from which the sections studied were made was hardened in F"lemming's Fluid and alcohol, stained in toto with hjematoxylin, imbedded by the chloroform process in paraffin, the sections therefrom placed in scries on the slide and stained with eosin and safranin, before being permanently mounted in balsam. The structures in question are so numerous that every second or third epithelial cell.for long stretches of the section, contained one of them. They are always placed in the outer half of the cell between the nucleus and the free border, and have a nearly uniform diameter (9-1 l/i, averaging lO/x) and an approximately spherical shape. They do not appear to have a definite or distinct membrane, and what takes its place appears to be a zone of homogeneous or faintly granular protoplasm which, in many cases, is denser and thicker at one side of the body than at any other. From this zone trabeculae of granular protoplasm pass inwards to ter- minate in a more or less centrally placed protoplasmic mass. In a number of these bodies sufficient to render the peculiarity prominent, the bulk of the protoplasm is collected at one side (Fig. 3), while the thicker portion of the protoplasmic rim occupies the opposite side with a large crescentic, oval, or round cavity intervening. The protoplasmic mass stains lightly but readily with eosin and contains a round homo- geneous nuclear body, which stains deeply with safranin and measures less than 2/A (i.S/^)- Sometimes the nuclear body is placed in a cavity in the protoplasmic mass and connected with the latter by a few fine strands. In a few instances, the nucleus was surrounded at a distance by I 1HK,re are not more than one or two forms that can be ranked as transitional. Two of such are represented in Fig. Sd and c. I have been led to consider them as stages in the formation of spores, because they present structures which resemble somewhat kary- okinetic figures. For example, in the form represented in Fig. 8d, the centrally placed stained body may be regarded as belonging to the dyaster stage and seen from one of the poles ; in it also structures, bear- ing a resemblance to individual chromatin loops, can be made out. This arrangement comes out well sometimes in preparations stained with ha;matoxylin and safranin, but oftener the safranophilous substance is collected in a ring form resembling, to a certain extent, the equatorial plate of nuclear division. Probably the explanation of Fig. 8^ is that it represents a multiple form of karyokinesis. The difficulty of de- termining the nature of such conditions will be readily understood, when it is remembered that the safranophilous bodies are usually not 2/a in diameter, and that, consequently, its metamorphic elements must be very small. >n i I TRANSACTIONS Of THE CANADIAN INSTITUTK. [Vou I. If the determination of the division of the nucleus is difficult, much more so is that of the full history of the spores. They are so small at first that, apart from the mother organism, they cannot be distinguished from other cellular contents, such as the swallowed portions of the debris of neighboring cells and the spore stages of other parasites. It is only in a few cases that circumstances favor the determination of some of the forms after they have escaped. In Fig. I, for example, is shown a cavity in the interior of a cell, evidently once occupied by the parasite in question, and in the neighborhood of the cavity is a number of bodies like plasmosomata, of similar, or nearly similar size. These are evidently the spores derived from the organisnr. which occupied the cavity. In a few instances, with the best conditions for observation, forms,, like those shown in Fig. ga, are seen. Here the structures are comma- shaped, and their resemblance to other forms in the same Figure, to that of Fig. \oa and to those in Fig. 2, is such as to suggest a developmental relationship. The probability, however, that very young forms of Sporo- zoan parasites arc similar to those represented in Fig. ga, is sufificicnt to invalidate any conclusion that might be drawn from this resemblance.* There is more certainty in regard to the larger comma-shaped forms, such as are shown in Figs. 2 and lo^n These are intensely safranophilous bodies, and measure from 3 to 6/j.. Their outlines are sometimes distinct, sometimes not, this depending on the way in which the organism is dis- posed in the field of the microscope. If the tail should happen to be above or below the head of the comma the organism may be recognised with difficulty. The connection between these and the spherulating forms can be seen by glancing at Fig. 10 a-h. In further development the head of the comma enlarges, the safranophilous substance collects into a small round mass, leaving the protoplasm which contained it more or less coarsely reticulated or