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He obtained his Ph.D. degree at the Sorbonne in 1902 and worked for many years at the University of Lyons. He came to Paris about 1921; at that time he was already famous for his work on the development, sexuality, tax- onomy and phylogeny of the Endomycetes and Saccharomycetes; the higher Ascomycetes; the cytology of the Cyanophyceae and Bacteria; starch formation; and especially on account of his research and theories on the chondriome and vacuome. He was appointed Chairman of the Department of Botany at the Sorbonne in 1935, and elected a Membre de I|’Institut in the same year. Most of his later papers and those of his students (among whom EICHHORN, PLANTEFOL, MANGENOT and GAUTHERET should be mentioned) have been published in his own journal, the Revue de Cytologie et de Cyto- physiologie Végétales. He married HELENE Popovicil, at one time his assistant, the daughter of Professor Popovict of Iasi in Rou- mania. His principal publications are being listed on pages 229/232 of this volume; cf. also “Titres et Travaux Scientifiques de M. A. GUILLIERMOND” (Lyon / Rey, 1921) and Chron. Bot. 11:128* (1936). LTE CYTOPLASM OF THE PLANT CELL BY ALEXANDRE GUILLIERMOND Professor of Botany at the Sorbonne, Paris - Membre de l’Institut AUTHORIZED TRANSLATION FROM THE UNPUBLISHED FRENCH MANUSCRIPT by LENETTE ROGERS ATKINSON, Ph. D. Foreword by WILLIAM SEIFRIZ Professor of Botany, University of Pennsylvania 1941 WALTHAM, MASS., U.S.A. P ublished by the Chronica Botanica Company First published MCMXLI By the Chronica Botanica Company of Waltham, Mass., U.S. A. All rights reserved New York, N. Y.: G. E. Stechert and Co., 31 East 10th Street. San Francisco, Cal.: J. W. Stacey, Inc., 236-238 Flood Building. Toronto 2.: Wm. Dawson, Ltd., 70 King St., East. Buenos Aires: Acme Agency, Diagonal Norte 567. Rio de Janeiro: Livraria Kosmos, Caixa Postal 3481. London W. 1.: Wm. Dawson & Sons, Ltd., 88a Marylebone High Street. Moscow: Mezhdunarodnaja Kniga, Kusnetzki Most 18. Tokyo: Maruzen Company, Ltd., 6 Nihonbashi; P. O. Box 605. Calcutta: Macmillan and Co., Ltd., 294, Bow Bazar Street. Johannesburg, S. A.: Juta and Company, Ltd., 43, Gritchard Street. Sydney: Angus and Robertson, Ltd., 89 Castlereagh Street. Made and printed in the U.S. A. FOREWORD = One cannot read this book by Professor GUILLIERMOND with- out being profoundly impressed with the thoroughness with which it has been written. No one has yet presented, nor is any one for a long time likely to present, so complete and authoritative an account of the mitochondria story. Most of us have been rather bewildered by the prolific, detailed, and contradictory literature on mitochondria, chondriosomes, chondrioconts, chloroplasts, amylo- plasts, plastids, etc. It is, therefore, a satisfaction to have it all assembled in a relatively condensed form. I remember the day, it was a meeting of the Royal Microscopical Society in London in 1921, when a number of cytologists admitted that they were at last convinced that mitochondria are not arti- facts. GUILLIERMOND handles the subject of artifacts very con- vincingly. He is forced to because much of his work has been on fixed material and much of it has been subjected to the usual cry ot “artitact’. GUILLIERMOND uses one bit of evidence in support of the reality of certain cell structures which I should like to apply to another part of the cell, the existence of which has also been much ques- tioned, namely, the spindle fibers. Proof of the reality of vesicles is to be had, says GUILLIERMOND, in the fact that they always appear at the same stage of development of the cell whatever the fixative employed. This is likewise true of spindle fibers. SHIMAMURA has recently offered evidence of the existence of spindle fibers by showing that if a cell in midmitosis is centrifuged and then fixed, the spindle with fibers is found to be centrifugally distorted. Centrifuging cannot distort an artifact before it has been formed by fixation. Though supporting the reality of mitochondria, Professor GUILLIERMOND in no way enthuses unduly over their possible sig- nificance in the life of the cell, as have some other workers. He regards as untenable the theory that chondriosomes are symbiotic organisms. He also discards the idea that chondriosomes are the means by which every synthesis in the cell takes place. In reject- ing the theory that chondriosomes are distinct organisms, a concept based on their superficial resemblance to bacteria and the fact that both are stained by the same dyes, GUILLIERMOND shows a breadth of mind characteristic only of the true scholar. He says, that though the theory is untrue, it gave rise to much good research. He concludes the section on the function of mitochondria by the courageous and laconic statement that “Nothing is positively known about the role of the chondriosomes”’. Controversial matters GUILLIERMOND handles with fairness and dignity. Each problem is dealt with unemotionally. He refers to SCHLEIDEN “as the promulgator of the cell theory”; to ROBERT HooKkE as the first to recognize the cellular organization of living things, but the significance of this structure was not understood for a long time thereafter. I anticipated a rather orthodox cytological handling of the sub- ject by GUILLIERMOND, and so was pleased to find him as awake to the contributions of the newer cytology as to those of the old. He takes DE JONG’s concept of cytoplasm as a coacervate and applies it to chondriosomes; vesiculation indicates that they too are coacervates. GUILLIERMOND deals with the question of the physical nature of the tonoplast. I should like to restate it somewhat differently, and what I say of it is also true of the outer surface layer of proto- plasm. All protoplasmic surfaces are probably coated with fats or other substances which are immiscible with water, but this does not mean that cell membranes are made of any substance other than protoplasm. Cell membranes are immiscible with water because protoplasm is immiscible with water. The immiscibility of protoplasm in water is not primarily due to an oily surface. Structural continuity is responsible. Protoplasm takes up water just as does a sponge, silica gel, or gelatine. Living matter is not a solution of salts, sugars, and proteins. Protoplasm holds to- gether. It could not be a living system if it did not do so. Such misunderstandings have arisen because protoplasm shows certain properties of liquids, such as rounding up and flowing. To certain students this can only mean that protoplasm is a liquid, and if it is a liquid it must be a solution. The colloidal viewpoint clarifies all this. Protoplasm does flow and therefore it is a liquid, but it is elastic, possesses tensile strength and contractility, and imbibes, that is to say, soaks up water. These are the properties of solids, their presence in protoplasm indicates structural continuity. Protoplasmic membranes, whether the inner tonoplast or the outer cell membrane, are of living matter, capable of the same physical and chemical changes as is the protoplasm which they bound. The cell membrane is not an inert layer of oil; it is a dynamic living system. When amoebae and slime molds move forward, the advancing surface is in a constant state of change. As the surface increases in area, material is added from the inner protoplasm, and as it decreases in area part of its substance is returned to the inner protoplasm. In short, the membrane is protoplasm. Convincing evidence of this is an interesting observation which led one of my students to flatly deny the existence of the tonoplast when it should have caused him to recognize that protoplasmic membranes are alive; that they are dynamic not static systems. He had observed a particle within the vacuole of a plant cell moving at the same rate and in the same direction as the streaming protoplasm. Close observation revealed that not only the mass of protoplasm but its surface, that in direct contact with the vacuolar sap, was also in an active state of flow. It was this streaming interfacial proto- plasm which caused movement of the vacuolar sap and the particle within it. There was therefore no quiet layer between protoplasm and sap, which meant to my student that there was no tonoplast. He failed to appreciate that the tonoplast and cell membranes in general are not inert skins but a surface layer of living proto- plasm. The tonoplast is protoplasm; in that sense is it formed of a substance immiscible with water. It is, as HUGO DE VRIES said, “a membrane differentiated and living”’. Much of the misunderstanding in regard to protoplasm arises from a failure to realize that protoplasm possesses both liquid and solid properties. Its liquid character is real but superficial. Its solid qualities are basic. That protoplasm is liquid is evident from the fact that it flows, but it is in no way comparable to a solution of salt in water. Viewing protoplasm as a liquid devoid of solid properties was an excusable fault in the classical cytolo- gists, but the modern physiologist is aware, as is GUILLIERMOND, of the true physical nature of living matter and the need of struc- tural continuity. GUILLIERMOND refers to this indirectly by attrib- uting to protoplasm such properties as torsion, elasticity, and immiscibility in water. WARREN LEWIS once expressed the need of structural continuity in protoplasm when he stated that were it not for the glutinous qualities, the tackiness of protoplasm, we should all fall to pieces. In short, protoplasm holds together, and this is as true of fluid protoplasm as of firm protoplasm. GUILLIERMOND concludes this book with the statement that the future of cytology lies in the union of morphology and physiology. In return for the admission by a morphologist that anatomy with- out physiology is sterile, let me say to Professor GUILLIERMOND that physiology is meaningless unless supported by structure and function. The present volume is the first addition, printed in the New World, to the list of books which Dr. FRANS VERDOORN is editing and publishing under the title of ““A New Series of Plant Science Books”. The book was especially written for the “New Series” by Professor A. GUILLIERMOND; it is the translation of an unpublished French manuscript and not merely an English version of a previous book by GUILLIERMOND. The translator is Mrs. ATKINSON of Amherst, Massachusetts (Ph.D. University of Wisconsin, sometime C.R.B. Fellow in Bot- any, University of Louvain). Mrs. ATKINSON has given us far more than a translation of the original manuscript, for there was much interpretation and rearrangement to be done. The translator is to be congratulated on accomplishing a difficult piece of work so well. Valuable help in editing the Ms. was also rendered by Dr. J. DUFRENOY of Louisiana State University. September 1941 WILLIAM SEIFRIZ CONE TS Chapter 1. INTRODUCTION :- HISTORICAL SKETCH — DIFFICULTIES IN THE STUDY OF CYTOPLASM. RECOMMENDED METHOD. .. . 1 Chapter 2. GENERAL FACTS ON THE STRUCTURE OF THE PLANT CELL, ITS CYTOPLASM AND MORPHOLOGICAL CON- STITUENTS:- THE CYTOPLASM AND ITS PERMANENT INCLU- SIGNS —— "THE SPARAPENSIn So Sir er 1 2 laren tee eee 5 Chapter 3. THE PHYSICAL PROPERTIES AND GENERAL CHARAC- TERISTICS OF THE CYTOPLASM:- ITS APPEARANCE IN LIVING’ FORM — VISCOSITY — Ricipiry — DENSITY — ECTO- PLASMIC LAYER — PHYSIOLOGICAL PROPERTIES OF CYTOPLASM — THE CYTOPLASM WITH REGARD TO VITAL DYES AND FIXED PREPA- PRACT IOING 2 CNAs hee SE ae ad oR ne ree fa eat ee Chapter 4. THE CHEMICAL CONSTITUENTS OF CYTOPLASM:- PROXIMATE ANALYSIS OF THE CYTOPLASM — THE CHEMICAL CON- STITUENTS OF CYTOPLASM. PROTIDES — LIPIDES — VARIOUS PRODUCTS AND MINERAL SUBSTANCES — WATER... . a3" Chapter 5. PHYSICO-CHEMICAL CONSTITUTION OF THE CYTO- PLASM:- ELECTRICAL CHARACTERISTICS OF PROTEINS — PHYS- ICAL CONSTITUTION OF CYTOPLASM — CELLULAR CONSTANTS AND EQUILIBRIA — IONIC REACTION OF CYTOPLASM — CELLULAR Chapter 6. THE PLASTIDS:- THE PLASTIDS — THE CHLOROPLASTS IN ALGAE AND BRYOPHYTES — THE CHLOROPLASTS IN VASCULAR PLANTS: THEORY OF SCHIMPER — CHEMICAL NATURE AND STRUCTURE OF PLASTIDS, — MOVEMENT OF CHLOROPLASTS.) =.) ste 40 Chapter 7. THE CHONDRIOME:- GENERAL CONCEPTIONS. WHAT IS MEANT BY CHONDRIOME IN ANIMAL CELLS — THE CHONDRIOME IN PLANT CELLS — THE CHONDRIOME IN FUNGI — DEVELOPMENT OF THE CHONDRIOME — PHYSICAL AND HISTOCHEMICAL CHARACTERISTICS OF CHONDRIOSOMES 24 ts. sicUh Ae Oh Rae? Salis ty eee 2 56 Chapter 8. THE CHONDRIOME (continued) :- THE CHONDRIOME AND ITS DEVELOPMENT IN THE PHANEROGAMS. RELATIONSHIPS BETWEEN CHONDRIOSOMES AND PLASTIDS. THE FACTS .... . 70 Chapter 9. THE RELATIONSHIP BETWEEN CHONDRIOSOMES AND PLASTIDS:- INTERPRETATIONS 85 Chapter 10. DUALITY OF THE CHONDRIOME:- THEORY OF THE AUTHOR — HISTOCHEMICAL AND HISTOPHYSICAL CHARACTERISTICS OF CHONDRIOSOMES AND PLASTIDS — DEVELOPMENT OF CHONDRIO- SOMES AND PLASTIDS AMONG THE PLANT GROUPS — PHYLOGEN- ESIS OF CHONDRIOSOMES AND PLASTIDS. .... ... 94 Chapter 11. HYPOTHESES RELATIVE TO THE ROLE OF CHONDRIO- SOMESUAND) PEASEIDS:.. 2 Gg) a a Sho. bw 6 Chapter 12. THE VACUOLES:- EARLY DATA. INSUFFICIENCY OF METHODS. TAERORY.OF LUGO. DEY VRIES. «5 2) ¢ os on Gye oo. web deo Chapter 13. VITAL STAINING OF THE VACUOLES:- COLLOIDAL SUB- STANCES IN THE VACUOLAR SAP — ACTION OF VITAL DYES ON THE CELLS. ADVANTAGES OF VITAL STAINING. . . rae hee Chapter 14. DEVELOPMENT OF THE VACUOLAR SYSTEM:- FIRST STAGES IN DEVELOPMENT — CHONDRIOSOME-SHAPED VACUOLES AND CHONDRIOSOMES. CHARACTERISTIC DIFFERENCES — PHYSICAL CHARACTERISTICS OF THE VACUOLES — CHEMICAL NATURE OF THE COLLOIDAL SUBSTANCE OF VACUOLES — VACUOLAR pH AND EET. RCS Bh SR I Meg mid "5° Ce nee Chapter 15. ORIGIN AND SIGNIFICANCE OF THE VACUOLES:- ‘ALEURONE GRAINS: THEIR FORMATION — REVERSIBILITY OF FORM IN THE VACUOLAR SYSTEM — THE PHENOMENON OF VACUOLAR CONTRACTION — ORIGIN OF VACUOLES — THE PRESENCE IN SOME CELLS OF SEVERAL DISTINCT CATEGORIES OF VACUOLES — DI- CUCRIVE MV ACUOUE St sac) cos. ls e. alkage el ay ot ee EO Chapter 16. THE ROLE OF THE VACUOLAR SYSTEM AND HYPO- THESES CONCERNING Rian tee oO oe BES Chapter 17. GOLGI APPARATUS, CANALICULI OF HOLMGREN AND OTHER CYTOPLASMIC FORMATIONS:- GOLGI APPARA- TUS AND THE CANALICULI OF HOLMGREN IN ANIMAL CELLS — POSSIBLE RELATIONSHIPS OF THE VACUOLAR SYSTEM WITH THE APPARATUS OF GOLGI AND OF HOLMGREN — RELATIONSHIPS BE- TWEEN THE GOLGI APPARATUS AND THE CHONDRIOSOMES AND PLASTIDS — THE SO-CALLED GOLGI APPARATUS IN PLANT CELLS = OTHER CYTOPLASMIC FORMATIONS 5. ef. cee ee ee EOL Chapter 18. LIPIDE GRANULES, MICROSOMES AND OTHER META- BOLIC PRODUCTS:- FATTY DEGENERATION — OTHER META- BOLIC’ PRODUCTS) o> oss oS aS cle ease ee ae Chapter 19. CYTOPLASMIC ALTERATIONS:- ALTERATIONS PRODUCED IN DYING CELLS — ALTERATIONS PRODUCED BY VARIOUS PHYSICAL AGENTS — ALTERATIONS PRODUCED BY PARASITES. . . . 208 Chapter 20. SUMMARY AND CONCLUSIONS:- CYTOPLASM — THE CHON- DRIOME — THE PLASTIDS —- VACUOLAR SYSTEM OR VACUOME 214 BIBTIOGRA PEWY 650 36 ibe fn 2 aa eee op eae ee AUTHOR. INDEX.) is, 2 > 2 ive can yeiehea es eee INDEX OF PLANT AND ANIMAL NAMES. .. . . 246 Chapter I INTRODUCTION Historical sketch:- It is well known that the cellular organiza- tion of living things was first recognized by the English engineer, ROBERT HOOKE (1665), who, toward the end of the seventeenth century, was looking at sections of cork with a view to finding out what applications could be made of the recent discovery of the microscope. He described the tissue as formed of alveoli resem- bling a honey-comb. These alveoli he called cells. For a long time, however, the significance of this structure was not under- stood. The French botanist, BRISSEAU DE MIRBEL (1833) thought that cellular tissue was composed of vacuoles hollowed out of a homogeneous substance, which corresponded to living matter. It was MOLDENHAWER (1812) who, for the first time, demonstrated the individuality of cells. Having succeeded in separating the elements of tissues by maceration, he proved that cells have a wall of their own and cannot, therefore, arise as cavities in a homo- geneous substance. Later DUTROCHET (1824), TURPIN (1827) and MEYEN (1830) considered cells as morphological entities but their attention had been centered rather on the walls than on the con- tents of the cells. In 1830 MEYEN had discovered chlorophyll grains, starch grains and crystals within the cavity of the cell. In 1831 the English botanist, ROBERT BROWN, who gave his name to Brownian movements, discovered the nucleus in the epidermal cells of orchids. Shortly after (1838), SCHLEIDEN, the promul- gator of the cell theory, attributed predominating importance to the nucleus to which he gave the name Cytoblast and which he considered as the generator of the cell. According to SCHLEIDEN, a cell is formed as follows: in a matrix, the cytoblastema, there appears the cytoblast on whose surface a membrane then becomes differentiated which lifts itself up like a watch crystal, grows, and bursts away from the cytoblast, leaving an empty space into which the matrix penetrates by filtration. DUJARDIN (1835), in the cells of Infusoria, first accurately described living matter, to which he gave the name sarcode. NAGELI (1866) perceived in addition that plant cells are occupied by nitrogenous matter and VON MOHL at the same time described it under the name of Protoplasma and attributed to it a primary importance. According to this observer, the plant cell, made up of the protoplasm, contains a nitrogenous primordial utricle, lining its wall on the inside and enclosing the nucleus. This primordial utricle is the seat of special movements already seen by B. CorRTI (1772) and TREVIRANUS (1807). The rest of the cell is occupied by cell sap. : Guilliermond - Atkinson —2— Cytoplasm A little later, COHN (1850), THURET (1850) and PRINGSHEIM (1854) perceived that the zoospores of the algae lack a wall and are made up exclusively of protoplasm. Max SCHULTZE and DE Bary (1859) finally established the fact, once for all, that the protoplasm of plants is the essential substance of cells and corre- sponds to the sarcode of DUJARDIN. LEYDIG (1857) defined the cell as “a mass of protoplasm furnished with a nucleus’. Max SCHULTZE (1861) defined it as “a mass or lump of protoplasm endowed with vital properties”. While these conceptions were being established, the works of VON MOHL, MEYEN and NAGELI were proving the inexactitude of SCHLEIDEN’s theory of cell origin and showing that cells multiply by division. Thus the word cell (French cellule, from the Latin cellula, little room) came to mean the contents of the cellular cav- ity, a significance quite different from that given it by HOOKE, who observed only the walls. From then on, the conception of the cell was enlarged upon but the knowledge of its structure, making only slow progress, was to remain obscure for a long time. The early cytologists observed only living cells. This presents serious difficulties, for, with the exception of unicellular organisms, observation of living material can be carried out only after tearing or sectioning tissue, operations which risk injuring the cells. Cells examined in a medium not their own — water, for instance — may, during observation, undergo serious alterations. Lastly, observation of living material never allows the study of cell structure to be pushed sufficiently far, be- cause, except for cases which are unusually favorable, the different elements which constitute the cell show too small differ- ences of refractivity for it to be possible to distinguish one from the other with clearness, and this becomes, moreover, well nigh impossible in embryonic tissue in which the cells are very small. The introduction of the paraffin method on objects pre- viously “fixed” has greatly facilitated the work of cytologists. This method consists in fixing, 7.e., coagulating, the cells by means of various chemical reagents, then embedding the tissues in paraffin, cutting them in thin sections with the aid of a microtome and finally staining them. Several stains may then be applied to the sections, which fix this constituent or that, according to its affinity for the stain, and superb preparations may be obtained and made permanent in Canada balsam. But this method, convenient as it is, has, nevertheless, the serious difficulty of reducing cytologists to the study of dead cells only. Fixation, 7.e., coagulation, of proto- plasm, modifies the structure of the cell and exposes cytologists to serious errors in interpretation. Finally, this method does not allow the physical character or biological properties of the cyto- plasm to be studied, although they are very important, and cytology is reduced to pure cellular morphology. The use of the paraffin method has led to the striking discovery of karyokinesis and has rendered very great service in the study of the nucleus and, in par- Chapter I — 3— Introduction ticular, of the chromosomes, whose form in the fixed material is preserved with a minimum of distortion. On the other hand, as far as the cytoplasm is concerned, the paraffin method has, over an extremely long period, given only mediocre results and cytologists have made the mistake of neglect- ing too greatly the study of living material. As a matter of fact, the most convincing data on the cytoplasm were for a long time obtained exclusively from living cells: on the one hand, the excellent discoveries of SCHMITZ concerning the chloroplasts of the algae and those of WILHELM SCHIMPER and ARTHUR MAYER in the study of plastids of higher plants, and, on the other hand, the classical experiments of HUGO DE VRIES on the vacuoles and their role in the osmotic phenomena of the cell. It is only since 1910 that knowledge of the cytoplasm has made rapid progress. At that date, the timely appearance of the ultra- microscope enabled A. MAYER and SCHAEFFER to make known some essential data on the colloidal nature of the cytoplasm of animal cells — data which can be applied to plant cells. At about the same