finelj' granular, and with feeble staining capacity. The tail still retains its safranophilous character and remains distinct for several stages. The space between it and the head tends to increase when its point becomes applied to the head (Fig. loc). At the same time it becomes somewhat elongated {d), and the safranophilous sub- stance in it condenses into a thin band bounding the convex side of the crescentic cavity. The head also undergoes further changes {e). The protoplasm becomes collected at its periphery as a rim to which the small round safranophilous mas.s, the nucleus, is attached by delicate proto- plasmic strands. In the next stage protoplasmic strands may stretch across the crescentic cavity, to the remains of the tail or the point of the •Compare with Steinhaus' Figures of the intracellular parasites in the pancreas of the Sala- mander, Ziegler's Beitrage Zur Path. Anat., Bd. VII., Taf. XI. 1889.y(».] MOHI'lIULOUV AM) I'll YsKlI.iiilY OK TilK CKLL. tail may fuse with llii; head ; in the latter case tin; crcsccntic cavity persists (/), The safranophilous substance ^railually disappears from the thin band representing the remains of the tail, till finally its staining capa- city is scarcely marked in some of the forms, althoujjh its density is notice- able. This sketch of the orj^anism developctl out of the comina-shapcil body explains thus the occurrence of a denser, frequently more tler|)ly stainintj zone at one side, the presence of a cresccntic cavity, or of a cavity next the zone, and the frequently excentric position of the nucleus in the adult orjfanism {h'igs. 3 and 4). In individual cases, in which these pecu- liarities are apparently wanting, it may be that they cannot be observed, because the organisms are not favorably placed in the microscopic field. We can, I think, now account for many of the forms shown in Fig. 9, especially those in which a deeply stained crescent occurs with a sphere in its cavity — they are merely comma-shaped parasites in the process of transformation into that stage in which sporulation takes place. In the same way we may explain some of the forms illustrated by Luk- janow,* especially his Figs. 14, 15, 16, 6ia and /;, 66, 72, 74 and 75, and probably al.so Figs. 7, 11, 13, 6.S, 69, jy and 94. His F"ig. 48 would seem to indicate that he saw the sporulating phase of the same organism. All his studies were made on the gastric mucosa of the salamander. I have found in the gastric mucosa of Diemyctiiliis very few abnormal structures of this character. If they are parasitic, their comparative absence from the stomach may be attributed to the digestive and resistent action of the gastric mucosa, and it is probable that the irregu- larity and atypical character of many of the structures drawn by Lukja- now may be due to the physiological action, during life, of the glandular elements in which they occurred. It is interesting to note the structure of the cytoplasm around the full-sized organisms (Figs. 3-7). It is constituted of very fine rodlets, each with a thick end directed towards the organism and passing in a radiating manner peripherally into a zone of what appears to be finely granular protoplasm, but which is, probably, a portion of the cytoplasmic reticulum condensed. The border of thickened points in many cases clcsely resembles a membrane. It depends, apparently, on the vitality of the cell whether the radiating arrangement of the cytoplasm occurs or not. It may be absent, as in Fig. 2, when the cell shows signs of degeneration. It is difficult to understand the function of this mechanism, but we may suppose it to act as a filtering apparatus. 'Op. cit. 6 TRANHACTIO.NH OF THE CANADIAN ISHTITUTE. [Vol,. I. II. On CUkOMATOI'MAdOUS AND OTIIKK I NTKACI'.LLUKAR PAKASITKS IN THK InTKSTINK OF NlX'TURUS LATKKALIS. Ill the intestinal epithelium of Necturus arc often found forms which, from their peculiarities, must be ret,'arcled as parasitic. When I observed tiicm first, I considered them to belong in a general way to that class of intracellular structures which Lukjanow* has described as occurring in the gastric mucosa of the .salamander, and of which there are not a few examples in the intestine of Necturus. They are well shown in prepara- tions made from recently captured animals, and their characters are pre- served well in the tissues fi.xed with Flemming's I'^luid, or corrosive sub- limate, and stained with alum cochineal, or haematoxylin or eosin. The chromatophagous forms have usually an irregular outline and the protoplasm extended in one or more long pseudopodial processes, which taper often to fine threads In some ca.ses the whole organism is thread- like (Fig. IS/). They are easily distinguishable in alum-cochineal pre- parations in the unstained, epithelial cytoplasm, in which they may be found, and by their stain being in every