time, the introduction of mitochondrial methods, new fixation tech- niques for certain cytoplasmic lipides, led to the discovery of the chondriosomes and made it possible to preserve the plastids, to stain them clearly and to follow them through their whole life history. A little later, the methodical use of vital dyes, which accumulate in the vacuoles of living cells, made it possible to follow the evolution of the vacuoles through all the stages of cellular development. Finally, the invention of the micromanipulator and of motion-picture photography, and the perfecting of methods of observation i vivo, have contributed in large part to making known to us the physical properties of the cytoplasm and of its various morphological elements. Difficulties in the study of cytoplasm. Recommended method :- As a result of work carried on during the last twenty years by means of the mitochondrial technique, or with the aid of the ultra- microscope, or by the use of vital dyes, it has been possible to solve definitely the problem of cytoplasmic structure. The discoveries in this domain are still too recent to be accepted by all cytologists. But if these are still being discussed, it is simply because they have been arrived at by methods very different from those usually em- ployed and because some cytologists are unable to verify them by their own methods. The study of the cytoplasm, a substance which is very easily injured, is infinitely more difficult than the study of the nucleus. It is necessary to use a long and delicate method whose general outline will be indicated here. This method, which will be called the analytical method, 1.e., analysis of the cell, consists of the following serial operations: First, this method requires the use of fixed and stained sections which are indispensable in bringing out the elements which are considered as entering into the constitution of the cytoplasm. But this method is always insufficient, for once these elements have been Guilliermond - Atkinson a Cytoplasm brought out by fixation and staining, their actual presence must be checked by observation of living material in such cells as lend themselves to it. This observation of living material permits a proper appreciation of the value of the above methods. As the very handling of living material runs the risk of producing altera- tions, every precaution to avoid them must always be taken. Fungi may be used which can be observed in their own environ- ment, or organs studied which are sufficiently transparent to permit their cells to be examined without any manipulation. Petri dishes of a special type (used at the International Bureau for the Culture of Fungi at Baarn) can be used if necessary. In the bottom of these is an opening of 3 cm. which can be covered with a slide sealed with asphalt cement (Fig. 88). In this way, seeds can be grown aseptically and their roots during development can be observed with an oil-immersion lens by turning the dish under the microscope. Vital dyes must be used to enable us to follow such elements as the vacuoles which are not well preserved by any other means. More- over, each element whose presence has been recognized by fixation and staining must be described by means of a systematic study of its behavior with most fixatives and stains; this study must be based upon histochemical reactions of the element in question. This method, called histochemical analysis, permits us to characterize the element, to distinguish it from others, and to inform ourselves as to its chemical nature. It permits us, besides, to distinguish some elements which are revealed only by certain methods of fixa- tion and staining. Histochemical analysis must be followed by an histophysical analysis, i.e., analysis of the physical state of the element, its viscosity, colloidal state, and so forth, an analysis in which will be employed the micromanipulator, the ultramicroscope, the polarizing microscope, the centrifuge, the plasmolytic method and, if need be, motion-picture photography. This histophysical analysis will supplement the description obtained by histochemical analysis. Then, too, the development of the element throughout the entire life of the cell must be followed in order to know whether it constitutes a permanent element or is only transitory, and in order to obtain an idea as to its significance by observing its be- havior. Finally, it will be useful to supplement this method called the analysis of the cell, which comprises the various operations just enumerated and which has, itself, all the value of an experimental method, by a further series of experiments designed to clarify the role of the element studied. Among these experiments are vivisec- tion, depriving a cell of an individual element by means of the micromanipulator, and a study of nutritional influences on the be- havior of the element under consideration. This is the cyto- physiological method which we will turn to only occasionally here, our aim being chiefly the morphological study of the cytoplasm. The procedure specified is the only one by which precise facts may be obtained on the morphological constituents of the cytoplasm: the plastids, chondriosomes and vacuoles. Chapter II GENERAL FACTS ON THE STRUCTURE OF THE PLANT CELL, ITS CYTOPLASM AND MORPHOLOGICAL CONSTITUENTS The cytoplasm and its permanent inclusions:- In agreement with STRASBURGER and HENNEGUY, the term cytoplasm! is here under- stood to mean all the living matter in the cell with the exception of the nucleus. By the term protoplasm is meant all living matter in the cell, z.e., both cytoplasm and nucleus. The cytoplasm occurs in living cells as a colloidal substance, hyalin and homogeneous, elastic and of a viscosity which is always superior to that of water. This substance holds permanently in suspension a certain number of small elements which resemble bacteria in form and dimensions and are distinguished in living cells by a refractivity slightly higher than that of the cytoplasm. These elements, which are called chondriosomes, appear in the form of granules, rods and threads. In addition to these elements there are, in green plants, the plastids, whose form is very variable in the algae and which, in higher plants, appear in green tissue as large globules, filled with chlorophyll, which are derived from small elements very similar to chondriosomes in form and histochemical constitution. The cyto- plasm also contains small fluid cavities called vacuoles, composed of water, containing crystalloid and colloidal substances. These vacu- oles, which are very small and very numerous in young cells, swell and usually run together little by little, to form, in mature cells, 1 Von Mou. designated under the name of protoplasm, the living substance of the cell, i.e., the nucleus and that which in the present volume is called the cytoplasm. But STRASBURGER, HENNEGUY, and most of the modern cytologists have reserved the term protoplasm for the cell contents (with the exception of the wall), including the nucleus. Within the protoplasm they distinguish the nucleoplasm or karyoplasm which is the nuclear substance, and _ the cytoplasm, which is everything else. The term protoplasm, however, is often used as a synonym for that which, in this volume, is termed cytoplasm. HARDY means by cytoplasm all that is not nuclear and he considers the protoplasm to be all living matter within the cytoplasm to the exclusion of the nucleus: the cytoplasm therefore includes the protoplasm and the products of its activity, various inelusions not pertaining to the living substance. Borrazzi, on the contrary, incorporated the nucleus into the protoplasm or bioplasm which, for him, includes all the living substance of the cell, i.e., the nucleus, the living ground substance, which is not nuclear, and the chondriome. He reserves the term cytoplasm for all the cell contents, i.e., the total protoplasm and all the products elaborated by it. Among these last, Borrazzi distinguishes: 1, the metaplasm including the products of cellular elaboration which are permanent (cell walls) ; 2, the paraplasm, represented by the reserve substances or waste products, which are only transitory in the cytoplasm (starch grains, fat globules, etc.), as well as soluble substances formed by cellular metabolism (glycogen, inulin) which may be detected by certain chemical reagents. Thus, for BoTTazzi,” protoplasm means all that is living in the cell including the nucleus and the chondriome, whereas the cytoplasm includes the protoplasm and the products of its elaboration (metaplasm and paraplasm). Guilliermond - Atkinson — 6— Cytoplasm an enormous single vacuole which occupies the greater part of the cell, forcing the nucleus to the periphery. This vacuole is often traversed by thin cytoplasmic trabeculae which radiate from the nucleus to join the parietal cytoplasm. Lipide granules are encountered in almost all, if not in all, cells. They are scattered in the cytoplasm in more or less consid- erable numbers according to the cells and their stage in develop- ment. One also finds, moreover, various inclusions in the cyto- plasm: reserve or by-products formed during cellular activity (starch grains, crystalline proteins, various crystals, etc.) which are localized either in the cytoplasm itself or in the plastids or in the vacuoles. All these substances, however, are only transitory products formed during cytoplasmic activity. The paraplasm:- We now turn to a new consideration. It has just been seen that in the cytoplasm there are in suspension some elements which are always present such as plas- tids, chondriosomes, vacuoles and lipide granules. Among these elements, a distinction must be made between those which can be considered as belonging to the living substance, to the architecture of the cell, and those which simply re- sult from its activity. Among the lat- ter whose chemical composition is more simple, there are some, like the starch grains, which form in the plastids, others, like many crystals, which are lo- Ue Eon or nad. calized an the wacuoles, others still,-lrke cae ag fea P, chloroe the crystalline proteins, which are con- any ; tained in the cytoplasm itself. Now, the cytoplasm is a substance which continues to exist permanently. It presents a chemical composition still not well known but which does not vary appreciably. It is a living substance. The plastids are elements which are never formed de novo but, like the nucleus, are transmitted by division from cell to cell. They, therefore, may also be considered as living substance. The chondriosomes seem to be of like nature. Such is not the case for the vacuoles. Although always present in the cells, they appear to arise de novo and to dis- appear only to be replaced by new ones. Furthermore, they enclose substances which, all of them, are products of cytoplasmic activity: reserve products, or waste products, or transitory products of metabolism. They do not, therefore, seem to belong to the living substances of the cell. This is true for the lipide granules which are present in almost all cells, but which vary greatly in quantity depending on the state of development of the cells: there are cells which contain only a few, others in which the granules accumulate in great quantity and fuse to form large globules filling up the cytoplasm. These are also products of metabolism. Co- existent with these visible products, there are others, like glycogen, Chapter II —7— General Facts which impregnate the cytoplasm and can be detected only by using certain microchemical reagents. All these products arise by cyto- plasmic activity and a distinction must therefore be made between them and the cytoplasm by which they were pro- duced. These products are grouped under the term paraplasm or deutoplasm and are separated from the cytoplasm, which, with the plastids and chondriosomes, constitute living substance. Among the products resulting from the activity of the cytoplasm are some which are of permanent char- acter, such as the cellulose wall. This is a cyto- plasmic secretion which persists during the entire life of the cells and can not, it would seem, be considered as belonging to living substance. These permanent formations of the paraplasm or deutoplasm are specified as metaplasm. Lastly, the name protoplast is used to designate all the contents of the cell except the cell wall, z.e., the protoplasm and paraplasm together. In the cytoplasm, then, we shall have to con- sider the cytoplasm itself, the plastids, the chon- driosomes and the paraplasm, the most important constituents of the last category being the vacu- oles and the lipide granules. Fic. 2. — Semidia- grammatie represen- tation of a living epidermal cell of Al- lium Cepa_ showing cytoplasmic trabecu- lae traversing the vacuole and uniting the parietal cytoplasm with the nucleus. These, and other paraplasmic formations not listed above, will be studied in the succeeding chapters. Chapter III THE PHYSICAL PROPERTIES AND GENERAL CHARACTERISTICS OF THE CYTOPLASM Its appearance in living form:- DUJARDIN, who first studied the cytoplasm in living cells of ciliated Infusoria, has given an abso- lutely exact description of this substance which he calls sarcode, a description which modern observations merely confirm. ‘This substance”, he says, “appears perfectly homogeneous, elastic and contractile, diaphanous and refracting light a little more than water and much less than oil. One can distinguish in it absolutely no trace of organization: neither fibre nor membrane nor an appear- ance of cellular form’’. There is nothing to add to this descrip- tion. The cytoplasm appears to modern observers just as it was described by DU- JARDIN. In living cells, it appears to be a homogeneous substance, as transparent as glass, viscous, a little more refractive than water and non-miscible with it. As has been already stated, modern research has shown that it does, however, always con- tain in suspension numerous granules (chondriosomes, lipide granules) and vacuoles, of which more will be said later. Hence the description of DUJARDIN can be applied only to the cytoplasm itself, aR “ Ca ATS omitting these elements which it contains. pidadiediced of Chondrioderma For the study of the physical proper- difforme. A, ingested foreign ties of the cytoplasm, the plasmodium of ees REL beas Whe atthe Myxomycetes has been much used. It is seen as a voluminous protoplasmic mass of irregular appearance, lobed in the most fanciful manner and enclosing numerous nuclei. This protoplasmic mass changes shape constantly by virtue of the amoeboid movements which control its displacement. It glides along the surface of its support and if this latter be of decaying wood, it worms its way into the interior of the wood, penetrates it only to come out again further on, then to re-enter it, and so on. The huge dimensions of the plasmodium make it a valuable object for the study of the cytoplasm. The classical experiments successfully performed by PFEFFER on the plasmodium of Chondrioderma difforme have shown that in order to alter a small portion of cytoplasm, it is necessary to exert a pressure of 8 mg. per sq. cm. The cytoplasmic strands of this plasmodium break when subjected to a tension of 120-130 mg. per mm. This indicates a rather strong cohesion which, it may be said, can be even stronger in other types of cytoplasm. Chapter III oa Physical Properties Viscosity:- There has been a good deal of discussion concerning the cohesive state of cytoplasm, i.e., its consistency. Most cytolo- gists consider that the cytoplasm more nearly approaches a liquid than a solid state. A few, however, believe it to be of a solid consistency. The plasmodium of the Myxomycetes is very fluid. A proof of this is in an experiment carried out on Badhamia utricularis by the English mycologist, LISTER. LISTER noticed plasmodia of this fungus on the trunk of an old hornbeam growing in his garden. The trunk was covered over with the fruiting bodies of Corticuwm puteanum. The plasmodia of Badhamia moved around on the sur- face occupied by Corticium, actually consuming the fruiting bodies and after their passage leaving the bark of the hornbeam as smooth and clean as if no fungus had ever grown there. But, although Badhamia assimilated the Corticium tissues, its spores, protected by a resistant brown membrane, were not attacked and accumulated within the plasmodium which took on a dark brown color. LiIs- TER collected one of these plas- modia on a glass plate, where it moved about, leaving behind it as evidence of its passage, a fine brown network, formed of the ingested spores. These had been progressively dropped, being poorly retained in the plasmodial cytoplasm. This demonstrates its weak viscosity. The plasmo- dium, however, still enclosed Fic. 4. — Two successive shapes, A, B, Many spores. LISTER then put in taken by the plasmodium of a Myxomycete as it moves in the direction of the arrow. its path a barrier of wet cotton. The plasmodium passed through rapidly, leaving in the cotton all the remaining spores, and emerged showing the yellow tint characteris- tic of it before it had taken up the fungus Corticium. LISTER thus brought about the filtration through cotton of plasmodial protoplasm and in this way succeeded in demonstrating its very fluid consistency. This somewhat crude evidence may be made more specific by a detailed examination of the plasmodium. It is composed of a network of opaque, anastomosing veins which, in certain species, may attain large dimensions: even a diameter of several milli- meters in the case of the principal veins. Toward one border, the network merges with a fan-shaped continuous layer of the same material. This entire body moves at about the rate of 1 cm. per hour and presents constantly changing contours; nevertheless the continuous layer is always in front and may be interpreted as protoplasm flowing slowly over the substratum. The substance composing the plasmodium is, in fact, fluid and motile but shows at its surface the ability to coagulate. A puncture in a vein allows a drop of the internal fluid protoplasm to escape. This drop is immediately coagulated on the surface. The internal protoplasm, Guilliermond - Atkinson — 10— Cytoplasm then, under its immobile surface, flows with extreme rapidity. In addition, the different veins and the flowing, continuous layers com- prising the plasmodium, show very curious, rhythmic but not syn- chronous, pulsations. These are rendered quite visible by motion- picture photography. Each vein shows an alternation of systole and diastole; each outer layer flows by jerks over the substratum, becoming alternately thicker and thinner (COMANDON and PINOY, SEIFRIZ). Under sufficiently high magnification the plasmodium seems to be formed of an homogeneous substance, holding in suspension in- numerable granules, in particular, lipide droplets and ingested debris, whose incessant displacement in different directions reveals with great clearness the existence of cytoplasmic currents. These currents are very irregular. They may be rapid or may even rush along in a vein and, at a given instant, \ they will immediately slow up, then change direc- { tion, accelerate, retard again, return to the orig- inal direction and so on. In each vein the same irregularities are observed but without any syn- chronism whatever. Thus, the movement of the entire mass of the plasmodium in one direction expresses the sum of all these movements in dif- } ferent directions and indicates that in this appar- ent disorder, the protoplasm flows more in one direction than in the other; namely, in the direc- ’ tion of the advance of the organism. Now these characteristics of protoplasm have “Wg. 5 — Diagram nOthing unusual about them. Microscopical ex- of the different direc. aminations of the contents of the most varied cells eee re Gren reveal that there, too, protoplasm behaves as a portion of the vein- fluid substance. The bodies which it holds sus- Ike eum of @ ended in it are in most cases more or less rapidly carried along in its multiple currents. Here, as in the extended body of the plasmodium, these currents run side by side, separated by calm borders. They ramify, anastomose and change constantly. These are the phenomena of cyclosis for which the cells of Elodea canadensis and the staminate hairs of Tradescan- tia or of Celandine are classic subjects for observation. These phenomena are found again in Spirogyra in which they appear with great distinctness and of which more will be said later on. All these facts indicate, therefore, that cytoplasm flows. This presupposes a mobility of molecules found only in a liquid state, which, in a word, is the essential property of a liquid state. These movements are, however, much less accentuated in some cases. They seem to be nearly absent in certain plant cells, among others in yeasts and various fungi, in which the cytoplasm appears to be much less fluid and shows no displacement of granules. This seems also to be the case in most animal cells. Other arguments resting especially on observations of plant cells have been brought forward in favor of the fluid state of cyto- Chapter III —11— Physical Properties plasm. HOFMEISTER and BERTHOLD showed for the first time that if a section is cut out of a filament of Vaucheria, a part of the cyto- plasm comes out, taking a spherical form by virtue of the law of surface tension which characterizes liquids (Fig. 7). Since that time, many similar experiments have been performed, notably by STRUGGER on Chara cells. And finally, an experiment by KUHNE on the cytoplasm of the Myxomycetes, can be explained only by a liquid state of the cytoplasm. This worker succeeded in obtaining an artificial muscle by enclosing in elastic tubes, fragments of the plasmodium (intestines of Hydrophilus piceus). All these facts, added to those obtained by microdissection, which will be spoken of further on, permit us to conclude that the cytoplasm possesses, in general, the properties fundamental to liquids: it flows and has a surface tension which tends to make it take the form of minimum surface, 1.e., spherical form. Research carried out in these later years with the aid of the microdissector, put into practice by CHAMBERS, has made great progress in the knowl- edge of the viscosity of the cytoplasm. This method consists in the use of a special instrument, the micromanipulator, or microdissector, provided with glass needles which can be moved mechan- ically with great precision. This permits the dissection of cells under high magnifications. The work of SEIFRIZ with the micromanipu- lator on various plants (Mucoraceae, Fucus, pol- len tubes, plasmodia of Myxomycetes) also dem- onstrates that the cytoplasm presents a consist- ency which is very variable: now very fluid and almost like water, now almost solid, even to the _ Fic. 6. — Cyelosis state of a gel with a consistency of bread dough ™ %,,st¢minate Tak or vaseline, in which the passage of the needle arrows indicate the direction of the cur- leaves a gaping hole. rents in the various 1 idj 7 cytoplasmic meshes. The cytoplasm is very fluid in the plasmodium i descr ci Sr RN of the Myxomycetes as long as the latter is in an van TircHem and active state. If the point of a needle of the micro- ©*8™N7™): manipulator is broken off in the plasmodium, the cytoplasm is rapidly aspirated (cf. p. 37). Its fluidity is, however, always greater than that of water, intermediate between that of water and that of oil of paraffin, but from the moment that the plasmodium ceases growth, 7.e., in the stages which precede sporulation, the consistency of the cytoplasm increases: it equals that of oil of paraffin, then that of glycerin and, finally, that of bread dough. In Rhizopus nigricans, the cytoplasm is also liquid in the young por- tions of the hypha. It is, nevertheless, less fluid than that of the Myxomycetes and presents approximately the consistency of oil of vaseline. On the contrary, in the older portions of the hypha of Rhizopus, the cytoplasm grows thick. Its viscosity becomes that Guilliermond - Atkinson —12-— Cytoplasm of glycerin and then that of bread dough and may even attain and exceed that of vaseline. In the egg of Fucus the cytoplasm becomes more and more viscous as the egg matures and its exchanges diminish. Immedi- ately after fertilization it again becomes liquid and remains in this state in the embryo. Areas seem to exist in the egg, there- fore, where fluidity is more accentuated,—sort of centers of activity where different chemical exchanges are produced. It appears from all these observations then, that the viscosity of cytoplasm is always superior to that of water and in numerous cases as great as that of blood. Yet in certain cells, especially in old organs, the viscosity can be much higher. Moreover in dehy- drated organs, seeds, for instance, the cytoplasm can be more or less solid. Many attempts have been made to measure directly the viscosity of the cytoplasm inside the cells. The easiest method to employ is that which makes use of the law for falling spheres established by the physicist STOKES. The cytoplasm of certain cells encloses starch grains which are more dense than the cyto- > plasm and which have a tendency, me ® because of their weight, to fall through the cytoplasm to the lowest point in the cell. By using an hori- ewe =e ee ee aa zontal microscope, the time required enidddciutects to break apts CtOr the, erainsto tall/is*determinend real wauniied, (ater wan ts, 204, by applying the physicist’s form- GHeM and COSTANTIN). ulae, the viscosity of the cytoplasm is calculated. By this process of determining the speed with which starch grains and certain crystals fall through the cytoplasm, WEBER was able to state that the viscosity of the cytoplasm varies with the cell under consideration. It seems to increase with the age of the cells. Certain influences (increase in temperature, the action of certain chemical substances, such as narcotics) can also cause variations. HEILBRONN has employed the following method: an iron needle, very fine but too heavy to be carried about in the cytoplasmic cur- rents, is introduced into a plasmodium. The needle being located under the microscope, an electro-magnet in which flows a current of increasing intensity, is brought near the preparation. When the needle in the field of the microscope begins to orient itself in the direction of the lines of force, the experiment is stopped. The intensity of the current necessary to produce a visible deviation of the needle is divided by the intensity producing the same effect on the same needle plunged into pure water. With the viscosity of water thus taken as a standard, the quotient measures the viscosity of plasmodial protoplasm. 5 O09 Chapter III a Physical Properties The centrifuge has also been used on living cells. This brings about a displacement of heavy inclusions, such as starch grains, or of light inclusions, such as lipide granules, with a speed and facility varying with the consistency of the cytoplasm. This method is much less precise since the exact densities of the gran- ules and cytoplasm are not exactly known. These various methods have shown that the most fluid cyto- plasm has a viscosity which is only 3-5 times that of water and that the most dense cytoplasm (that of animal cells) reaches nearly 10,000 times the viscosity of water. Thus from the results obtained by means of centrifuging, microdissection and other techniques, an essential and very general conclusion may be drawn: the cytoplasm of plants does not present at all times the same viscosity, for this varies essentially with the physiological state of the organ under consideration. Rigidity:- The cytoplasm possesses, at the same time, a property which, as will be seen later, is tied up with its physical state and which is characteristic of cells, namely, a certain rigidity which gives to it an elasticity of torsion. Rigidity expresses the physical ties between the particles of the system in question, ties which are lacking in true liquids. This rigidity can be brought out by micro- manipulation. Thus, in displacing cellular inclusions within the cell, it was observed that sometimes these return to their places when pressure is released, the rigid surroundings acting as a spring, and sometimes the inclusions are displaced as from a liquid without rigidity. The stability or instability of form of these inclusions after being deformed, for instance being drawn out between two needles, also teaches us something of their rigidity and their varia- tions. In this way, SCARTH, for example, demonstrated the elastic- ity of the cytoplasm of Spirogyra. The nucleus of one cell of this alga, when pushed by a microneedle from one side of the cell to the other and then left alone, was observed to return of itself to its original position. Like viscosity, rigidity seems to be variable in the cell. Microneedles sometimes penetrate very easily into a fluid cytoplasm without reaction, and sometimes with difficulty into a thick gell. There is no method for measuring the rigidity of the cytoplasm. Density:- By a micropycnometric method, LEONTJEW succeeded in measuring the density of the plasmodium of Fuligo septica and has shown it to be, on the average, 1.040 for individuals collected in dry weather while it does not surpass 1.016 for those collected in wet weather, but eleven hours after sporulation it rises to 1.065. Ectoplasmic layer:- The cytoplasm is not miscible with its ex- ternal surroundings but remains always very sharply separated from them. In cells with no skeletal walls, the cytoplasm is sur- rounded by an external zone presenting a consistency greater than that of its central part. This zone is called ectoplasm, or ectoplas- Guilliermond - Atkinson —14— Cytoplasm mic layer, or plasmalemma, in opposition to the rest of the cyto- plasm which is designated as endoplasm. This is the only mem- brane which exists in the plasmodium of the Myxomycetes, in the Myxamoebae, as well as in various zoospores and spermatozoids of the algae and fungi. In other material, especially in the lower organisms, the equilibrium forms of the protoplasm when in the presence of water consist of lobes or pseudopodia, irregular and changing, blunt or spiny, perhaps in accordance with temporary and local modifications of surface tension. There again everything goes on as if there were an elastic layer around the cytoplasm. Figure 8 represents the division into two tiny plasmodia of Vampyrella. The individuals in the process of separation remain for a long time united by a protoplasmic strand which becomes increasingly thin. Then suddenly it breaks in the middle and the or | fe trations above 0.005% the dye stains \ IVE FA: 6) ( the chondriosomes at first but rapid- AA, pea f | ly causes them to become vesiculate, * I) ( A S then accumulates in the vacuolar system as well, and later brings sepricynia, showing the similarity of about the death of the fungus. Most ee ine ae es wae: In of the other dyes, among them not visible. Gr skoadceenernee lip. methyl violet and Dahlia violet, are ee Seer N, nucleus. Regaud’s more toxic and stain living chondrio- somes only in concentrations not exceeding 0.005%. At greater strengths they stain the cytoplasm and nucleus as well as the chondriosomes, which become vesiculate, and then very rapidly bring about the death of the filaments. The attempts to grow cultures of Saprolegniaceae in media to which vital dyes have been added, has demonstrated the great toxicity of the dyes. It has been possible, for example, to make one culture of Saprolegnia grow in peptone bouillon containing up to 0.008% Janus green. Under these conditions, it first reduced Janus green to its rose derivative, which seems less toxic, and then developed without showing any coloration whatever in its chondriosomes. With a concentration of 0.004% Janus green, the fungus ceased to grow. Dahlia violet and methyl] violet proved even more toxic for this same fungus which did not develop at all in a 0.001% solution of this stain. The reagent iodine-potassium iodide preserves the chondrio- somes perfectly. It makes them more yellow than the cytoplasm Guilliermond - Atkinson — 68 — Cytoplasm and renders them very apparent. A 1-2% solution of osmic acid also preserves them and does not make them brown. The chon- driosomes of fungi, like those of animals do not, therefore, reduce osmic acid unless followed by a treatment of pyrogallol. On the contrary, the methods of osmic impregnation recommended for bringing out the Golgi apparatus, blackens the chondriosomes very strongly and usually makes them vesiculate (GUILLIERMOND). The comparative study of living and fixed hyphae of the Saprolegniaceae has also enabled us to demonstrate that all the ordinary fixatives containing acetic acid or alcohol profoundly alter the chondriosomes. Careful observation, however, shows that they continue to exist in the cytoplasm, sometimes in a very contracted state, sometimes vesiculate, in which case they are more stainable than the cytoplasm. The mitochondrial fixatives, 2.e., those of BENDA, MEVES, REGAUD, and formaldehyde as well, preserve the chondriosomes, on the contrary, as faithfully as possible in the forms they show when alive. After the action of these last fixatives, the chondriosomes stain clearly with iron haematoxylin, acid fuchsin, and crystal violet. They behave, therefore, exactly as do the chondriosomes of animal cells. Investigations of REGAUD, then of FAURE-FREMIET, and of MAYER and SCHAEFFER have proved that the chondriosomes of animal cells are made up of a lipoprotein complex in which lipides (phosphoaminolipides) predominate. Of these workers, the last three named based their conclusions on the belief that mitochondrial fixatives are all oxidizing agents which transform the unsaturated fatty acids into hydroxyl acids. These are only slightly soluble in alcohol and xylol, and are capable of being strongly stained. The chromaticity of the chondriosomes, there- fore, is due to the lipide substance which they contain. The work of GIROUD has shown, on the other hand, the presence of proteins in the chondriosomes. This author was able to obtain within these elements all the reactions of proteins. These facts apply as well to the chondriosomes of fungi as to those of animals. As a matter of fact, the presence of lipides in the chondriosomes is established in several ways: not only by their characteristics of fixation and staining but also by their reduction of osmic acid after treatment with pyrogallol or after prolonged infiltration in the oven; by the fact that the chondriosomes stain by the method of DIETRICH-SMITH, considered as characteristic of phos- phoaminolipides; by their reaction with indophenol blue, a lipide indicator. MILOvIDoV has also demonstrated that the chondrio- somes of the Saprolegniaceae give all the reactions characteristic of proteins. It may, therefore, be concluded from their behavior, which is quite analogous to that shown by the chondriosomes of animal cells, that the chondriosomes of fungi are, like those in animal cells, composed of lipoproteins much more rich in lipides than is the cytoplasm. More recently, MILOVIDOV has established the fact, by a study of the Plasmodiophoraceae and Myxomycetes, Chapter VII — 69 — The Chondriome that these chondriosomes do not give the nuclear reaction of Feulgen and furthermore, that their protein substance has nothing in common with chromatin, contrary to the opinion expressed by P. A. DANGEARD who has called them chromatinosomes. It is seen, therefore, that the study of the histochemical and histophysical characteristics of chondriosomes in the fungi have completed and made more accurate those carried out on animal cells and have shown that, in both cases, the chondriosomes behave in identical fashion. Chapter VIII THE CHONDRIOME (Continued) The chondriome and its development in the phanerogams. Relationships between chondriosomes and plastids. The facts:- The first investigations on this subject were those of PENSA (1910). Applying the Golgi method to various tissues of phanero- gams, 7.e., impregnating sections of living phanerogam tissue Fic. 33. — Development of chloroplasts in a young leaf of the plumule of barley. 1, chondriome in the basal meristem; 2, cells beginning to differentiate with some elements of the chondri- ome showing thickening, and 38-5 their transformation into chloroplasts; 7, cells in older regions showing chloroplasts. c, young chloroplasts; gc, mature chloroplast. Regaud’s method. with silver nitrate followed by treatment with a reducing solution of hydroquinone, he noticed that the chloroplast has the property of reducing silver nitrate and appears strongly blackened by a deposit of metallic silver on its substratum. Now, while studying the chloroplasts in differentiating tissues, PENSA stated that these elements appear first as very small bodies which present the form characteristic of chondriosomes in animal cells. Yet this author never reached the point of specifying the origin of these chon- driosome-shaped elements. He did not find them in tissues lack- ing in chlorophyll and stated that their property of reducing silver nitrate is correlated with the presence of chlorophyll in the substratum. PENSA, however, put forth the hypothesis that the chloroplasts are derived from chondriosomes on which chlorophyll accumulates, giving them the property of reducing silver nitrate. Chapter VIII ii The Chondriome (cont'd) Without knowledge of PENSA’s work, LEWITSKY, a student of STRASBURGER, was working at the same time with mitochondrial technique (method of MEVES). LEWITSKy (1911) showed in the bud of Asparagus officinalis that the chloroplasts are built up from minute elements looking like the chondriosomes of animal cells. This investigator concluded therefore that the plastids, contrary to the opinion of SCHIMPER, do not keep their individuality but arise from chondriosomes which LEWITSKY considers originate, in turn, from a differentiation of the cytoplasm. At the same period (1911) and a little later, in cells of plants belonging to very diverse groups (phanerogam seedlings, nucellus, embryo sac, pollen, asci of Pustularia vesiculosa), we were demonstrating by Regaud’s method, the existence of chon- i Re ae We /op ene Ye AN Eee ~O “YIGe Se oO OO Oe Fic. 34. — Various types of starch formation. 1, within mito- chondria in young potato tuber; 2-4, compound grains within chondrioconts in the meristem of a young root of castor bean; 5-7, compound grains within chondrioconts in bean root; 8, within fusiform leucoplasts surrounding the nucleus; 9, leucoplasts show- ing successive stages in starch formation. 8, 9, from the root of Phajus grandifolius. Regaud’s method. driosomes quite similar in form, as well as in histochemical be- havior, to those of animal cells. Our investigations led us to consider, contrary to the opinion of LEWITSKY, that the chondrio- somes are permanent organelles, being transmitted by division from cell to cell and incapable of forming de novo. We were demonstrating besides, by a study of the plumule of barley, that chloroplasts arise by the differentiation of some of the chondrio- somes in cells of the meristem. Finally by a study of the potato tuber and of roots of various seedlings, notably those of castor bean, we were able to prove that starch never forms in the cyto- plasm but is always the product of the activity of chondriosomes. Our later investigations (1912-1923) as well as those of PENSA (1912) and LEwitsky (1912), followed by many others, confirmed these facts and if interpretations still differ, it nevertheless seems Guilliermond - Atkinson —2— Cytoplasm that the great majority of cytologists are at present in agreement in recognizing that the plastids of SCHIMPER are derived from elements presenting the same forms as those of the chondriosomes. The life history of the chondriosome in phanerogams will now be studied in detail by first following the formation of chloroplasts. As the phenomena are the same in all buds, it is sufficient to choose a single example. The most favorable, because of the dis- position of foliar primordia, is the bud of Elodea canadensis, first investigated by LEwWITsKy. Afterward, it was the object of inten- sive study for us and our reports were confirmed by FRIEDRICHS. If a longitudinal section of a bud of Elodea canadensis be ex- amined after being fixed by Regaud’s method (fixation by a mixture of potassium bichromate and formaldehyde, and staining with iron haematoxylin), there may be observed in the meristem of the stem and in the youngest foliar primordia, a chondriome exactly like that of many animal cells, composed of a mixture of chondriosomes and granular mitochondria. These elements have a diameter of about 0.5-lp. (Fig. 36). By following successively developed foliar primordia, there may be seen with the great- est accuracy, all the developmental stages of the chondriome and it may be observed that the chondrioconts differentiate into chloro- plasts. The differentiation, manifested by a thickening of the chondrioconts, begins in those foliar primordia which are about 160, long. In those measuring about 200, in length, the chondrioconts form little swellings on their PM edi ee ane long axes in which a small starch grain is son (A) of the chondri: Sometimes elaborated. As this grain is not oot of Blniea owe stained by iron haematoxylin, it looks like a sis with (B) that of the vesicle. Starch grains thus formed are only gaud’s method. % 3,000, ‘transitory and soon disappear. The swellings then gradually separate by rupture of the slen- der portions between them. They increase in volume and, in ma- ture cells, take on the appearance of large, rounded or ovoid, chloro- plasts about 4-8, in diameter. These are distinguished from the chondrioconts, from which they arose, by the modification which they have undergone in their chemical qualities which gives them a special resistance. They are preserved by all the fixatives which destroy the chondriosomes. Henceforth these chloroplasts often elaborate large starch grains. During the differentiation just described, the granular mito- chondria elongate first into rods then, in mature cells, generally become typical chondrioconts. In the axil of each leaf primordium there is found, as is known, a small scale made up of a group of cells in which there is no pro- Chapter VIII a The Chondriome (contd) duction of chlorophyll. In these cells, the chondriome always keeps the characteristics which it shows in the meristem. It, therefore, remains undifferentiated, made up of a mixture of mitochondria and thin chondrioconts. These phenomena may be verified in fresh material by studying Fic. 36. — Development of the two categories of chondriosomes in the bud of Elodea canadensis. 