respect similar to, and as deep as, that of the chromatin bodies of the epithelial nuclei. With high- powered objectives the stain is seen confined to the fine granules which densely crowd the cytoplasm of these organisms. There is sometimes a quantity of unstained protoplasm at the thicker end (Fig. 14/), or a more or less curiously shaped mas.s may lie in its neighborhood (Fig. \6 pr). Sometimes the bodies are found in the interior of nuclei, but, as a rule, they are not easily recognizable in this position, unless they show amoeboid outlines or are fixed in the act of migrating from the nucleus. One is shown in the latter condition (Fig. 15/}. The nucleus is in this case partially deprived of its chromatin by the parasite, which owes its staining capacity to the chromatin it absorbs or invaginates. An explanation of the relations of such structures as are shown in Fig. 13 (/) can be at best only problematical. Here two parasites, each in a separate cavity in the cytoplasm, have their prolongations hooked around one another. This is only one of several instances observed of such a condition, but the preparation drawn shows the process most distinctly. It may be a case of conjugation. There are a number of forms which are either wholly unstained by the coloring reagent, or which possess one or more stained spherules or granules (Fig. 1 3 p). These may, in some cases at least, represent young stages of the chromatophagous forms. •L. c. I lf*8l)-90. MORPIIOLOOY AND PIIY8I0L0RY OF TIIR CKLI,. In Fig. 12 is shown a cell from the base of the tpiihclial layer, which has certain peculiarities worthy of note. In one of its two nuclei is a cavity containing an cosinophilous, dumb-bell-shaped structure. The chromatin of this nucleus is very much condensed, but a portion of it is extended into the cavity in the form of doubly-beaded roi n METHODS OF STUDY. I used several methods at the outset of this research but finally gave the preference to one mode of preparation which included either Flcm- ming's Fluid or corrosive sublimate as the hardening reagent. This mode of preparation was as follows : The animal {Diemyctylus viridescens, Amblystoma punctatiim, Pletlio- don glutinosiis) was decapitated, the abdominal cavi' 't opened, the pancreas snipped away and immediately dropped into a saturated solu- tion of corrosive .sublimate, where it remained ten to fifteen minutes, or into a quantity of Flemming's Fluid, wnere it was left from one to twenty- four hours, according to the need. The operation of removal was usually done within twenty seconds, this interval including the decapitation process al.so The object of this was to prevent any post-mortem *Untersuchuni;cn Uber einige intra- and cxtranucleare Gebilde im I'ankreas der Siliigethiere auf ihre Dezieliung zu der Secretion. Von C. Melissinos. Mitgetheilt von K. Nicolaides. Arch. fUr Anat., und Phys., Phys. Abth., 1889, p. 317. f ii\ !5 12 TRANSACTIONS OF TIIK CANADIAN IN8TITUTK. [Vol. 1 changes in the pancreatic cells and I believe that it was attained in every case. The piece of tissue was after removal from either of these fluids, vva.shed for a few seconds in distilled water, then transferred to 70% alcohol for three hours, in the case of the corrosive sublimate prepara- tion, and for twenty-four hours, when the Flemming's Fluid was used. When the latter was allowed to act longer than one hour, the alcohol was changed as often as it presented a trace of chromic acid coloration. The harden ng was completed by a stay of twenty-four hours in 95 per cent alcohol. The organ was now transferred to the staining fluid, alum hiumatoxylin, (a few drops of a saturated solution of ha;matoxylin in ab- solute alcohol to a saturated .solution of pure ammonia alum in distilled water: allowed to stand one month in summer sunlight before using, and kept from deterioration by crystals of thymol), for ten to fifteen hours. In order to prevent overstaining, I found it advi.sable to dilute the original ha;matoxylin solution with twice its volume of distilled water, in which dilution, after the time allowed, there is only a pure chromatin .stain in the nuclei of the pancreatic cells and a faint shade of purplish blue in the nebenkerna. The objects are now washed in distilled water to remove the alum and the excess of the staining fluid, and are then put in a quantity of a i per cent solution of eosin in 30 per cent alcohol for from two to three hours. Washed in 95 per cent alcohol, till the latter ■was but faintly colored with the eosin after one hour's action, the object was placed in absolute alcohol for five minutes, then in pure chloroform for fifteen hours on the average, after which it was kept in a saturated solution of paraffin in chloroform at 3S°C. for about eight hours, and finally placed for a like period in melted paraffin {melting point 52°C). The