1, diagram of longitudinal section of bud; 2, mitochondria and chondrioconts in a foliar primordium at level (A); 3, 4, chondrioconts transforming into chloroplasts in a slightly older foliar primordium; 5, mature chloroplasts in cells of a nearly mature leaf taken at (B). Some of the mitochondria have be- come rods or chondrioconts; 6-11, details showing the same sequence of events. C, level at which chloroplasts develop in the stem, also chon- driosomes in Fig. 11. Regaud’s method. the living bud. The tip of the bud may be seen, without any al- teration taking place within its cells, by stripping it of its oldest leaves and putting it in water under a cover slip which is pressed gently so as not to injure the vegetative point. It is seen that the meristem of the stem and of the youngest foliar primordia do not contain chlorophyll. In addition to a large nucleus, these cells show only a confusedly granular cytoplasm in which it is possible Guilliermond - Atkinson — 74 — Cytoplasm to distinguish the chondriome. It is only in foliar primordia in which the chlorophyll is beginning to appear that the chondrioconts are visible. They are here impregnated with chlorophyll and all the forms can be followed in sequence from these elements to the large chloroplasts of mature cells. The other chondriosomes, how- ever, are difficult to distinguish. The cells of the mature leaves are, on the other hand, very transparent and very favorable for 5 bh. P< . : Fic. 87. — Development of the chondriome in the castor bean root. 1-6, meristem; 7-11, differentiating cells, plastids forming starch; 12, differentiated parenchyma cell of the central cylinder. Regaud’s method. the study of living cells. In them can be seen all that Regaud’s method brings out and it is possible to distinguish with great clearness within the hyalin, homogeneous cytoplasm, large chloro- plasts, often in the process of dividing, interspersed with chondrio- conts whose slightly higher refractivity distinguishes them from the cytoplasm. It is easily possible to follow the formation of chloroplasts by a study of the development of the chondriome in other buds, among them barley which was the object of our first research (1911). Here also, the chloroplasts are derived from chondrioconts which thicken, and on their long axes form small swellings which after- Chapter VIII — 75 — The Chondriome (cont'd) wards separate and then elaborate a grain of transitory starch. It is not until this is absorbed that the swellings increase in vol- ume and take on their characteristic appearance of large chloro- plasts. The living root of Hlodea does not lend itself to study. On the other hand, Regaud’s method brings out in the meristem a chondri- ome entirely similar to that of the vegetative tip. During the differentiation of tissues all that can be observed is that a certain number of elements of the chondriome, especially the chondrioconts, without modifying their form or chemical quality, elaborate little starch grains along their long axes, but this elaboration is not very active. When the root is exposed to light, on the contrary, there are formed in the course of cell- ular maturation and by differen- tiation of a part of the chondrio- somes, chloroplasts similar to those in the stem and leaves. A study of the root of the castor, bean. (Figs. 37, 38) 1s more profitable and will serve as anexamplehere. Thechondriome of cells of the meristem is, here also, composed of a mixture of granules, rods and _ chondrio- conts. In the central cylinder, a part of these elements, especial- ly the chondrioconts, elaborate small starch grains directly. On the long axis of the chondrio- Fic. 38. — 1,2,3A, the chondriome in the conts, there are seen to form small castor bean root. 3B, in the bean root. 1, portion of a parenchymatous cell of the vesiculate swellings occupied by cenit eripdens howe’ Pe nee oe a sort of vacuole which corre- jiasts (A): 2. amyloplasts containing) come sponds actuallystovastarch eraim.poand starch. (s) inf simiae. eh oe ee left colorless by Regaud’s tech- ee Pe cern neiated Geioplaas nique. Soon, around this small starch grain, others are seen to appear which give a spongy ap- pearance to the swellings and thus a compound starch grain is produced. This increases in size little by little while remaining surrounded by a thin mitochondrial layer prolonged to a sort of tail, the remaining portion of the chondriocont. In the cells of the cortex, on the contrary, some of the elements of the chondri- ome differentiate by thickening slightly and it is not until after this is accomplished that they elaborate starch as first described. Thus, some of the elements of the chondriome elaborate starch and play the réle of amyloplasts either immediately, or after thick- ening slightly. It is easy to obtain the characteristic reaction for starch by treating the preparation obtained by Regaud’s method with the reagent iodine-potassium iodide. The chondriocont is stained by the haematoxylin while the starch grain becomes yellow- ish brown, due to the action of the xylol which turns yellow the Guilliermond - Atkinson — 76 — Cytoplasm starch grains stained by the iodine. Moreover, simultaneous stain- ing of the starch and the chondriocont may be obtained by various more complicated processes. MILOVIDOV, especially, has shown how to make such permanent preparations. These methods are much more delicate and do not give constant results. Starch formation takes place in the same way in the greatest variety of tissues which are without chlorophyll: roots, tubers, epidermis. Yet there are cases, as in the tuber of the potato, in which the chondriome is represented only by mitochondria which elaborate starch after having undergone a slight increase in vol- ume. In such cases they take on the appearance of vesicles, due to the production in their interior of a starch grain which mito- chondrial methods do not stain. Sometimes the chondrioconts which will later elaborate starch may acquire, before its production, a much more marked increase in volume which makes it possible to distinguish them very clearly from the other chondriosomes in the mature tissue. This is seen, for example, in the root of Phajus grandifolius in which by following the meri- stem to the region of differ- entiation, it is seen that some of the chondrioconts take the form of rods or _ spindles. These chondrioconts are very clearly bigger than the chon- driosomes which continue to exist side by side with them oT ne ee €© (@ but without increasing in size. Fic. 39. — Stages in starch formation in These enlarged elements cor- potato tubers. respond to the amyloplasts described by SCHIMPER and MEYER, through the agency of which the grains of starch arise. It seems that the increase in volume is due to the formation in the chondriocont of a needle-shaped protein crystal lying along the long axis of the element, whose contours follow that of the crys- tal. In other cases the amyloplasts assume the appearance in mature cells of rather long rounded bodies (hairs of Tradescantia virginiana). It may be added that the simple or compound starch grains instead of arising in the center of the swelling of the chon- driosome, chondriocont, or mitochondrium, may form on its periph- ery. The chondriosome then bears a vesicle whose wall is much thicker on one side than on the other. The starch grain which occupies the vesicle increases in size and ends by bursting out of the chondriosome which is thus reduced, little by little, to a thin cap, covering the starch grain in the region most distant from its hilum (potato tuber, root of Phajus grandifolius). The starch grain thus formed no longer remains surrounded by a continuous mitochondrial layer as in the preceding case (Figs. 40, 41). Chapter VIII — 77 — The Chondriome (cont'd) It is difficult to check these phenomena by observation of living material. Roots in general do not lend themselves to this type of investigation. On the other hand, in the course of our research, we have found exceptionally favorable examples in which the entire process of elaboration of starch can be followed with remarkable accuracy in living cells. In a fragment of the epidermis of the anther of a young flower of Iris germanica examined in Ringer’s solution, a chondriome is observed with great clearness, composed of thin, elongated, and undulating chondrioconts which sometimes branch, interspersed with granular mitochondria and short rods. In some cells there is no elaboration of starch; in others there may be seen several stages in the formation of small, compound, very refractive starch grains on the long axis of the chondrioconts. Similar phenomena may be observed in epidermal cells of the leaves, of the bracts, and of all very young floral parts. At later Fic. 40 (left). — Successive stages in the formation of leucoplasts in the root of Phajus grandifolius. Regaud’s method. Fic. 41 (right). — Leucoplasts from the root of Phajus grandifolius showing starch. 1, central cylinder; 2, cortical parenchyma. Regaud’s method. stages of development it is seen that the starch grains are absorbed within the chondrioconts which persist after the disappearance of the starch (Fig. 44). It can also be seen in these same cells that the chondrioconts are, at certain stages, the seat of a production of small osmium-reducing lipide globules, clearly visible on the long axis of these elements because of their strongly refractive power. These granules which often completely fill the chondriocont are very frequent in the monocotyledons. They can not be considered as formed of a-8 hexylene-aldehyde (MEYER), for they present char- acteristics of lipides and not those of aldehydes. The fact that they are stained by Dietrich’s method suggests that they are made up of phosphoaminolipides. These granules, very numerous in the young stages of development of leaves, bracts and floral parts, disappear from the plastids as soon as the starch grains and pig- Guilliermond - Atkinson — 78 — Cytoplasm ments begin to form, except, however, in certain regions where they persist during the entire life of the cells. Do they represent an intermediate product from which starch and pigments are built up, or do they result from a breaking down of the lipoprotein com- plex which makes up the plastids (lipophanerosis)? It is difficult to say. In any case, these granules reappear in large numbers in the plastids at the moment when the flower begins to form. They are in this case products of disintegration of the plastids and mark the beginning of plastidial degeneration. The epidermal cells of perianth parts of the tulip are also par- ticularly favorable objects for observation of the living chondri- ome and in them it is possible to follow the formation within the chondrioconts of a yellow pigment, xanthophyll. In the white tulip, for example, the chondriome can be observed very clearly in a frag- AP ay As LAN 2 (@} ooo ° I Fic. 42 (left). — Epidermal cells of living young anther of Iris germanica, showing refracting mitochondria, chondrioconts and strongly refracting lipide granules. The two lower cells contain chondrioconts with small compound starch grains (A) on their long axes. Fic. 43 (right). — Epidermal cells of leaves of Iris germanica. A comparison of the chondriome in (b) a living cell with (a) one fixed by Regaud’s method, showing that the chondrioconts in (a) correspond to the plastids in (b); that the rod-shaped and granular chondriosomes are similar in the two cells; that the lipide granules appear only in (b). c, d, e, similar portions of the cell showing (c, e,) starch bearing plastids in living and fixed material (Regaud’s method) respectively; d, plastids not forming starch; f, g, successive stages in the vesiculation of living plastids. C, chondriosomes; P, chondriocont, plastid; Gl, lipide granules; A, starch. ment of the epidermis of the perianth. It is made up of a con- siderable number of very elongated chondrioconts and granular mitochondria. The bases of the perianth parts are almost always yellow and, on examining the epidermis in this region, it is seen that it is the chondrioconts which serve as substratum for the xanthophyll pigment and consequently represent the chromoplasts. The mitochondria, on the contrary, remain colorless. In yellow flowers, however, the chondrioconts in all parts of the epidermis appear yellow because of the xanthophyll. In living epidermal cells of the perianth and those of the exo- carp of fruit of monocotyledons, the chondriosomes can be observed with greatest ease and the formation of carotinoid pigments fol- Chapter VIII —= 19 == The Chondriome (cont'd) lowed. A study of the latter can hardly be made from preparations where the mitochondrial technique has been used, for, although the plastids are stained, there is no indication as to what pigments they contain. So in the flower of Clivia nobilis, it can be seen that the orange-red pigment, carotin, arises directly from chondrio- conts. Small starch grains are first elaborated and, at the moment when these are absorbed, the carotinoid pigment arises in the in- terior of the chondrioconts as small grains or more especially as long needle-shaped crystals. In the flower of Sternbergia the chondrioconts form several acicular crystals of carotin which give them the appearance of thick spindles. In other cases, the chondrio- somes in which the pigment will form are always chondrioconts Gi) oe } pee ERY Yel 3 Fic. 44 (left). — Development of leucoplasts in living epidermal cells in leaves of Iris pallida. 1, lipide granules (GG) within the leucoplasts (chondrioconts) in a young leaf; 2, detail of leucoplasts; 3, later stage, leucoplasts containing starch (A); 4, absorption of starch, diminution in lipide granules; 5, leucoplasts without starch containing lipide gran- ules in adult leaf. Fic. 45 (right). — Living epidermal cells of a petal of white tulip. At left, from a young flower; at right, from a mature flower. C, chondriosomes; Chr, chromoplasts; GL, lipide granules; O, fatty body. enclosing small starch grains. When these are absorbed, small vesiculate swellings enclosing a watery liquid are formed on the long axis of the chondriocont. Small grains of carotin appear on the walls of these vesicles which later become isolated by rupture of the slender regions of the chondriocont which separate them. They then appear as small rounded vesiculate chromoplasts (peri- carp of the fruit of Asparagus officinalis and of Arum italicum). In the epidermis of the perianth of Ivis germanica the phenomena are a little more complicated. The yellow pigment, xanthophyll, first appears in a diffuse state in the chondrioconts which contain small Guilliermond - Atkinson — 80 — Cytoplasm starch grains. Then, when the starch is absorbed, the chondrio- cont thickens at the same time that large vesicles appear along the element. These may disjoin by a rupture of the more slender por- tions of the chondriocont which connects them, so that vesicular chromoplasts are formed with tails of varying lengths. In other cases the pigment begins to appear in chondrioconts which increase their dimensions proportionally as the pigment develops, until they have been transformed into large chromoplasts of the same form and dimensions as chloroplasts. Fic. 46 (left). — Transformation of chondrioconts into chromoplasts in living epidermal cells. 1, formation of starch in very young petals; 2, starch being absorbed and replaced by small granules and needle-shaped carotinoid pigment in older petals; 4, the same in an open but young flower; 5, chromoplasts in an older flower. 1, 2, Clivia nobilis. 4, 5, C. cyrtanthi- flora. Fic. 47 (right). — Development of chromoplasts in living cells of the fruit of Asparagus officinalis. 1, chondrioconts forming starch; 2, starch being absorbed; 8, carotin granules forming on the borders of vesiculate swelling in the plastids; 4, fragmentation of chondrioconts to form round vesiculate plastids containing carotin granules which tend to fuse; 5, chromo plasts and chondriosomes in a cell of the pericarp of a nearly ripe fruit. Xanthophyll always seems to be diffuse in the substratum of the plastid or else in the state of indistinct granules. Its iso- mer, rhodoxanthin, on the contrary, appears as isolated, clearly distinguishable granules. This is true of carotin and lycopin if they are not in crystalline form. When crystalline, the crystals give widely-differing shapes to the chromoplasts. These facts show that whenever the chromoplasts do not arise by metamorphosis of the chloroplasts as in the parenchymatous tissue, studied especially by SCHIMPER, MEYER, and COURCHET, they arise from chondrio- conts which have first elaborated starch. Chapter VIII — 81 — The Chondriome (cont'd) The very best material for the study of living cytoplasm is to be found in the epidermal cells of flowers and various organs of the monocotyledons, those of Iris and tulip among others, as well as in the bulb scales of Allium Cepa, which will be taken up later, and in the Saprolegniaceae which have just been studied. On these forms we have been able to make the most accurate observations of the chondriosomes that it has been possible to make up to the present time. We have been able to show, by a comparison of these observa- tions with those on fixed and stained cells, that the mitochondrial methods preserve the cytoplasm and its morphological constituents, the chondriosomes and plastids, in a manner as faithful to the form they present in life as it is possible to have it done. These observations permitted us, also, to specify the histo- RRS SEES . chemical and histophysical WiEZRSAS) GF AD) characters of the elements. This will be taken up later. We have studied the chondriome very accurate- ly during the formation of the embryo sac and of pol- len grains in the Liliaceae and, in particular, in Lili- um candidum. In the young ovary, all the cells of the nucellus present a chondri- ome made up of a mixture of chondrioconts and of Fic. 48. — Development of chromoplasts in cells of mitochondria. The embryo a petals 8 ae ables eet me ee oe oe sac, which arises from a Srannle® chonuciosomear 2, the satereh-bearing. plas: cell of the nucellus first tids begin to fill with xanthophyll; 3, the starch is : Lr Eee absorbed as the chromoplasts increase in size; 4, shows a chondriome simi-_ mature cell with variously shaped chromoplasts, most lar to that of other cells of of them showing vesiculate swellings. the tissue, then, in the course of its differentiation, at the mo- ment when synizesis begins, it is observed that a part of the chondrioconts thicken and form small swellings on their long axes. These grow little by little, often detach themselves from the chon- driocont in which they rise by rupture of the thin portions which connect them, then enlarge greatly, and take on a crystalline ap- pearance. This seems to be due to the production in their interior of protein crystalloids. These plastids, which we have called proteo- plasts, because of their ability to elaborate protein, then appear to be digested in the cytoplasm and their protein is thus utilized as a reserve product. In the synergids, the egg and the antipodal cells, on the contrary, no elaboration of protein is noted and the chondri- ome remains about as it is in the embryo sac at the beginning of differentiation, except for the appearance of small granules slightly larger than the other elements of the chondriome. These granules are the leucoplasts. In the tulip no proteoplasts are observed and Guilliermond - Atkinson — 82 — Cytoplasm the chondriome, consisting of a mixture of chondrioconts and mito- chondria, undergoes no modifications during the development of the embryo sac!. (Figs. 49, 50, 51, 52). In the sporogenous cells of the pollen grains of Lilium can- didum, the chondriome is seen clearly as short rods and granules. In the pollen mother cells, only mitochondria are to be found. Beginning with the period of synizesis, some of these mitochondria which are to become amyloplasts, undergo a slight increase in size, then, at the time of the heterotypic mitosis, they elongate into chondrioconts and afterwards, in the pollen grains, break up into mitochondria. The remainder of the mitochondria are unchanged from the beginning. When the pollen grain is mature, only gran- aS Kae Fic. 49 (left). — Embryo sae of Lilium candidum at the beginning of differentiation. >< 1500. Regaud’s method. Fic. 50 (right). — Formation of proteoplasts in the embryo sac of Liliwm candidum at the end of the second mitosis. Regaud’s method. ular mitochondria are found, among which a few larger than the others elaborate compound starch grains. Other investigators of the development of the chondriome dur- ing the formation of pollen in other plants have produced data more or less analogous (WAGNER, MASCRE, KRJATCHENKO-DOUZE, PROSINA, Mrs. LUXEMBURG, KRUPKO, Miss Py). Recently there has appeared LEWIS ANDERSON’s very good work on the develop- ment of pollen in Hyacinthus orientalis. Investigations of NICo- LOSI-RONCATI, WAGNER, and others have shown that the chondrio- somes in the spore mother cells of some species may collect in a compact mass which surrounds the spindle as a sort of mantle dur- ing the heterotypic division and divides (chondriocinesis) at the same time as the nucleus. The significance of this grouping of chondriosomes is not clear and one wonders if it does not corre- spond to an alteration. Chondriosomes have been observed in all the cells of the em- bryo before the maturation of the seed and in the seed in the dor- 1LEWIS ANDERSON finds this is also the ease for the embryo sae of the hyacinth. Chapter VIII — 83 — The Chondriome (cont'd) mant state (GUILLIERMOND, WAGNER). Some of the chondrio- somes later, at germination, form the amyloplasts of the root and the chloroplasts of the chlorophyll-bearing organs (leaves, etc.). It has been proved by our research that chondriosomes exist per- manently in all phanerogam cells and that they are transmitted by division from one cell to the next. Fic. 51. — Portion of the embryo sac of Liliwm candi- dum. 1, 2, stages in the development of the plastids (P) and the mitochondria (M); 3, digestion in the plastids. The origin of plastids, which for so long remained obscure in the phanerogams, is now well known, through the use of mito- chondrial techniques, by means of which a chondriome has been demonstrated in embryonic cells analogous to that in animal cells. The entire life history of this chondriome has been followed and it has been shown that events take place as if the plastids arose by differentiation of some of the elements of the chondriome. Fig. 52 (left). — Development of the chondriome during the formation of pollen grains in Lilium canadense. 1, sporogenous cells; 2, spore mother cell in synizesis, leucoplasts appear slightly larger than other chondriosomes; 3, metaphase, leucoplasts have become chondrioconts; 4, anaphase; 5, pollen grain, leucoplasts slightly larger than other chondriosomes, various stages in development of compound starch grains. Fic. 53 (right). — Chondriome in pollen of Helleborus foetidus. 1, synizesis; 2, accumula- tion of chondriosomes about the nuclear figures of the first division; 3, pollen grain; 4, generative cell. (After Miss Py). There is still one gap in our knowledge. This is the behavior of chondriosomes during fertilization. It is still not known whether the chondriosomes of male origin participate in this phenomenon. In a recent work, however, LEWIS ANDERSON reports having ob- Guilliermond - Atkinson — 84 — Cytoplasm served that in Hyacinthus orientalis the facts are in favor of a passage of the chondriosomes of male origin into the egg. Ktyo- HARA, almost at the same time, described in certain phanerogams the passage of plastids from the pollen tube into the oosphere. More recently still, MANGENOT in gymnosperms (Pine) was able to follow the course of the chondriosomes of male origin because of their size which is greater than that of the chondriosomes of the oosphere. All the chondriosomes of the pollen tube and oosphere are in the form of mitochondria but those in the pollen tube are larger, and can be followed during their penetration into the oosphere during fertilization, and can be recognized after fer- tilization has taken place. During the development of the em- bryo, however, the chondriosomes of male origin remain in that portion of the oosphere which does not contribute to the formation of the embryo and which will later degenerate. Therefore there does not seem to be any mixing of chondriosomes of male and of female origin. Chapter IX THE RELATIONSHIP BETWEEN CHONDRIOSOMES AND PLASTIDS Interpretations:- It was logical to admit from the facts already displayed, which have been verified by many observers, that the plastids described by SCHIMPER arise by differentiation of some of the elements of the chondriome during cellular development. This opinion, which had been maintained from the beginning by many workers, notably PENSA (1910), LEWITSKY (1912-19138), FOREN- BACHER (1912), Maximov (1913), and which we ourselves were among the first to formulate in our early work, is still held by LEWITSKY and his school (1925-1927), ALVARADO (1918-1925), Morte (1928), GATENBY and his collaborators (1930), JUTTA VON Lour (1930), CHALAUD (1923), LEWIS ANDERSON and others. It was adopted by WILSON in his book, The Cell in Development and Heredity (1925). Still the opinion has been variously expressed. Thus, for certain investigators such as LEWITSKY and RANDOLPH, the chondriosomes are not permanent components of the cytoplasm but form de novo from it. Our first interpretation was quite con- trary to this. Not having recorded any fact which would permit us to think that the chondriosomes can arise by differentiation from the cytoplasm, we had from the beginning considered them as permanent components of the cell, incapable of forming other- wise than by division of pre-existing chondriosomes. Therefore at that time, we considered the plastids of the phanerogams as a variety of chondriosome, differentiated in the course of cellular development and having a special function. The plastids, there- fore, we believed belonged to a much more general category present in every cell, whether plant or animal. Then too, the theory that we had formulated was only an extension of that of SCHIMPER and MEYER and in no way contradicted it. It is this same point of view which MEVES and ALVARADO adopted. Although based on incontestable facts, this theory, however, raises very serious theoretical difficulties, for it can only be applied to higher plants. In fact, although in the phanerogams the origin of plastids had been for a long time only imperfectly known, this was not so for the algae. In many of these plants, as has already been said, the chlorophyll is present in all stages of plant develop- ment and in that case chloroplasts are observed in all cells. These chloroplasts are transmitted by division from cell to cell, beginning with the egg. This has been well known since the work of SCHMITZ. We have seen, besides, that in many algae there is in each cell only a single, voluminous chloroplast which divides at each cellular division. This chloroplast, however, can not be considered as different from the chloroplasts of the phanerogams, for it offers the same histochemical characteristics. There are found, more- Guilliermond - Atkinson — 86 — Cytoplasm over, in the algae, bryophytes, and pteridophytes, all the interme- diate stages between this chloroplast, of special form and chloro- plasts such as exist in the phanerogams. Now the research of RANDOLPH has demonstrated in Vaucheria, which contains chloro- plasts similar to those in phanerogams, that these chloroplasts are found in all parts of the thallus at the same time as the chondrio- somes. Our work on Spirogyra showed the chondriosomes to be constantly present and distinct from the single permanent chloro- plast which is characteristic of this alga. The work of SAPEHIN, of SCHERRER, and of MOTTIER showed that in the bryophytes, too, chlorophyll seems to persist in all stages of development, and that all cells, even the egg and apical cell of the vegetative shoot, con- tain both chloroplasts and chondriosomes. There even exists, in this group, the genus Anthoceros in which each cell contains only one single crescent-shaped chloro- plast adhering to the nucleus. Coexistent with this organelle there are, however, numerous chondriosomes. There can not, therefore, be found in these plants any ge- netic relationship between the chloroplasts and the chondriosomes. It is to be added that in centrifuging the cells of various algae and cells of Hlcdea canadensis in the process of division, BOROVIKOV was able to obtain cells without chloroplasts but containing chondriosomes. He was never able to observe in these cells any trans- formation of chondriosomes into chloro- plasts. These contradictory facts there- fore must be explained. TO Cees BION eas RUDOLPH (1911), SCHERRER (1913), amen ot Vankers; aves | SAPERIN: (1913), ARTHUR Muyver (1914- Neale as ar ee 1921) and Noack (1921) protested, not ory altogether disinterestedly, against the new results obtained in the phanerogams by mitochondrial technique which, at least in appearance, seem to invalidate the classical theory. They did not hesitate to consider the chondriosomes and plastids as inherently different formations. To explain the phenomena in the phanerogams, RUDOLPH, SCHERRER and SAPEHIN report that in the meristematic cells of these plants, the plastids and the chondriosomes stain in the same way with mitochondrial technique. According to these investigators, the plastids appear as small grains and the chondriosomes as rods or filaments. Now, as the plastids divide actively, RUDOLPH, SCHER- RER, and SAPEHIN believe they become dumb-bell shaped and are thereby confused with the chondriosomes (chondrioconts). But from the moment that the cells differentiate, the plastids enlarge and appear as large bodies which it is no longer possible to confuse with the chondriosomes, since the latter keep their original form Chapter IX —87— Chondriosomes & Plastids and dimensions. SCHERRER and SAPEHIN think, furthermore, that chondriosomes are not permanent elements of the cytoplasm but that it is more probable that they are merely reserve products. MEYER, who was the instigator of this opinion, attributed to the chondriosomes a ferronuclein-like constitution and called them Allinantes. NOACK has carried this idea further and maintains that there is not the least morphological or histochemical resemb- lance at any time between ¢chondriosomes and plastids. He finds chloroplasts even in the meristematic cells of buds of Elodea cana- densis and shows them to be different histochemically from the chondriosomes, for they are preserved by all the fixatives which destroy chondriosomes. JARETZKY, in his German edition of SHARP’s book, Hinftihrung in die Zytologie appears to be of the same opinion, an opinion ex- pressed, moreover, with insufficient knowledge of the question. GEITLER takes the same stand (Grundriss der Zytologie) and also Fic. 55. — Anthoceros. C, chondriosomes; P, plastid, (After SCHERRER) . Kuster. The latter, particularly in Die Pflanzenzelle, tends to con- sider the chondriosomes as heterogeneous formations resulting from cellular metabolism. P. A. DANGEARD, having made observations exclusively on liv- ing material, first thought, as will be shown further on, that the formations described as chondriosomes belonged to the initial forms of the vacuolar system (vacuome, p. 149) and that the refractive granules corresponded to the microsomes of early authors. These are encountered in all cells and will be discussed later (p. 203). According to DANGEARD, the plastids, therefore, bear no relation to these dissimilar formations, the chondriosomes. How- ever, after more profound studies with mitochondrial technique, DANGEARD was obliged little by little to renounce his former opin- ion. He had to recognize that the chondriosomes, which he at first had found impossible to distinguish from the leucoplasts, are discrete elements corresponding neither to young vacuoles nor to microsomes, and that they evidently very much resemble the leuco- plasts?. 1p, A. DANGEARD, who, however, is not convinced as to the individuality of the chondrio- somes, says in his last re-statement of the question with reference to the multiplication of the chondriosomes, “If this multiplication were to take place by division or fragmentation, the chondriosomes would be akin to the plastids but distinguishable from them’. Guilliermond - Atkinson — 88 — Cytoplasm MorTTiER (1918) finds that plant cells containing chlorophyll constantly enclose plastids and chondriosomes which stain in the same way. In meristematic cells of phanerogams he finds these two categories of elements have the same form and are very diffi- Fic. 56. — A. Development of chloroplasts in aerial root of Chloro- phytum Sternbergianum (After MEVES). 1, meristematic cell, the chondriosomes represented by chondrioconts (P) only, the granules (Gm) MEveEsS believes to be metabolic products; 2-5, successive stages in the transformation of chondrioconts into chloroplasts, granules unchanged. B. Development of amyloplasts in pea root (After Morrirr). 1, meri- stem, amyloplasts (P) shaped like chondrioconts, mixed with small, filamentous or granular elements (C) the only elements which MOorTtiER believes to be chondriosomes; 2, mature root cell, amyloplasts (P) form: ing starch, the chondriome (C) unchanged; 3, detail, amyloplasts (P) forming starch (A), division figure of chondriosome (C) at Cd. cult to differentiate, still the plastids would always be recognizable because of their slightly greater size. The two classes of elements correspond to permanent components of the cell, incapable of aris- ing de novo and multiplying only by division. Nevertheless this American investigator considers them as radically different forma- Chapter IX —89— Chondriosomes & Plastids tions, but without, however, bringing forth the slightest histochem- ical proof in favor of this idea. Later (1921) he seems to have abandoned his opinion. He states that chondriosomes can not always be distinguished from plastids in meristematic cells of plants and seems to admit that the chondriosomes are plastids whose functions are multiple, the plastids of chlorophyll-bearing plants being only a special variety. Texte ‘} Fic. 57. — Chondriosomes and plastids in leaf cells of Elodea canadensis, I, in embryonic cells, II, at the beginning of differentiation. a, the chondriome; b, plastids and c, chondriosomes drawn separately. III, later stage, a, plastids; b, chondriosomes. On the basis of research carried out exclusively in the phanero- gams, MEVES (1918) expressed a theory which is the exact opposite of that held by most of the authors just discussed. This eminent cytologist observed that, in the meristem of buds, mitochondrial technique brings out both chondrioconts and granules, and that all the chondrioconts are transformed into chloroplasts in mature cells, Guilliermond - Atkinson — 90 — Cytoplasm whereas the granules persist after the differentiation of the chloro- plasts. He considers that only the chondrioconts correspond to the chondriosomes and the granules are not mitochondria but simply metaplasmic granules. Thus, according to MEVES, all the chondrio- somes are transformed into plastids in the mature cells of the Fic. 58. — Elodea canadensis (fig. 57 cont.). I, Il, further differentiation. a, plastids; b, chondriosomes changing shape. III, in a differentiated cell. a, the chondriome; b, plastids and ec, chondriosomes. phanerogams and it is those elements considered by most authors to be plastids, which MEVEs believes are represented by the chon- driosomes, whereas those described by others as chondriosomes have nothing in common with them. BOWEN (1926-1929) at first adopted the theory of MEVES and considered the plastids as corresponding to the chondriosomes of animal cells. In later research he was led to modify his opinion and to admit the existence in the phanerogams of two categories of Chapter IX —91— Chondriosomes & Plastids elements which are different, but whose form is similar: the plas- tids, peculiar to chlorophyll-bearing plant cells; and other elements, which, hesitating for some unknown reason to liken to the chon- driosomes of animal cells, he groups into a category which he calls the pseudo-chondriome. WEIER (1930-1933), having obtained impregnation of the chlo- roplasts of Polytrichum commune by Golgi technique, felt justified in likening the plastids of plant cells to the Golgi apparatus de- scribed in animal cells. This theory was adopted by Dusposcg and GRASSE who, in a recent treatise, maintain that in animal cells there are two sorts of permanent, closely allied constituents of a lipoprotein nature, namely, the chondriosomes and the Golgi mate- rial, or dictyosomes, the latter being comparable to the plastids of chlorophyll-bearing plants. Finally, KIYOHARA (1936), after mak- ing observations of living material car- ried out under improper conditions, thought he noticed that all the plastids normally appear as vesicles and that it is the mitochondrial technique which al- ters them and makes them appear as chondrioconts. But this Japanese inves- tigator obtained plastids of vesicular form by osmic impregnation, the tech- nique used for the detection of the Golgi apparatus. He thinks that chondrio- somes do not exist in plant cells and that all forms described under that name cor- respond to images brought about by Fic. 59. — The chondriome. 1, 2, alterations: the: plastids: Sie vreports,. Roc o Cente ere ee however, that in mature cells there al- parenchyma, some thicker chon- ways exist, as well as the large vesicular _dzicconts (FP) form starch, short plastids, other much smaller vesicles, ceptibly changed; a few elongate but these he believes to be plastids in the oe gen aroronts. 8. young act of degenerating. 4, frog’s liver. All these theories, aside from being essentially contradictory are, unfortunately, at variance with the facts. They are the result of hasty generalizations, founded on observations limited to certain types of cells and carried out, most often, with defective techniques. They give evidence of an insufficient knowledge of that which in animal cells has been designated as chondriosomes and which have been recognized in all the fungi. It is now demonstrated by our research that in mature cells the plastids without chlorophyll generally keep the shape charac- teristic of the chondriocont. For example, it is in this form that they appear in the epidermal cells which we have already described (Iris, tulip, Allium Cepa). It is therefore impossible to attribute, as do MEYER and SAPEHIN, the form of chondrioconts to division figures of plastids which have for the moment stopped dividing. It has also been proved in the most evident fashion, by our re- Guilliermond - Atkinson — 92 — Cytoplasm search (1912-1923), as well as by that of FRIEDRICHS (1923), that the meristem of the bud of Hlodea canadensis does not contain chloroplasts. This is contrary to the findings of NOACK, whose error can only be explained by supposing that in his preparations he confused tissues already differentiated and containing chloro- phyll, with the meristem. It has been proved, besides, that among the elements which constitute the chondriome of meristematic cells in the phanerogams, it is impossible to distinguish those which will later become plastids, from those which will remain chondrio- somes. Both have the same shapes and histochemical character- istics. Cytological work on animals (LEVI), as we have said, has established the fact that the chondriosomes are indeed permanent and clearly characterized elements of the cytoplasm. This is dem- onstrated, furthermore, by the research that we have done on the Saprolegniaceae, in which the absence of plastids makes this study more easy than in chlorophyll-bearing . ; %, CAs plants, and in which we have been LES able to follow the chondriome in liv- ing material during the entire de- velopmént of these fungi. It is there- fore not possible, for the time being, to consider the chondriosomes as dis- similar elements, or as products of cellular metabolism (Allinantes). The theory of ARTHUR MEYER, who labels the chondriosomes Allinantes, has never been verified, and is today definitely invalidated. It has not been confirmed, either, that the plastids and the chondrio- somes of embryonic cells of phanero- Fra. 60, — Similarity of the chon. gams can be differentiated by their driome in A, the basidium of Agaricus dimensions, as was thought by Mot- HY Sa aia gat meal gates pe’ TIER who, without doubt, observed Pe eee sy pata pes only cells already in the process of CARD). differentiation. Furthermore, our research has shown that the chondri- osomes which are not transformed into plastids are in no wise exclusively shaped as granules, as MEVES believed. It sometimes happens, on the contrary, that in embryonic cells, the chondriosomes exist as chondrioconts, whereas the plastids are represented by mitochondria. This is so much the case, that when the chondrio- somes appear as granules in meristematic cells, they almost always have the appearance of typical chondrioconts after the differentia- tion of the plastids has taken place, as in the leaves of EHlodea canadensis. Therefore they are obviously chondriosomes. (Figs. 57, 58). There is no basis, on the other hand, for the opinion of WEIER, for if it is true that the plastids are blackened by osmic impregna- tion because of their lipide constitution, it is also true that the Chapter IX — 93 — Chondriosomes & Plastids chondriosomes coexistent with them behave in the same manner. In the fungi, especially in the Saprolegniaceae, where there are no plastids, it is easy to demonstrate that the chondriosomes are in- tensely blackened by osmic impregnations, just as are the plastids of chlorophyll-bearing plants. Furthermore, the Golgi material in animal cells may be distinguished from the plastids by the fact that it does not stain with mitochondrial techniques. The opinion of KIYOHARA results from an initial error of ob- serving living material under defective conditions. This author leaves out of consideration that characteristic property of chondrio- somes of becoming vesiculate during alteration (cavulation). He observed cells in which the chondrioconts were already transformed into vesicles and misinterpreted this phenomenon, mistaking the vesicles for normal shapes of the plastids and the chondrioconts for their altered shapes. Chapter X DUALITY OF THE CHONDRIOME The first data obtained in our laboratory by EMBERGER and MANGENOT on the pteridophytes and the algae as well as the very meticulous study of the development of the chondriome in certain phanerogams, notably in the bud of Hlodea canadensis and in the root of Cucurbita Pepo, led us, as early as 1920, to formu- late a new theory which removes all the difficulties and accords with all the facts drawn from the development of plastids in the plant kingdom. It has been seen that among the elements which constitute the chondriome in meristematic eells of the bud of Hlodea canadensis, it is possible to distinguish between the chondrioconts which be- come chloroplasts during cellular differentiation, and the granular mitochondria which do not participate in this phenomenon but elongate into rods and later into chondrioconts. Now it has been known for a long time that chloroplasts have the ability to divide. Our work has shown that it is only by this process that they in- crease in numbers and that in differentiated cells the chondrio- somes which persist along with the plastids are incapable of becom- ing chloroplasts. This shows therefore that the two categories of elements which constitute the chondriome of cells of the meri- stem develop separately and seem independent, one of the other. The study of the development in the chondriome in the root of Cucurbita (Figs. 59, 61) will furnish a similar example. Here again, there is observed in the cells of the meristem a chondriome com- posed of two categories of elements: chondrioconts and mitochon- dria or short rods. If, in the course of cellular differentiation, the development of these elements is followed, it is seen that in the cen- tral cylinder, where elaboration of starch is not very active, the appearance of the chondriome does not change and one witnesses the production of small starch grains only within the chondrioconts. In the cortex, on the contrary, the chondrioconts undergo a consider- able thickening and may be subdivided into rods or granules. The other chondriosomes keep their original size but sometimes take the form of chondrioconts which differ from the other chondrioconts by being thin. Our first impression is that there exist at this mo- ment in the cells, two categories of chondriosomes, the one consist- ing of