sections were made of a thickness not ex- ceeding 5m with the Thoma-Yung microtome and fixed by the ribbon method in series to the slide with a diluted Sch'allibaum's clove oil- collodion mixture, (clove oil i volume, collodion 3, equal parts of absolute alcohol and ether 3). I used, sometimes in the case of the corrosive sublimate preparations, the Gaule method oi' fastening the paraflfin sections to slide, but, as the process of staining on the slide was not employed, except when the action of saffranin was required, it did not present any points of advantage over the other, which was the quicker. The paraffin was removed with benzole and '^'le sections mounted in benzole balsam. The staining of the object as a whole with haematoxylin and eosin has the advantages of giving a regular and uniform depth of reaction in the various sections and the different parts of each, and of preventing the loss of important elements entailed by the process of staining on the slide. I found that a little practice enabled one to judpe of the length 188J [Vol. 1 1889-90.1 MORPHOLOOY AND PUYSIOLOOY OF THE CKLU IJ of time necessary to give the tissue its proper depth of stain, and I determined that a stay of eight or ten hours longer than usual in the diluted hematoxylin solution, did not seem to increase the depth of the stain, or to make it more diffuse. Probably the explanation of this is that the equilibrium between the coloring matter in the diluted solution and that deposited in the tissue is reached when the chromatin is saturated. This, of course, is merely an application of the principle, that length of time and degree of concentration are elements in the right employment of staining methods and that these are, roughly speaking, in inverse proportion to one another. In order to determine if the nebenkerne contribute in any way to the elaboration of the secreted elements of the pancreas I resorted to the use of pilocarpin. I had a large number of Diemyctyli at my disposal, and on these I studied the action of the drug, so far as the nebenkerne are con- cerned. Batches of ten, twenty and thirty were taken, and into the ab- dominal cavities of each of these less than 2 mgrm. of pilocarpin was injected. Three of these were, at certain periods after the injection, decapi- tated, the pancreas of each removed, hardened with corrosive sublimate, and treated as described above. These periods were usually : i, 2, 3, 4, 5, 7, 9, [2, 17, 22, 36,44, 52 and 60 hours, and these were chosen in some cases for convenience. I took three at each period, because, if I depended on one, misleading results might be obtained. It was found that the averages of the results obtained from each three agreed with each other in presenting an unbroken outline of the history of the nebenkerne. I treated very young forms of Amblystotna punctattim also with pilo- carpin, the method of employment of the latter in this case being to dis- solve twenty to fifty milligrams in about half a litre of water and placing the animals therein for a period of five to twelve hours. As they mea- sured between thirty and thirty-five millimetres in length, it is obvious that an intra-abdominal injection of a solution of the drug was out of the question. The specimens of Necturus kept in the laboratory aquarium were not used for this investigation, since, owing to their not having been fed for a long time, the pancreas presented a more or less atrophied condition. It was found impossible to stimulate the gland in these to activity, or even to make it secrete at all. There is a great advantage to be obtained from the concurrent use of the two hardening reagents, corrosive sublimate and Flemming's Fluid. The former fixes thoroughly and quickly the zymogen granules as well as the cellular and nuclear structures in the pancreas, while A^ith Flem- tl Vl y i H THANHACTIONS OF THE CANADIAN INSTITUTK. [Vol. I. mine's Fluid, though the cell structure and nucleus arc well preserved, the zymogen is dissolved out of all the cells except those at the immediate periphery of the organ. This removal of the zymogen is due to the acetic av 'd in the fluid, which penetrates '"here another constituent of the same mi.xturc, osmic acid, is unable to ditl'use. The action of acetic acid in this reagent enables us to distinguish between zymogen and other granules which have the same staining capacity with cosin. The osmic acid, fur- thermore, gives a dark tinge to the nebenkerne and unusual bodies in those cells near the periphery and thus brings them out in clear contrast to the other cytoplasmic structures. OnSKKVATION.S. In sections made from the pancreas of Dieinyctyliis, which has been hardened with P'iemming's Fluid and stained with hematoxylin and eosin, one observes in addition to the nucleus and cell protoplasm and, some- times, zymogen granules, other structures