large chondriosomes, the other of little ones. The large chondriosomes represent the amyloplasts. At certain periods they are the seat of active elaboration of compound starch. Once having reached maturity, the starch grain formed in the interior of the larger chondriosomes considerably mcdifies the appearance of the latter. The starch is seen as a large grain surrounded by a thin mitochondrial layer. This layer is often prolonged as a tail, the Chapter X —95— Duality of the Chondriome remnant of the elaborating chondriocont. When the starch is util- ized, its absorption takes place within the amyloplast. The grain diminishes in volume while the outer mitochondrial layer grows and regenerates a chondriocont which will function again later. Fic. 61. — Chondriome. A-D, Root of Cucurbita Pepo. A, meristem; a, amyloplasts; a’, inactive mitochondria. B, cortical parenchyma; a, starch; }, amyloplasts; b’, inactive mitochondria, in some _ cases (b’’)appearing like typical chondrioconts. C, parenchyma of central cylinder; a, starch; c, amyloplasts; c’, inactive mitochondria. D, corti- cal parenchyma of hypocotyl; a, compound starch grain in chloroplast, d; d’, inactive mitochondria. E, liver of frog. F, basidium of Psalliota campestris. In E and F, the mito- chondria form vesicles of unknown significance. Regaud’s method. The chondrioconts, therefore, are not destroyed during elaboration and it is always the same elements which function in the formation of starch. Here again there are found the two categories of ele- ments observed in Elodea canadensis and their shape generally makes it possible to follow them separately during their entire development. (Figs. 57, 58). Guilliermond - Atkinson — 96 — Cytoplasm But these are rare and almost diagrammatic examples. In most cases, it is absolutely impossible among the elements which consti- tute the chondriome in cells of the meristem to distinguish those which will become plastids from those which will remain inactive, ‘\ ~ Q.9, O! <2 Se J ex 1S Pa pd ai SEV Sy Wars dct rll ee act STANT) IZ f Ud ~ al é aN E ss a Wwe ww Re, SOA Nh Ny <\ , Oo S Vi 4, = — Van \ ee A B Fic. 62 (left). —- Chondriome, showing morphological similarity of the meristem of pea root (A) and of pancreas cells of. guinea pig (B), each fixed by Regaud’s method and stained with iron haema- toxylin. A, plastids and chondriosomes indistinguishable; B, chon- driosomes and Claude Bernard granules. (After Cowpry). Fic. 63 (right). — Detail of chondriosomes (A) in pea root and (B) in mouse pancreas. X 1687. (After Cowpry). for they are of similar form. Although in most cases it is the chondrioconts which become plastids, there are numerous excep- tions, and cases are found in which the granules, as well as the chondrioconts, become plastids. It may even happen in some cases that the granules alone form the plastids, whether the other ele- ments present have the form of chondrioconts or whether they also have the form of mitochondria. In the tuber of potato, for example, only granular mitochondria are found (Fig. 39) and it is through the agency of some of these that starch is elaborated. Furthermore, the distinction which we have made between the two categories of elements, 7.e., chondrioconts and granules, in the bud of Elodea canadensis and in the root of Cucurbita Pepo, is far from being general. Whether we can differentiate between the two cate- gories depends, in Elodea, upon the state of activity of the bud and the period at which it was collected. There are buds in which this distinction is much less clear, others where it can no longer be made. The difference in size which we have noticed between the starch-forming plastids and the chondriosomes in parenchyma cells of the pumpkin root may itself diminish or disappear. For ex- ample, in cells in which the starch grains have just been digested, the chondrioconts which elaborated them, grow thinner and are in- distinguishable from the other elements of the chondriome. Cells in the meristem of Hlodea and of the pumpkin are derived from other embryonic cells in which the two categories are not dis- tinguishable. , Chapter X —97— Duality of the Chondriome Theory of the author:- From the series of facts which we have just related, however, there arises the idea that the chondriosomes of cells of the meristem do not all have the same significance, al- though morphologically and histochemically similar. One is led to think that the chondriome of embryonic cells in phanerogams, although appearing homogeneous, is composed of two categories of chondriosomes, maintaining their individuality throughout cellular development. One of these categories corresponds to the plastids and may take on much larger dimensions during the course of development, by virtue of its active elaborative power. The other of these categories, which kept its original size after the plastids had become differentiated and to which we have provisionally given the name inactive chondriosomes, seems to have functions which are as yet not definitely determined. Fic. 64 (left). — Chondriome in (1-4) differentiated colorless root par- enchyma of Athyrium Filix-femina and in (5, 6) frog’s liver. Regaud’s method. (After MANGENOT and EMBERGER). Fic. 65 (right). — Detail of chondriome in (A) Saprolegnia and in (B) epidermis of tulip perianth. X 3000. Regaud’s method. These two categories have the same shape in the phanerogams and it is almost always impossible to tell them apart in the meri- stems and usually, also, even in mature cells which do not have chlorophyll. They are, however, always perfectly distinct in some lower plants (bryophytes, algae) in which chlorophyll is present in all stages of development. Thus considered, the plastids are not differentiated chondrio- somes; they are a special type of chondriosomes. In fact, when the life history of the chondriosomes is followed in the phanero- gams, one is struck by the chondriosomal characteristics which the plastids always maintain. The amyloplasts are usually typical chondriosomes and are only occasionally a little thicker than the other elements of the chondriome. They are not actually distin- guishable from the inactive chondriosomes coexistent with them, until they become chloroplasts.. In this case they appear as thick- ened bodies which, in the last analysis, are only hypertrophied chon- driosomes containing chlorophyll. As for the variations in plas- tidial shape, we do not yet know whether they are caused by a growth of these elements or merely by imbibition. Guilliermond - Atkinson — 98 — Cytoplasm Figure 60 gives a very exact idea of these facts. Here, in the pumpkin seedling, are shown side by side all the forms assumed by the two categories of elements in the course of cellular differ- entiation. It is seen that the two types of chondriosomes have the same shape through all the stages of cellular development— granules, rods, filaments—but these shapes are not always identical for both types at a given stage in development and there are stages in which one type appears as granules and the other as filaments. This makes it possible to distinguish them at every stage. Further- Fic. 66. — A-D, Epidermal cells of petal of tulip. c, leucoplasts; m, chondrio- somes; gg, lipide granules. B, beginning of change in leucoplasts. CC, vesicula- tion. D, cells fixed by Regaud’s method. E-H, filament of Saprolegnia. n, nu- cleus; c, chondriocont. F, beginning of change in chondriosomes. G, vesiculation, v, vesicle. H, filament fixed by Regaud’s method. more, the two categories of elements are capable of division and frequent stages in division are observed. If these two categories of elements are compared with the chondrioconts in liver cells of the frog or with those in fungi, as represented in Figures 60 and 61 (cf. also pp. 85 and 118), it is seen in a general way that it is the plastids which most resemble the animal chondriosomes and those of the fungi. In a general way also, the inactive chondriosomes are a little smaller than animal chondriosomes and are less frequently found as chondrioconts. The plastids in general have the same dimen- sions as the chondriosomes of animal cells but in certain phases become much more voluminous. Chapter X —99— Duality of the Chondriome One may object to the above theory on the ground that the two categories of elements do not have the same origin, that they develop separately and do not possess the same functions. This seems to imply that they are of different nature and that they correspond to radically different formations. This would bring us back to the first opinion of MOTTIER (1918). We shall see, indeed, that in cytology one must be suspicious of analogies in shape, since elements as different as young vacuoles and chondrioconts may show in certain phases of cellular development entirely analogous forms. These forms, moreover, are the forms of chromosomes as well. The histochemical point of view must therefore be the decid- ing factor. Fic. 67. — Epidermal cells of bulb scale of Allium Cepa, as seen under the ultramicroscope. 1, lipide granules (Gl). 2, granules, and the faintly luminous contours of chondriosomes (C) and plastids (P). We shall see, however, that this objection is not valid here. Even before it was known that the chondriome of embryonic cells of the phanerogams was composed of two types of different ele- ments, COWDRY (1918) made a meticulous comparison between the morphological and histochemical characteristics of the chondrio- somes of pancreatic cells of the guinea-pig and those of the pea root. In the latter, the two categories are blended in the meristem and were not differentiated by the author. Cownbry concluded that the chondriosomes are identical in the two cases, including, of course, those which are transformed into plastids. (Figs. 62, 63). Similar comparisons (Fig. 64) were undertaken by EMBERGER and MANGENOT (1920) between the chondriosomes of fern roots (including the amyloplasts) and those of various organs of the frog (kidney and liver). These observations led to the same results. Guilliermond - Atkinson — 100 — Cytoplasm Histochemical and histophysical characteristics cf chondriosomes and plastids:- We proceeded ourselves to make a comparative histo- chemical study of the two chondriosomal categories in chlorophyll- containing plants with respect to the chondriosomes of the Sapro- legniaceae. In the latter, there exists only one category of chondriosomes which can be unquestionably homologized with the chondriosomes of animal cells as we have seen earlier in these pages. This study consisted first of a comparative examination, made as accurately as possible, of the chondriome of epidermal cells from portions of a tulip perianth (white variety) and of the chondriome of Saprolegnia, both of which are particularly favorable for ob- servation of living material. (Figs. 65, 66). The living chondriome in these two different tissues has a sim- ilar morphological appearance and refrac- tivity. It is represented in the tulip by long, thin, undulating and sometimes branching, chondrioconts which correspond to the plas- tids, and by the inactive chondriosomes in the form of granules or short rods. In Saprolegnia, except for the region at the tips of the hyphae, the chondriome, as we have seen, is formed exclusively of long and some- times branched chondrioconts entirely sim- ilar to the tulip plastids. MEYER, who had observed them before the discovery of chon- driosomes in the fungi of the same group, did not hesitate to liken them to plastids. These elements arise from mitochondria by growth and elongation, just as do the tulip plastids. It is in the mitochondrial form that they appear in the extremities of the Fic. 68. — Epidermal cell Honhae of Saprolegnia and in epidermal of Iris germanica. At right, - 4 ‘ modifications in form ob- cells in perianth parts of very young tulip served when a chondriocont fl moves as from (A) to (B) Owers. QA ae »>—> <_<“ 1K <7 > Poy en oacrar a Enea This study was completed later by sim- where arrows indicate direc- . 5 s tion of current. ilar observations which we made on epider- mal cells from the leaves of Iris germanica and especially on epidermal cells from the bulb scales of Allium Cepa. In these bulb scales, the plastids and the chondriosomes present the same forms. They are both composed of a mixture of mitochondria, short rods and chondrioconts. As the plastids do not elaborate starch, it is very difficult to tell the two categories of elements apart. The plastids, however, are often recognizable because they are slightly thicker than the chondriosomes and are longer when they are in the chondriocont stage. We will therefore review briefly here the principal results which we have obtained from this comparative study and add those reported by other authors. The two categories of chondriosomes, plastids and genuine chondriosomes (Fr. chondriosomes proprement dits), of epidermal Chapter X —101— Duality of the Chondriome cells which we have studied and the chondriosomes of Saprolegnia show exactly the same refractivity. Slightly superior to that of cytoplasm, this refractivity, although very slight, still permits the chondriosomes to be adequately seen. Under the ultramicroscope the two categories of elements of epidermal cells and the chondrio- somes of Saprolegnia are distinguishable only under very favorable conditions. When visible, they always have the same appearance and are seen only because of their very faintly luminous contours. With the Zeiss micropolychromar they are made to appear very clearly with a different color from that of the cytoplasm, green on a red background, for instance, or yellow on violet. Chondriosomes and plastids of epidermal cells, as well as the chondriosomes of ’ Saprolegnia, behave like extremely delicate elements which the least change in osmotic equilibrium, or the least pressure on the cover glass of a prep- aration, suffices to change into vesicles (cavulation). In a hypertonic medium they keep changing shape as long as the cell is liv- ing but as soon as it dies, they become vesiculate (Fig. 67). We have already seen that in Saprolegnia the chondrioconts are moved about slowly by the cytoplasmic currents and that dur- ing these displacements they change shape, passing through the most varied forms. They are even able to branch by growing a kind of pseudopodium which afterwards is retracted. In epidermal cells of tulip in which cytoplasmic movements are very slow or do not exist, nothing of this sort is ob- served. In cells of I7vis germanica and of Allium Cepa, however, which are very favor- able objects for study, we have observed for the plastids these same displacements and Fic. 69. — Cells from a tuber of Ficaria ranuncu- loides (A) before and (B) after centrifuging. A, chon- driosomes with and without starch dispersed in the cyto- plasm; B, at one side of the cell are found the nucleus and the starch-bearing chon- driosomes; those without starch remain dispersed. (After Mtnovipoy). the same instability of form. (Figs. 68, 70, 150). During the movement from place to place the plastids are capable of taking the most irregular shapes. They are capable of shortening by becom- ing thicker or of elongating by stretching out. They may form swellings along their long axes which are sometimes vesicular or they may put out transitory ramifications which are later retracted. Analogous observations have been made by EMBERGER for the leuco- plasts in the epidermis of the bulb of Asphodelus cerasiferus. This proves that the plastids and chondriosomes are composed of a semi- fluid, very plastic substance, are in the same physical state and possess the same viscosity. During these observations, moreover, we could follow under the microscope, the process of division of the chondriosomes in the leaf cells of Elodea canadensis, that of the plastids in the epidermal cells of Allium Cepa and of the tulip, in which the displacements of the plastids and the cavulation (vesicu- Guilliermond - Atkinson —hO2-— Cytoplasm lation) which they undergo as they are altered can be observed (GUILLIERMOND, OBATON, GAUTHERET). It may be added that MILOVIDoOV and ORTIZ PICON have demon- strated that the chondriosomes and plastids have a specific weight rather like that of cytoplasm. After centrifuging, the plastids and chondriosomes remain scattered throughout the cytoplasm and it is only when the plastids contain starch grains that they are carried with the nucleus toward one extremity of the cell (Fig. 69). Recent work of FAMIN has proved that, contrary to previous opinions (POLICARD and MANGENOT), the plastids of epidermal cells of tulip petals and the chondriosomes of Saprolegnia are both resistant to high temperatures. Although their visibility in living form is diminished and their chromaticity with mitochondrial tech- niques is lost, they are not destroyed by these high temperatures. The leucoplasts and chondriosomes of epidermal cells of bulb scales of Allium Cepa and tulip = we petals behave as do the chondrio- Ae yy, eta pa somes of Saprolegnia in regard to = BF WS = vital dyes. They stain selectively S ZS GS rm. with Janus green, methyl violet 5B, as Ma Dahlia violet and a certain number > 3 mee Ne of dyes recently mentioned (GUIL- XS ey Sw ~~~ LIERMOND and GAUTHERET). Em- = ( as Las ployed in 0.0005-0.005% solutions, a> : ~< ~\ Janus green stains only the chon- = S Les driosomes and leucoplasts, giving ae | ps = them a pale bluish green color in 2S ( aps cells which are living and showing 2 ey. ‘ z cytoplasmic currents. In 0.01-0.02% ae x solution of the dye, the chondrio- = ens somes and leucoplasts are stained 1 : B but in a manner clearly more ac- s centuated in the former than in Fic. 70. — A, Saprolegnia. Forms taken by a chondriocont observed for the latter and at the same time half an hour. B, Epidermis of Allium the dye accumulates in all the va- Gene.) Forms taken ‘by: a leuconis! >) cuales whichsicontain » phenol ;come= pounds (oxyflavanol and tannin compounds). At higher concentrations it produces only sublethal staining with vesiculation of the chondriosomes and leucoplasts, resulting after a short while in the death of the cells. The other dyes, which for the most part are very toxic, behave somewhat differently. In low concentrations (0.0002-0.0008% ) they stain only the leucoplasts and the chondriosomes which are colored in exactly the same way. The cells remain living for a long time and show very active cytoplasmic currents. In solutions of 0.001% and above, the dyes accumulate at the same time in the vacuoles containing phenol compounds and finally stain the nucleus and cytoplasm to which they give a diffuse color in cells showing active cytoplasmic streaming, but in which they rapidly cause death. Staining is therefore sublethal. Chapter X —103— Duality of the Chondriome As is seen, a single difference is shown between the chondrio- somes and leucoplasts. Janus green stains the former more in- tensely than the latter with a 0.01-0.02% solution of the dye. In recent work, Miss SOROKIN has maintained that Janus green did not stain the leucoplasts. This is inexact, but it is certain that under some conditions Janus green stains the chondriosomes more intensely than the leucoplasts. The chondriosomes and plastids of epidermal cells as well as the chondriosomes of Saprolegnia are preserved with the reagent iodine-potassium iodide which makes them brown and renders them much more distinct than in living material. Both these elements of the chondriome are preserved with a 2% solution of osmic acid which does not turn them brown but, if the preparation is treated with pyrogallol after being in contact with osmic acid for half an hour or an hour, the chondri- osomes and plastids appear gray. Both become even intensely black after being for a long time in a 40% solution of osmic acid (method of osmic impregnation used to reveal the Golgi mate- rial). Lastly, the chondriosomes and plastids of epidermal cells and the chondriosomes of Sa- prolegnia behave in exactly the same way in regard to fixatives. They are strongly modified and lose their chromaticity when treated with fixatives containing alcohol and acetic acid, and are preserved in their shapes and are stained clearly with all mitochondrial techniques (meth- ods of REGAUD, BENDA, MEVES, HELLY, TUPA, VOLKONSKY, AL- Fic. 71. — Chondriosomes and plastids in VARADO’S modification of RIo- the prothallus of Adiantum capillus-Veneris. 3 1, vegetative cell; P, plastid. 38, very young HORTEGA, etc.). They are stained egg. 4, mature egg. 2, young embryo after by DIETRICH - SMITH’s method eee Pacers egg. Regaud’s method. (used for the detection of leci- thins) and after sufficient time by indophenol blue. This behavior, together with that described above, proves that the chondriosomes and plastids have a similar lipide constitution. MILOVIDOV was able, moreover, to show in the chondriosomes and plastids all the protein reactions, just as GIROUD had demon- strated them for the chondriosomes of animal cells. The two categories of elements, therefore, have the same lipoprotein con- stitution. More recently, M1ILovipov demonstrated that the chon- driosomes and plastids in the roots of pea and of Alliwm Cepa do not give the Feulgen reaction and in consequence do not contain thymonucleic acid. Chloroplasts, nevertheless, offer much greater resistance to fixatives containing acetic acid and alcohol than the other plastids and the chondriosomes. Furthermore, in regard to Guilliermond - Atkinson — 104 — Cytoplasm the vital stains for chondriosomes (Janus green, Dahlia violet, methyl violet, etc.) the chloroplasts do not behave like the leuco- plasts, in that the chloroplasts are not stained as long as the cells are alive. On the contrary, as STRUGGER has shown, living chloro- plasts are stained by rhodamine B which, at a sufficient concen- tration, gives them, with time, a very characteristic yellowish color in cells which show cytoplasmic currents and which remain alive for a very long time. (Rhodamine B also stains the chondriosomes and leucoplasts but very faintly.) The chloroplasts are distin- guished from other plastids by their property of reducing silver nitrate. This property was noted first by MOLISCH and, as we have seen earlier, is manifested only in living cells. RUHLAND and WETZEL found it possible, with this reaction, to demonstrate the presence of chloroplasts in the chondriosomal state in generative cells of Lupinus luteus and of some other plants. GAVAUDAN in his Fic. 72 (left). — Chondriome of antherozoids of Adiantum capillus-Venerts. 