which can be ranged in two groups at least. One of these groups comprise forms whose fundamen- tal structure elements are thick or thin fibrillar, either in sheaf shape, or wound in a ball fashion (Fig. i). Sometimes the fibrillae may be so thick as to merit the designation threads (Fig. 8). These forms are usually but not always, placed between the nucleus and the membrana propria, and they frequently sit, cap-like, on the nucleus, or the latter may be indented by them. In the second group, which are, at the outset, unlike the first, in that they are placed in cavities of the cell, are structures which present a varied form and composition. They are sometimes eosinophil- ous, sometimes chromophilous, and at times they present both characteis, They are numerous in the pancreas of a freshly captured animal, but are not so much so as the members of the first group. The members of these two groups of intracellular elements have been confused by other observers, and Ogata describes them as derived from the plasmosomata migrated from the nucleus, while Steinhaus appears to believe they are all parasites. In order to show that the views of these observers are hasty generalizations from a limited number of results, I propose to go fully into the description of the structure origin, mode of production, and history of each group. As plasmosomata, migrated, or extruded from the nucleus, are sometimes present, and as they have a different history, they merit special attention as a third group. These three groups may then stand in the order of description as follows : 1. Parasites. 2. The remains of broken down cells and nuclei swallowed by healthy adjoining cells. 1889-90] MOKPHOLOOY AND Pll YSIOKOOV OF TIIK I'KLt. 15 3. Plasmosomata, migrated, or extruded from the nucleus' into tlio cell protoplasm. I. Parasites. These arc, as already said, usually, but not always, placed between the membrana propria and the nucleus of the cell. They vary in size, measur- ing in their extreme limits i/x and 9/i, and their shape, usually oval, may also be oblong, spherical, elongated, club-like, or crescentic in section. They are not very sharply .separated from the protoplasm of the cell and if the latter is dense, their outlines arc distinguished with difficulty. Their structure varies also, but there are certain features in this respect which arc tolerably constant for the great m,<•//;;/ and \2ri/n). Nor arc these bodies confined to the pancreas, for I have found them in the cphithelial cells of the intestine, in the liver, the kidney and cutaneous epithelium of Diciiiyctylus and Nictiiriis. , They indicate, however, how little of a tissue is normally lost to itself and how it husbands its waste material. It is, of course, on first view, surprising that the pancreatic cells .should exhibit am'i'boid properties, but it is less so when we remember that the hcp;itic cells, wiiich in sections have a definite and apparently fi.ved form, manifest in the teased out scrapings from the cut surface of the fresh liver annnboid movements. 3. Mi(;ii7 and Ranatra the chitin necessary for each process is elaborated in a cavity between and surrounded by two epithelial nuclei, and the only legitimate conclu- sion from such a circumstance is that the chitin is derived from a nuclear substance. I may also here refer to the fact that my own observations have definitely shown that the h.emaglobin of the red corpuscles in Xeciiirus SiXid Aiiiblystoiiia \s derived from the chromatin of the nucleus both of the fully formed as well as of the developing red cell, and that the ha;maglobin so formed diffuses through the nuclear membrane and be- comes fixed in the cytoplasm. All these facts point definitely to the prominent part played by the nucleus and if everything in connection therewith is carefully studied, it will be admitted, I believe, that the inter- pretation which I have given of the changes occurring in the nuclei of the pancreatic cell during the various phases of glandular activity, is not a strained or a far-fetched one. APPENDIX. After the foregoing was written, a paper containing the observations of Eberthij: on the pancreatic nebenkerne in salamander came into my hands. In this is advanced a new view of the relations of these bodies, or pseudo-nuclei, as Eberth prefers to call them. He states that they are developed out of the reticular fibrillcTe of the cytoplasm, the latter at spots apparently becoming swollen, or thickened by fusion with their neighbors, and at the same time altered in composition, whereby their * See a resum^ of such researches in Strasburger : " Ueber Kern-und Zelltheilung im Pflan- zenieiche nebst einem Anhang iiber Belruchtung," Jena, 1888, pp. 194-204. t" Ueber einige interessante Vorgange bei ('er liildungder Insecteneier." Zeit. iiir wess, Zool. Bd. 45. JNeber Einschlussein Epithelzellen. Fortschritte der Medicin, Sept. i,, 1S90. 