1, 2, antheridial initial. 3, 4, sperm mother cell. 5, 6, stages in the formation of the antherozoid. Regaud’s method. (After EMBERGER). Fic. 73 (right). — Behavior of the chondriome during the life cycle of a fern. f, leaf; starch-forming chloroplasts and chondriosomes. 2-3, formation of spores; decreasing activity of plastids. 4, spore mother cells; inactive plastids indistinguish- able from chondriosomes. 5, mature spore; plastids become active. 6, prothallus; starch-bearing chloroplasts. 17, 8, sexual cells; second cessation of activity of plastids. 9, egg; homogeneous chondriome. 10, developing embryo; certain chon- driosomes secrete starch. 11, 12, adult plant; 11, amyloplasts of the root. 12, chloroplasts of the leaves. (After EMBERGER). study of the hepatics claimed that this property is common to all plastids, even those lacking in chlorophyll, and considers it a means of distinguishing plastids from chondriosomes. But our later re- search, as well as that of GAUTHERET and of MIRIMANOFF, did not confirm this assertion and proved that plastids without chlorophyll do not reduce silver nitrate in living cells any more than do chon- driosomes. This property which chloroplasts have of reducing silver nitrate in living cells has nothing in common with the black coloration of the plastids and chondriosomes in cells treated by ALVARADO’s modification of RI0-HORTEGA’s method, or with that sometimes taken on when they are impregnated with silver (Golgi method). It does, however, explain why PENSA found that only the chloroplasts were stained when he treated living tissue with Golgi’s technique (silver impregnation). Chapter X —105— Duality of the Chondriome All these facts lead us to the conclusion that the two categories of cytoplasmic organelles in chlorophyll-containing plants both show the characteristics of chondriosomes and it is evident that there is no criterion, unless it is the ability of the plastids to form starch and chlorophyll, for including the inactive chondriosomes rather than the plastids in the formations known in animal cells as chondriosomes. On the contrary, the plastids by their elongated chondriocontal forms sometimes resemble the chondriosomes of animals even more than do the inactive chondriosomes of plants. Fic. 74. — Fern sporangia. Successive stages in the return to a homo- geneous chondriome by resorption of starch and loss of pigment in the chloro- plasts. 1-3, Asplenium Ruta-muraria. 1, sporogenous cells; 2, spore mother eells; 3, tapetum and spore mother cells. 4, Pteridium; tetrad and tapetum. Regaud’s method. (After EMBERGER). The plastids are sometimes, however, slightly larger. Further- more, the inactive chondriosomes unquestionably have the charac- teristics of chondriosomes from which it is impossible to separate them, as MEVEs does, for they, too, show in a great number of cases the form of typical chondricconts. These two categories of elements, therefore, fit the definition of chondriosomes. They correspond to organelles seeming to be incapable of forming other than by division, they have the shape Guilliermond - Atkinson — 106 — Cytoplasm of granules, rods or chondrioconts, are able to change from one of these forms to the next, and are characterized by a number of well determined physical and chemical properties. The theory of the duality of the chondriosomes has been remarkably confirmed by a study of the life history of the chondriome throughout the plant kingdom and is particularly well supported by the investigations of MANGENOT and EMBERGER. Development of chondriosomes and plastids among the plant groups:- E/MBERGER (1920-23), in investigating the pteridophytes (Figs. 71-74), found that the egg cell in the ferns contains a chon- driome exactly like that in the animal cells, in which chondriome it is not possible to distinguish the plastids from other chondriosomes. However, in those prothallial cells from which the egg cell is de- rived there are both large chloroplasts and small chondriosomes. As these cells differentiate in the course of the formation of the egg, EMBERGER has shown that their chloroplasts lose chlorophyll and Fic. 75 (left). — Selaginella Kraussiana. 1, vegetative tip of the stem, each cell containing chondriosomes (C) and a slightly larger plastid (P) appressed to the nucleus. 2, Diagram of developing plastid during cellular differentiation. (After EMBERGER). Fic. 76 (right). — Selaginella Kraussiana. Plastid (P) in living parenchyma cell of the stem. (After EMBERGER). at the same time diminish progressively in volume, with the result that they take on, little by little, the appearance of small elements which it becomes impossible to distinguish from the chondriosomes. The same phenomena occur in the formation of the antherozoids. The fertilized egg resulting from the fusion of these cells shows, therefore, a homogeneous chondriome. In the embryo, some of the elements of this chondriome differentiate anew. In the leaves they become chloroplasts, in the stem and root they become large chon- drioconts which represent the starch-forming plastids. In the epidermal cells of leaves which will produce sporangia, both chloroplasts and inactive chondriosomes are encountered. In the young sporangia, however, the chloroplasts again lose their chlorophyll and appear as typical chondriosomes, indistinguishable from the inactive chondriosomes. From the time that the spore begins germination, these typical chondriosomes grow larger, be- come impregnated with chlorophyll and take on again the character of chloroplasts. Chapter X —107— Duality of the Chondriome Analogous phenomena were found later (CHOLODNY, 1923) in the submerged leaves of Salvinia natans which, as is known, look like roots, as they have no chlorophyll and are reduced to veins. The chloroplasts which are present at first in these leaves lose their chlorophyll and take on the appearance of chondriosomes abso- lutely indistinguishable from the genuine chondriosomes. Investigating the Selaginellas (Figs. 75, 76), EMBERGER ob- served that in cells of the meristem and in the spores where the chondriome contains above all long chondrioconts, there is only a single colorless plastid, which also appears as a chondriocont but is a little larger than the others and is pressed against the nucleus. This organelle, already pointed out by HABERLANDT, SAPEHIN and P. A. DANGEARD, which is at first scarcely distinct from the other chondrioconts, grows little by little during cellular differentiation until in each cell of the leaf and stem there is a single chloroplast. It is composed of a series of large swellings united by thin filament- ous portions which are brought about by uncompleted divisions of the initial plastid. This fact is particularly interesting from two points of view. First, in Selaginella and Anthoceros (in which there is also in each cell only one chloroplast, crescent-shaped in this case, more or less appressed to the nucleus but always larger than the small colorless plastids of embryonic cells of Selaginella), there are found the intermediate steps between the plastids of the phanerogams and the large chloroplasts of some algae. This shows that there is no reason to consider the chloroplasts of the algae as different from ordinary plastids. Secondly, the presence of this solitary plastid, which can be followed through all cells of Selagi- nella and which divides when the cell does, furnishes undeniable proof that the plastids maintain their individuality during the course of cellular development and arise always from the division of pre-existing plastids. The behavior of the plastids in the phaner- ogams makes this seem likely but does not sufficiently demonstrate ike The investigations of MANGENOT (1922) brought out that the algae behave differently, from the point of view of plastidial de- velopment, depending on whether the chlorophyll persists in all stages of development or disappears in the sexual organs. When it persists, the plastids are distinguished from the chondriosomes in all stages of development, including the egg, by their size, their shapes and their colors. Consequently chloroplasts and chondrio- somes are coexistent at all times. MANGENOT demonstrated the presence of large chloroplasts and small chondriosomes at all stages in the Siphonales. These had already been encountered in Vaucheria by RUDOLPH. These two categories of organelles, in spite of the difference in their dimensions, have analogous shapes and divide at the same time in some phases of development. Such is also the case in the Fucaceae, in which, however, the chlorophyll and fucoxanthin lose their intensity in the oogonium and in the apical cells. There the phaeoplasts take on the form of small rod, or spindle-shaped Guilliermond - Atkinson — 108 — Cytoplasm organelles, differing only slightly from the chondriosomes., Fur- thermore, the chlorophyll and fucoxanthin disappear in the mother cell of the antheridium and the phaeoplasts in this cell come to look like chondrioconts and are distributed among the antherozoids in such a way that each encloses a single phaeoplast. This plastid later is filled with a carotinoid pigment and becomes the stigma. The stigma, then, is simply a structure derived from a phaeoplast. Plants in which the chlorophyll does not persist but rather disappears in the sex organs are found in the Rhodophyceae (Flo- yideae) and the Characeae. In the Rhodophyceae, for example in Lemanea, the cells of the thallus (Fig. 78, I and the upper portion of A) enclose large, ribbon-shaped rhodoplasts, sometimes anasto- Fic. 77. — Fucus vesiculosus. Fusiform and rod-shaped plastids (pl) with mito- chondria (m) and fucosan granules (p.p.) in Fucus vesiculosus. 1, apical cell; 2, two celled embryo. N, nucleus. Regaud’s method. (After MANGENOT). mosing to form a network, together with small chondriosomes. In those portions of the thallus containing little chlorophyll (Fig. 78, I, and lower portion of A), these elements grow thinner and appear somewhat like chondrioconts. In the rhizoids (Fig. 78, 1), in which neither chlorophyll nor phycoerythrin exists, the plastids become very small and look so like the inactive chondriosomes that it becomes impossible to tell them apart. The trichogyne and other cells of the carpogonial branch (Fig. 78, A) develop from an ordinary cell of the thallus containing large rhodoplasts. A regres- sion of chlorophyll and of phycoerythrin may be observed in these cells. The plastids lose their color and are transformed into small rods becoming like the chondriosomes which are present with them in the cell. Then the carpogonium shows a chondriome in which all distinction between plastids and chondriosomes is impossible. This chondriome persists in the first cells of the gominoblast fila- Chapter X —109— Duality of the Chondriome GES) (EE) Fic. 78. — Rhodophyceae. Life history of the plastids in Lemanea. A, fragment of wall of cystocarp with two carpogonial filaments. B, Id. with gominoblast fila- ments. C, detail of filament. D-G, carpospores. H, germinating spore. I, assimilat- ing filament. i, filament near substratum. Is, rhizoid. J, cystocarp. (After MANGENOT). Guilliermond - Atkinson — 110 — Cytoplasm ment (Fig. 78, B, C) and then there can be followed in these cells (Fig. 78, D-F) a differentiation of large rhodoplasts from some of the elements of the chondriome. In the mature carpospores (Fig. 78, G, H) there are found fairly large, well differentiated, disc-shaped rhodoplasts. In the Characeae, MANGENOT found small chloroplasts and chondriosomes in the apical cells but in the oosphere there is no chlorophyll. In the cells which give rise to the oosphere, MANGE- NOT observed a regression of the small chloroplasts. They lose their chlorophyll and are transformed into mitochondria or short rods which can not be distinguished in the young oospheres from the inactive chondriosomes. In the course of development of the oosphere, some of the mitochondria and rods representing the former chloroplasts, elongate and take on the shape of typical chondrioconts, whereas the inactive chondriosomes persist in the form of mitochondria. The chondrioconts then elaborate numerous. starch grains in the usual way. They cor- respond therefore to amyloplasts. Information on the development of the plastids is still scarce in the bryophytes. It has already been- seen that RUDOLPH, SCHERRER, SAPE- HIN, and MOTTIER believed that chlorophyll persists in these plants in all stages of development. They state that bryophytic cells always contain chloroplasts and chondrio- somes at the same time. The fact are aL Oe ee a Ran is well demonstrated for Anthoceros ment of the chondriome in the egg. A, but is questioned for the other bryo- Onn ee See ee Diytes.. Whereas (b 7A. DANGEARD, chondrioconts. _D, mature egg; chon- 12. DANGEARD, GAVAUDAN and WEIER ager forming starch. E, detail of tend to confirm it, ALVARADO, SEN- JANINOVA, MOTTE and CHALAUD Op- pose it and believe that the chloroplasts are derived from the chondriosomes. ALVARADO has stated that in young paraphyses of Mnium cuspidatum there are no chloroplasts but only chondrio- somes of which some afterwards become transformed into chloro- plasts. According to MOTTE, most mosses contain in the apical cell of the stem both small lenticular chloroplasts and chondrio- somes, while in some species (Grimmia crinita) only chon- driosomes are found, a part of which develop into chloroplasts in cells arising by division from this apical cell. MorTTeE described a regression of chloroplasts in the sperm mother cells. In the forma- tion of these cells, the chloroplasts lose their chlorophyll and be- come transformed into long chondrioconts. These fragment to form granules that it is impossible to distinguish from the chon- driosomes, which are coexistent with them. According to MOTTE, Chapter X —11l1— Duality of the Chondriome the archegonium is formed from an initial cell containing only chondriosomes and he finds that the egg also lacks chloroplasts. These facts make it seem extremely probable that, at some stages in development in the bryophytes, the chloroplasts are capable of regression and of taking on mitochondrial form just as in the pteridophytes and in certain algae. However this may be, it follows from data presented for the first time in the splendid work of EMBERGER and MANGENOT, that the chloroplasts may, under some conditions, lose their chlorophyll, become considerably smaller, and take on again the size and shape of typical chondriosomes. The form typical of chondriosomes and the form typical of chloroplasts are therefore reversible and the chloroplasts may be considered as chondriosomes containing chlo- rophyll. The chloroplast is derived from a chondriosome and may under certain conditions lose its chlorophyll and revert to the state of the chondriosome, the state which is characteristic of the functionally inactive phase of plastids, exactly as the amyloplast resumes its initial form after the absorption of its starch. If this reversibility of chloroplasts is not ordinarily observed in phanero- gams, it is doubtless because, according to the research just dis- cussed, the chlorophyll-containing tissues in these plants achieve a state of differentiation too advanced for a regression to take place such as occurs in plants less evolved. Therefore the fact that chlorophyll is elaborated in a continuous or discontinuous manner influences very notably the appearance taken by the chondriome in chlorophyll-containing plants. In the first case, the cells contain constantly and at the same time, both large chloroplasts and small chondriosomes; in the second case, on the contrary, there are found, during the periods when chlorophyll is lacking, chondriosomes which all together constitute a chondri- ome analogous to that encountered in cells of animals and fungi, and in which it is not possible to distinguish the plastids from the genuine chondriosomes, and it is only during phases of elabora- tion of chlorophyll that some of the chondriomal elements grow and become large chloroplasts. These facts will be illustrated by a study of saprophytic or parasitic phanerogams in which chlorophyll is formed only in very small quantities or, in some species, not at all. Among these, Limodorum, a saprophyte which is poor in chlorophyll, contains only very small chloroplasts. In the genus, Orebanche, in which chlorophyll has disappeared, plastids elaborating starch are still observable which in some portions of the plant also contain carotin- oid pigments. In the stem of Monotropa, in which chlorophyll is also lacking, there is no longer production of starch, except in the endodermis, and in that region only, can the plastids be dis- tinguished from the chondriosomes. This distinction is impossible in the tissues of Cytinus Hypocistis, a plant more completely adapted to parasitic life. This plant is also lacking in chlorophyll and has lost its power of forming starch (EMBERGER and MANGE- NOT, unpublished observations). Guilliermond - Atkinson — 112 — Cytoplasm Considering this from a different angle, one of our students, GAUTHERET, has shown that the production of chlorophyll may be experimentally obtained in most roots when they are grown under certain conditions (in the presence of light and in media contain- ing a certain quantity of sugar). Thus, for example, in the root of the barley, whose cells normally contain a chondriome composed of a mixture of chondrioconts, rods and mitochondria, in which plastids and genuine chondriosomes are indistinguishable, GaAu- THERET has succeeded in obtaining large chloroplasts, entirely com- parable to those encountered in the leaves of the same plant, by a differentiation of some of these elements. The series of investigations undertaken with the aid of mito- chondrial methods, either by us in the phanerogams or in our laboratory by EMBERGER and MANGENOT on the pteridophytes and algae, have permitted us definitively to solve this problem which has remained obscure for so long, namely, the origin and life his- tory of chlorophyll-containing plastids. The progress made since the work of SCHIMPER and MEYER may be judged by comparing the state of this question when there were no methods for preserving plastids in stained preparation and when the investigator had to be content with incomplete observations of living material, to the present status of the question with its very accurate data obtained by mitochondrial technique, completed by observation of living material. It is now evident that the chlorophyll-containing plants possess two categories of organelles which are permanently found in every cell, both of which show all the characteristics of chondriosomes in animal cells. The first category, whose roéle it has not been possible to define completely, corresponds to the chondriosomes found in ceils of animals and fungi. Its elements may be called inactive chondrio- somes or even genuine chondriosomes. The second category, peculiar to chlorophyll-containing plants, corresponds to the plastids of SCHIMPER. These are to be dis- tinguished under the name plastids. These two categories of organelles seem each to keep their individuality in the course of cellular development and seem to form only by division of pre-existing elements. This behavior, difficult to demonstrate for the plastids in the cells of phanero- gams, is absolutely proved by what is known regarding the chloro- plasts of green algae, by the study of Anthoceros, and especially, by the observations of EMBERGER on Selaginella. It is extremely probable, also, that these characteristics are shared by the genuine chondriosomes, since these elements are present in all cells, since they have never been observed to form de novo or to disappear and since they are capable of division. Indirect arguments in favor of this opinion may be drawn, moreover, from the behavior of the plastids which are very similar to them. The two categories of elements have (except in many algae in which chlorophyll persists in all stages), the same characteris- Chapter X —113— Duality- of the Chondriome tic forms: granules, rods and filaments, capable of changing from one shape to the other. They offer, moreover, and this is much more important, the same histophysical characteristics (same re- fractivity, same viscosity, same process of alteration) and the same histochemical characteristics, even to their behavior with a great number of chemical reagents and dyes. For these reasons, it is generally impossible to tell them apart in embryonic cells of plants in which chlorophyll is not continuously elaborated. The plastids, therefore, are distinguished from other chondriosomes only by the fact that they are the centers of very active elabora- tions which considerably modify their shape. This is true in cells lacking in chlorophyll in which plastids elaborate starch, and espe- cially true in green cells in which the plastids become voluminous because of the chlorophyll which they accumulate. Moreover, these modifications of form may be only transitory. The amyloplasts in cells without chlorophyll, as soon as the starch has been absorbed, go back to their forms of typical chondriosomes. The chloroplasts themselves may in some cases lose their chlorophyll and return to their original state as chondriosomes. In a word, the chondrio- somal form is the form taken by these organelles during