1 889-90. j MORPHOLOGY AND I'lIYSlOLOCiY OF THK CKLl,. capacity for absorbing staining reagents is inc/cased. Later several of sucii bent fibrills approach one another and acquire the shape of a sickle, semi- circle, or circle. The latter show all possible stages of transformation into the laminated bodies and spherules, which possess a very irregular fibrilla- tion appearing to consist of loose threads, while they may at times resemble laminated colloid bodies. The pseudo-nuclei disappear during hunger, while becoming gradually paler and less easily stainable. As to the process and manner of disintegration Eberth could offer no cxplanat' p He compares these bodies with structures described by Czcrmak as occurring in the ethmoid cartilage of the calf, and with those found b\- Solger in the cartilage cells of the shoulder-girdle of the pike. Ebcrtii believes these structures to be normal, and in a sense, compar.ible to the nodules of the nuclear network. Eberth states that the employment of corrosive sublimate as a harden- ing reagent and of paraffin for imbedding produces contraction and shrinkage in these objects, and that then one obtains the peculiar shapc'A which possess a certain resemblance to Cytozoa. He accordingly recom- mends Rabl's Fluid or Flemming's Fluid for hardening and celloidin for imbedding. Now I have carefully gone over the whole of my preparations since last October, and have during this winter made a number of new prepara- tions from Dieviyctyli and young Ainblystoiiiata, using for this purpose each of the three hardening reagents mentioned, frequently on pieces of the pancreas from the small animal. I have found that Rabl's Fluid often gives the appearance of coarse, parallel fibrillation in the pancreatic cells, when neither Flemming's Fluid nor corrosive sublimate demon- strated the presence of a single nebenkern in the parts of the pancreas hardened with either of these reagents. Such a parallel arrangement of coarse fibrillae is probably artificially produced. It appears also to cause a swelling of the cytoplasmic fibrilkt, whereby these are rendered more distinct, and I think that to this property is due the advantage obtained by the employment of Rabl's Fluid in demonstrating the elements of the achromatic spindles in dividing nuclei. My later observations strongly confirm my view that the nebenkern c are parasitic elements. In eight Amblystomata, killed during Januar)- and February, there were nebenkerne in only one, and here very abun- dant 'y. There could be no doubt about the sharply outlined form, as Stc haus has figured it, often homogeneous but as often fibrillated. I have seen quite distinctly the thickened portion ot the organism which simulates a head. As the Ainblystoiiiata kept in the laboratory tank were m 28 TKANSACTIONS OF THE CANADIAN INdTITUTE. [Vol. I. 'I not rcffularly fed, I attribute the intact form possessrd by many of the parasites to the lowered vitality of the host produced by want of food. Kbcrth's views are directly oppo.sed to mine. He considers the fibril- lation of the structures in question not as an evidence of their degenera- tion, but as a stage in their formation. His observations, confined as they were to one form, cannot, I think, be held as conclusive by any one who has studied the changes in the pancreas of Amphibia as exhibited throughout the year. I cannot share Eberth's views as to the action of corrosive sublimate on the form of these bodies and that it does not produce a contraction or shrinkage, as he maintains, is shown by Figs. I, 2, 9, and lo^, n/>, which were drawn from preparations made with this reagent. I would call attention to Fig. lo/;, )i/>, which shows a form not at all uncommon in the specimen oi Aiiiblystoina re- ferred to in the last paragraph and which is very like some of the specimens of Drepanidiuin figured by Gaule. I have, in this connection, made further observations on the elabo- ration of the pancreatic ferment. The results of these are confirmatory of the views already advanced by me and may be summarized as fo lows : — 1. In the gland cell filling up with zymogen granulesj the latter are largest at the border of lumen of the gland tubule, while the smallest are found at that edge of the granular area nearest the nucleus. This serves to show that the granules are increased in volume by the depo- sition of a substance from the " protoplasmic " area of the gland cell. 2. While the eosinophilous substance disappears from the nucleus, the "protoplasmic" zone becomes eosinophilous at a time nearly coin- ciding with the commencement of the deposit of granules in the cell In other words, the eosinophilous (or safranophilous) substance diffuses out the nucleus to the protoplasmic zone of the cell, from which it is ai)parently removed to be fixed in some way in the zymogen granules. 3. In the gland cell after exhaustion and when a restoration of its active condition commences there is an absorption, apparently from without, of chromatin, or ot a chromatin-like substance, by the protoplas- mic zone, and it would seem that the nucleus increases its quantitj^ of chromatin from this source. [Vol. I. any of the t of food. 3 the fibril- r dcgenera- jonfined as by any one IS exhibited le action of it does not shown by itions made _ „/;, which iblystoma re- some of the n the elabo- confirmatory arizcd as fo :he latter are e the smallest ucleus. This by the depo- - eland cell. e nucleus, the nearly coin- 2S in the cell stance diffuses )m which it is logen granules. iteration of its pparently from ,' the protoplas- its quantity of '.^'^^'O :| /"■ mmt^4 nil '{»' ^ ^ .5. V.>; .'>■•' ' r. ■' '! ;..,■* rf . ■• ,-.■ jl /' •■isif^'o^;^ •V'- ■ , 'I. •r' •■■■■:' I'i/l "lt.w V r t;:;.v/)l;.f7?'V: , (•■Ml;,::.. nb /"■ /.>. '^ ..3) /4. /' 7 ">;: ?;."" »/'(. '1 r.iii.'.iu Ikiii.-.. (.'.m. Ii isl A'ol. I I'latc I. > n ■■•I /' J« -. ■ .•I.'. A* ■?,',•;;■ '■•.•■.'■■<-,"-V>-vi ■•^;.r;, *.^ ,® ^ i)c- 9 9 > /'/, „ ,T.i'n'''T""''"^L-'.r . '.v t' ■' '\ I /' /.5. /(). ^^•'\ty^\TiKr^V- .1 -.J JiV /b> LMW'-rrri.lf^i—r .-• v xhi-':-: l¥' 1889-90.] MOKPnOI.OCiV AMI l'IIV>ililI,(i(JV OK TllK ( KLL. •2\) EXIM.ANATION 0|- ri.ATI I. The illustrations arc dreiwn with the Ahbc camera lucida, ccmbiiied with the 3111m. or the 2mm. apochroinatic objectives (Zei-is), and compen- sation ocular 4 or K. I'"igs. I- 1 1 are from the intestine of Dieinycty/i(s ^in'i/rsrri/s. ]'\ represents the cavity in uhicli, ap[)arently, a parasitic element matured and whose spores are seen in the atljacent cytoplasm. In the central cell are probably both spores and inva^inated. cjtolyseil material, while in the cell to the left there are structures which from their sliape appear to be parasitic. X720. Fi{^. 2. Two epithelial cells in one of which the nucleus is degenerated. In both cells arc seen structures exemplifying two stages in the develop- ment of the same parasite. In the degenerated cell the parasite, /», is ma- tured, but in th(; cytoplasm of the other cell, they are, apparently, all comma-shaped. Xiooo. Fig. 3. A single epithelial cell containing a fairly typical specimen of the parasite, it, the cellular nucleus, fi/>, the nucleus of the parasitic organ- ism. There is present a cavity and a rim of thickened protoplasm,/;'. Fig. 3(?. An epithelial cell in which the parasite, p, is in the stage of transition from the comma to the adult form. Xicoo. Fig. 4. In this the parasite, /, possesses a central mass of protoplasm in which is imbedded the nucleus and which sends processes toward the periphery. The remains of the tail of the comma are still recognisable in the denser portion of the periphery. Y 1000. Figs. 5, 6, and 7. The sporulation stages of the parasite with the tnibecular arrangement of the cell protoplasm pc, well marked The horseshoe form of the spore is clearly shown in 6rt!. Xiooo. Oa. X2250. Fig. 8. Represents five stages in the development of the sporulation phase of the parasite. In a the thickened band of protoplasm at one side represents the remains of the tail of the comma stage ; in l> the two central rings probably represent a stage of mitosis which is further ad- vanced in c; \n d the spores are formed each in a cavity of the ptoor plasm and these are further developed in e. X 1000. i< 1*1 M 30 TRANHACTlD.NS (IK IIIK CANADIAN INSTITt'TK. [Vol. I. « I I''K- 9- Represents illustrations of comma forms met witli in tlic epi- tliclial cells. In n coiled form is shown. Xiooo. I'i^;. lo (i-(/. are illustrations showing,' tlie way in which the comma is tr.uisAjrmcd into the adult parasite; <•-/ represent forins which show the v.irious ways in wliich the nucleus, cavity and tail arc disposed in the ailult or developing,' form. X lOOO. I'ifj. I \(i. Represents a section of an epithelial cell in a cavity of which arc eni;,miatical structures, the larjjer one probably bcid}^ parasitic, the others may be cither parasitic or protoplasmic mas es with chromatin spiicrulcs. Xiooo. Fig. I \/>. In this cell arc a number of i»tructure^ all of which arc evi- dently parasites. Xiooo. Fig. 12. A cell found in the epithelial layer of the intestine of A\rtii- yiis,ii, the nucleus,/, plasmosomata-like masses which may be parasitic. n\ a nucleus in which the chromatin is principally massed at one side and continued into the cavity in the form of doubly beaded rodlets ; a dumb- bell shaped body, deeply eosinophilous, is shown in the act of migration from the cavity. X66o. Fig, 13. Epithelial cells of the intestine of Mr/wr/zj'; ;/, the epithelial nuclei ; /, the nuclei of leucocytes ; /», two intracellular parasites lying in cavities of the cell. XG60. Fig. 14. Intestinal epithelial cells of .Vtr///r//j ; /, parasitic elements ; /, the nucleus of a leucocyte, x 660. Fig. 15. Intestinal cells of Nccturns ; p, parasites migrating from from nucleus ; /', either in vaginal ed, cytolysed material or stages in the development of the parasite. X600. Fig. 16. A single epithelial cell of the intestine of AVf///r«j, showing a large cavity in its proximal part occupied by a parasite /, and proto- plasmic remains, /r. X600. Explanation of Plate II. The outlines of all the figures were made with Abbe's camera lucida in combination with 2mm. apochromatic objective and compensation ocular, 4 or 8. In the case of Fig. 8 the drawing was made at the foot instead of at the level of the stage of the microscope, hence the difference in the magnification. be, blood corpuscle. ci)t, cytolysed masses. [Vol. I. in the epi- - comma is h show the •scd in the ty of which rasitic, the chromatin chare cvi- of AW///- parasitic. e side and a dumb- Ill i{iratio 11 epithelial lying in lements ; ng from s in the showing d proto- ucida in 1 ocular, stead of e in the I ■\ 'in Ill) t .5. •\'i L" 9 ■till nh iih nh o^^^'r' 'u jtnii'ij "p^ •<^^^ III l>. t III ill III •, « r/?;// iriic I'll III »f# *''• -r^ -f^ Rx y (■/;/ j cm ^^ chm- /^ r\Wt ■■(■ cm *. jiiii( II h irlii- ]l.s (). nil ^■!l .^'■< nb'~^.' /(/; /(/. J ll*\'Jl Oil ■/(/^ /'(■/(( .*/,^■ w l/fl^ .* ■^.'; CO /i?. 4#^ / li&.Ar'f ff H^'r." V .*{ Jt%Vif. r;,7J*.^»^M/'>''rf. ! I V 18«!)-90.] MOKPHOLOGY AND IMIYSIOI.OOY OF THK CELL. 31 dim, chromatin masses or bodies derived from chromatolysed nuclei. pmeg, protoplasmic bodies loaded with eosinophilous granules like zymogen, but insoluble in acetic acid. ul\ ncbenkcrn, relic, remains of chromatolysed nuclei and cells. "St zymogen granules. Fig. I A resting pancreatic cell from Diciiiyctylus; iil, a large irregu- lar plasmosoma ; the chromatin is very abundant. Corrosive sublimate. l[;em., cosin. Xiooo. Fig. 2. Two resting pancreatic cells from the same preparation as the last. In the right hand cell the elasticity of the fibrils of the degenerated nebenkern has sprung out the cell wall. X looo. Fig. 3. From the active pancreas of Dieviyctylus. Illustrates the invagination by normal cells of cytolysed material. The cavity in the centre occupied by the round mass, rchc, was probably the site of the cylo- l)'scd cell, and from this the cytolysed products have passed to the sur- rounding cells. The part represented occupied the centre of the section and the meshes of the cytoplasmic network were filled with zymogen granules which were dissolved out by the acid hardening reagents. It is to be noted that the nuclei here are large and rich in chromatin. Flem- ming's Fluid. Hicm., eosin. Xiooo. I'ig. 4. Taken from near the margin of a similarly prepared section and therefore showing zymogen granules ; a, enlarged nuclei ; b, a nucleus with a sickle-shaped clement, half within and half without the cell. Xiooo. I"'ig. 5. I'Vom the resting pancreas of Dieuiyctyhis. The part drawn was from near the margin of the section. In the centre of the illustration is shown a cavity or intercellular space partially occupied by cytolysed material and the chromatin derived from it is found in the adjacent cell (dim), whose nucleus is greatly enlarged. The other nuclei are somewhat irregular and rich in chromatin. Flcmming's Fluid. Ha;m.,eosin, safra- nin. Xiooo. I'ig. 6. From the central part of a section from the pancreas of a freshly captured specimen of Diemyctylus. Here also are shown free intercellular masses, and in the adjacent cells spherules of chromatin and cytoplasm ; a represents a single cell from the same preparation. Flem- mings Fluid. Iliem., eosin. Xiooo. Fig. 7. Three pancreatic cells from Diemyctylus. The formation of zymogen has advanced somewhat, the chromatin is abundant and the I %\ ft ki.\ ^H 32 TRANSACTIONS OF THE CANADIAN INSTITUTE. [Vol. I. karyosomata numerous and sometimes large (ks). The plasmosomata of which there are two to each nucleus are usually large and irregular in shape. Corrosive sublimate. Ha;m., eosin. x looo. Fig. 8. From the pancreas of a specimen of Dieniyctylus deprived of food for five weeks. Corrosive sublimate. Haem., eosin. Hg- 9i From the pancreas of a specimen of Dicmyctylus removed forty-five hours after an intra-abdominal injection of o.4mgrm of pilo- carpin. Corrosive sublimate. Ha;m., eosin. XlCX)0. Figs. lo and 1 1 . From the pancreas of specimens of Amblystoma pnnc- tatum (developing into adult condition). Fig. lo, a-f, drawn from the same pancreas. Corrosive sublimate. Haim., eosin. XIOOO' Fig. 12. Cells lining the pancreatic ductlets of Dicmyctylus, showing in their interior cytolysed and chromatolysed products a and b, from the pancreas 24 hours and one hour respectively after the intra-abdominal injection of pilocarpin. Corrosive sublimate. Ha^m., eosin. Xiooo I !