the func- tionally inactive period. The only distinction, therefore, that exists between the animal and fungal cell on the one hand, and the cell of the green plant on the other, is the presence of plastids, the second category of chondriosomal elements. This distinction is re- lated to the existence of the chlorophyll function which charac- terizes green plants. These facts, now exactly demonstrated by our work and that of our students, carried on over a period of thirty years, have led us to formulate the theory of the duality of the chondriome in chlorophyll-containing plants. This consists in stating that the chondriosomes of chlorophyll-containing plants are composed of two categories having between them the same relationships which the heterochromosomes bear to the autochromosomes, the first cate- gory (the genuine chondriosomes) being very similar to the chon- driosomes of animals and fungi, the second category (the plastids) composed of a supplementary line of chondriosomes related to photosynthesis, which characterizes these plants. It is obvious that the two categories must, after all, possess differences in chem- ical constitution. Otherwise it could not be explained why one has functions which the other does not have. Nevertheless these differences, probably very slight, do not appear during histo- chemical analysis. In any case, both categories of elements have very closely allied lipoprotein constitutions and form in the cyto- plasm a disperse lipoprotein phase. They are differentiated only by one of them manifesting a function which is lacking in the other. The question is therefore definitely solved as far as the facts are concerned and it is demonstrated that the chondriosomes and plastids are two individual cellular components with the same lipoprotein constitution, capable of presenting identical shapes but Guilliermond - Atkinson — 114— Cytoplasm developing side by side without any genetic bond between them. This is undeniable and cytologists are more and more inclined today to admit it. There is only one point which still remains hy- pothetical and this is the identification of plastids with chondrio- somes which many cytologists still refuse to accept. In order to settle the question definitely we should have to know more pre- cisely the chemical constitution of these two categories of elements and this is impossible in the present state of science. We should also have to obtain information on the phylogeny of plastids and chondriosomes which now escapes us. That which is certain is that the chondriosomes and plastids can only be regarded as very closely allied formations. It is illogical to deny these incontestable relationships as so many cytologists still do, for it is a much greater assumption to consider them as essentially different formations than it is to put them both together under the heading of chondrio- somes. Also, our theory, which nothing has contradicted since we formulated it nearly twenty-five years ago, seems to be the only interpretation possible in the present state of knowledge. It has, furthermore, the advantage of suggesting a series of working hy- potheses, not only with respect to the réle of the genuine chondrio- somes which is at present almost completely unknown, but also with respect to the physiological functionings of the plastids in the elaboration of chlorophyll, their functioning in photosynthesis and in the condensation of hexoses into starch. It is possible to imagine that a day will come when the physico-chemical study of the cyto- plasm will show us that the chondriosomes have a very general function of which that manifested by the plastids is only one specific part. Phylogenesis of chondriosomes and plastids:- Nothing is known about the phylogenesis of these two categories, chondriosomes and plastids, whose chemical constitutions are so closely allied. The fungi, which many botanists consider to be derived from the algae, show, however, no trace whatever of plastids. There is only one line of chondriosomes in them. In the Cyanophyceae (p. 41), which by reason of the primitive structure of their nucleus, may be considered as the most inferior algae known, it is impossible, as we have seen above, to detect the presence of chondriosomes and of plastids. The chlorophyll is diffuse in the cytoplasm of these algae. It has sometimes been thought that the lipoprotein sub- stance of plastids and chondriosomes was also diffused in the cyto- plasm. This, however, is only an hypothesis based on the absence of plastids and on the fact that chlorophyll can hardly have as sub- stratum any other than a lipoprotein substance. In all flagellate algae, another inferior group thought to be the common ancestors of algae and protozoans, there exist very varied forms, some with chlorophyll, some without. All contain chondriosomes (CHADE- FAUD). We have seen that in chlorophyll-containing forms the plastids sometimes look like the chloroplasts of phanerogams (Euglenas, Peridiniaceae), sometimes appear as a single voluminous Chapter X —115— Duality of the Chondriome chloroplast (certain Chrysomonadales). According to CHADEFAUD, the forms possessing only a single chloroplast represent the most primitive types. Among the forms without chlorophyll, VOLKONSkKyY has reported in Polytoma uvella the existence of a single leucoplast per cell, which appears as a fine network spread throughout the cytoplasm. This leucoplast elaborates starch. More recently Miss RABINOVITCH found the same organelle in Polytomella coeca. It has been possible to suppress the chlorophyll by various cultural processes in the Euglenas but it has not been possible previously to understand what became of the chloroplasts in the forms deprived of chloro- phyll. In the phylogenetic series, above the flagellated algae are placed the green algae, such as the Chlorophyceae, which often possess only a single voluminous chloroplast. We then progress through the Rhodophyceae, the Characeae and bryophytes to the pteridophytes and phanerogams, in which the plastids are always fragmented into numerous small elements similar to chondriosomes. Chapter XI HYPOTHESES RELATIVE TO THE ROLE OF CHONDRIOSOMES AND PLASTIDS It has just been seen that cytological investigations, carried out during recent years, have shown that the cytoplasm always contains in suspension various inclusions, among which the most important are the chondriosomes to which, in chlorophyll- containing plants, are added the plastids. The latter present a close analogy to the chondriosomes but may be considered as a line dis- tinct from these elements. Immediately, this raised the question as to the rodle of the chondriosomes and plastids. We find ourselves here on uncertain ground, for it must be recognized that if, at the present moment, our morphological knowledge is very advanced, we are still ex- tremely ill-informed as to the role which must be attributed to these various elements in the functioning of the cell. As DEVAUX says, “The fact is that we do not sufficiently know the inner organ- ization of the cell, for all that a microscopic study makes possible is a first approximation, manifestly incomplete. We should, for complete knowledge of the cellular mechanism, be able to reach the molecules themselves, that is, the elementary particles of the cell, and study them from the triple point of view of structure, of molecular attractions and movements, as well as from the point of view of reciprocal relations.”’ Perhaps some day the progress of physical chemistry will give us precise information on this point, but for the moment we must be content with hypotheses which are still very vague and which we will take up here as briefly as possible. Investigations at the beginning of the study of chondriosomes led various authors, REGAUD in particular, to attribute to the chondriosomes of animal cells, an important role in the phenomena of secretion. According to this opinion the chondriosomes are organelles through whose agency are elaborated very diverse products of cellular activity: zymogen granules, fats, pigments, i.e., the chondriosomes have a role exactly like that of the plastids in chlorophyll-containing plants. Thus the chondriome appears as the secretion apparatus of the cell. Although the in- vestigations in plant cytology have demonstrated the very curious fact that it is precisely the plastids of chlorophyll-containing plants which show exactly the same forms and the same histochemical re- actions as the chondriosomes, we have seen, however, that more accurate research on animal cells and on the thallus of fungi, employing the control methods of direct observation of living ma- terial and of vital staining, have not been able to confirm this secretory réle except in exceptional cases (NOEL). The direct par- ticipation of chondriosomes in phenomena of secretion seems to us therefore to be hypothetical and in reality we know nothing as to Chapter XI — 117 — Role of Chondriosomes the réle of these organelles. Nevertheless, the fact that the chon- driosomes usually show no morphological evidences which can be related to their participation in secretory phenomena does not exclude the possibility that they may have a réle in these phenom- ena, and it is possible that later studies may succeed in demon- strating this rdle which is suggested by the close parentage of chondriosomes and plastids. So all cytologists are at present agreed in attributing to the chondriosomes some important réle in cellular metabolism. The role of the plastids in chlorophyll-bearing plants is, on the contrary, very clear, for it is manifested morphologically by the production within these organelles of chlorophyll, carotinoid pig- ments, starch grains and so forth. However, we do not know at all by means of what physico-chemical processes these phenomena are brought about. At first an essential réle in the elaboration of these different products, as well as participation in the phenomenon of photosynthesis, was attributed to the plastids. The plastids were considered as small laboratories which were the seat of the most important synthesis in the plant cell. At the present time there is a tendency, rightly or wrongly, to react against this way of thinking and to consider the plastids as perhaps only the accumulation centers for certain substances manu- factured by the cytoplasm itself. In any case, the work of modern physiologists caused it to be thought that the plastids contribute only in part to photosynthetic phenomena. Actually, we still do not know how much to attribute to the chlorophyll, how much to the substratum of the plastids and how much to the cytoplasm. Nevertheless, it seems that the plastids do indeed play an important part in photosynthesis. In any case, there is a rdle which cannot be denied the plastids. It is the ability to condense the hexoses into starch. In this case it is a question not of accumulation but of actual synthesis. The influence of plastids on the form and growth of starch grains is manifested by the fact that these prop- erties are determined by the place where the grain appears in the plastid. If the grain arises in the middle of the plastid, the hilum, z.e., its oldest part, is the center and concentric layers, which are the outcome of growth by apposition, develop regularly about it. The starch grain in this case remains entirely enveloped by a thin mitochondrial layer. When, on the contrary, the growing grain forms on the periphery of the plastid, it very soon bursts out of the plastid which then covers it as a sort of cap at only one extremity. In this case the hilum is situated at the pole opposite to that occu- pied by the cap, and the layers of growth do not form any longer, except in those regions which are still in immediate contact with the plastid. The grain then is eccentric in structure. The fact that the starch grain is formed and. is hydrolyzed in the interior of the plastid led certain investigators to believe in the existence in the substratum of the plastid of a diastase of rever- sible action, capable of bringing about both synthesis and hydro- lysis of starch (MEYER, SALTER). MAIGE, on the contrary, thinks Guilliermond - Atkinson — 118 — Cytoplasm that there is in the plastid only a synthesizing diastase and that the hydrolyzing diastase has its seat in the cytoplasm. The ex- istence of diastase is, however, not necessary, if the hypotheses are accepted as formulated by NAGEOTTE and DEVAUX who con- sider the plastids and chondriosomes as catalysts. It is known, moreover, that plastids in the embryo sac of Lilium candidum may enclose protein crystalloids which are used as re- serve products. Some experiments seem to indicate that the plas- tids have the ability to accumulate proteins and it is even possible that they may be very important synthesizing centers (ULLRICH, GRANICK, etc.). Recently VOLKONSKY seems definitely to have furnished proof that the reticulate leucoplast of Polytoma uvella undergoes considerable variations in volume depending on the na- ture of the nutriment furnished it. It expands greatly in media rich in assimilable nitrogen. The leucoplast seems, therefore, to be the region of the cell to which nitrogenous nutrients most readily go, especially the amino acids, which are there transformed into more complex products. This phenomenon is to be considered in connection with the observations of NOEL on chondriosomes in livers of mammals, and seems to confirm the hypothesis of ROBERT- SON-MARSTON, of which more will be said later. Yet VOLKONSKY says that this synthesis does not go beyond polypeptides and that the formation of proteins is completed in the vacuoles. It is seen that, in reality, we are still very insufficiently in- formed on the réle of plastids. The close relationship of the plas- tids and chondriosomes leads us to suppose that the two categories of elements must have a single function which is very general and that the function manifested morphologically by the plastids is only a special example of it. Thus, while admitting with the majority of cytologists that the chondriosomes have an important role in metabolism, we can, at the same time, examine the various hypotheses which have been proposed to explain the role of plas- tids and that of chondriosomes and which may apply to both categories of elements. Purely for historical interest, the theory formulated in France by PorTIER (1919), then in America by WALLIN (1922) may first be mentioned. This theory held that the chondriosomes and plas- tids represent symbiotic bacteria which are found present in all cells and by means of which all syntheses take place in the cell. It was based solely on the morphological resemblance of the chondriosomes to bacteria and on the fact that with mitochondrial technique the symbiotic bacteria which are encountered in certain cells, notably those in the bacteria-containing root nodules of the legumes, stain like the chondriosomes. The theory is untenable, for even if it is true that the symbiotic bacteria stain as the chon- driosomes do by these techniques, it signifies nothing since these stains are not specific and since symbiotic bacteria show histo- chemical behavior which makes it impossible to confuse them with chondriosomes (resistance to alcohol, acetic acid, etc.). This the- ory, successfully opposed by LAGUESSE, REGAUD, COWDRY and OLIT- Chapter XI — 119 — Role of Chondriosomes SKY, DUESBERG, LEVI, and MILOVIDOV, is today abandoned by Por- TIER himself. Nevertheless it had the merit of initiating investi- gations which have produced methods by which chondriosomes can be distinguished in cells from symbiotic and parasitic bacteria. Cowpry and OLITSKY, DUESBERG, and MILOVIDOV have described methods by which, in the cells of nodules of legumes and in the adipose cells of cockroaches, symbiotic bacteria can be distin- guished from the chondriosomes by means of differential staining. By these methods MILovipov found that the symbiotic bacteria and the chondriosomes, including the plastids, are both distributed to the daughter cells during mitosis but not in the same manner. He has demonstrated, besides, that centrifuging brings about a dis- placement of the symbiotic bacteria in the direction of the centri- fugal force but has no influence on the chondriosomes and plastids. The symbiotic bacteria, therefore, are heavier than the cytoplasm and are heavier than the chondriosomes and plastids. (Figs. 80, 81). This work on animal cells led REGAUD to consider the chondrio- somes as “organelles having an eclec- tic and pharmaceutical function in the cell” 7.e., as “electosomes”. Ac- cording to this theory, the chondrio- somes by means of a physico-chemical mechanism still unknown, draw from the surrounding medium the mate- rials necessary to the life of the cell, ey Peele oer ar ene transform them and finally release teria and chondriosomes differential- the product, of elaboration, sO that it) 5 fee eS Pine beet may be excreted or kept in reserve. _ the chondriosomes (black) surround An analogous theory was applied by ‘he } 1) | Fic. 92. — Teeth of young, living rose leaflets containing anthocyanin pigment which makes visible the vacuolar system developed from filamentous vacuoles which swell, anastomose and fuse to form one large vacuole per cell. A, D, cells at tip; B, C, older cells. D, after PENSA. the formation of anthocyanin may be followed. Now, we ob- served that in the youngest cells, namely, those at the tip, the pigment appears as minute, numerous, filamentous elements very like the chondrioconts. These elements, taken all together, are exactly like a chondriome. In the region nearer the base, these elements seem to swell and be transformed gradually into small vacuoles, which, by their fusion, finally form a single enormous vacuole, occupying the major part of the cell and enclosing the Chapter XIV — 147 — The Vacuolar System pigment in solution. By reason of the great resemblance between the initial shapes in which anthocyanin first appears and the shapes of the chondrioconts, we were led to think, originally, that this pigment arose in the elements of the chondriome which then be- came transformed into vacuoles. This interpretation was founded also on the fact that these shapes were preserved by mitochondrial techniques. With the method of Regaud, for example, we ob- tained at that time, both typical chondriosomes and elements of the same form as the chondriosomes, but a little larger, stained in the same way, but for which the staining was less stable and which, if destaining was prolonged, lost all the dye and took a yellow color. This color we attributed to the action of the potas- sium bichromate on the anthocy- anin. Now, between the typical chondriosomes and these elements which resemble them, there seemed to exist all intermediate stages. We thought at that time, therefore, that the larger = elements, to which the potassium bichromate gives a yellow color, corresponded to chondriosomes impregnated with anthocyanin. Our interpretation was a natural one at the moment, for the chon- driome was not yet well known, the origin of the vacuoles was not . PO sap Se understood, and it was thought that most of the pigments of ani- mal cells were of mitochondrial origin. This observation was immedi- ately investigated by a certain number of workers among whom Fic. 93. — Oil glands of a walnut leaf. 1, 4, preparations prop- (M) | stained Regaud’s method. erly stained; chondriosomes black, filamentous vacuoles (VA) containing an anthocyanin-tannin complex stained yel- low brown. 2, 3, preparations insufficiently destained; 2, both elements black; 3, only vacuoles visible. Preparations such as 2, 3, might lead observers to think the chondrio- somes are transformed into tannin-contain- ing vacuoles. some agreed with our interpreta- tions (MOREAU, MIRANDE) and others contested them. Among the latter ARTHUR MEYER thought, but without having proved it, that the chondriosome-like elements which mark the beginning of the formation of anthocyanin, represent filamentous vacuoles. LOw- SCHIN, on the other hand, expressed the opinion that these fig- ures correspond merely to anthocyanin itself. According to him the anthocyanin is deposited in the cytoplasm in this form and consequently there is merely a fortuitous similarity in shape be- tween these figures and that of the chondriosomes. PENSA, stressing the fact that the chondriosome-shaped ele- ments of anthocyanin always appear colored yellow by mitochon- drial methods — probably in preparations too much destained — was led to think that they have no relation to the chondriosomes. Taking up LOWSCHIN’s theory and drawing upon his own observa- tions, PENSA concluded that these figures correspond merely to an Guilliermond - Atkinson — 148 — Cytoplasm aggregated state of anthocyanin and that this pigment might, according to the conditions in which the cell is at the time, assume two different colloidal states in the cytoplasm: an aggregated state characterized by the chondriosome-shaped elements scattered throughout the cytoplasm and a state of dispersion, 7.e., a state of pseudosolution in the cytoplasm. By treating with alkaloids those cells containing anthocyanin in the state of a pseudosolution in the cytoplasm, PENSA claims to have obtained a return of the pigments to the aggregated state, characterized by the produc- tion of anthocyanin granules assembled in little chains or in a network. Thus, according to PENSA, the chondriosome-shaped ele- ments of anthocyanin do not always coincide with the stage of the formation of pigment. But PENSA’s interpretation is erroneous, for the author did not understand that the pigment is dissolved in the vacuoles and not in the cytoplasm, and that the phases which he attributed to the state of pseudosolution of the pigment corre- spond to the dispersion state of the anthocyanin within a single enormous vacuole, occupying the major part of the cell and sur- rounded only by a thin layer of parietal cytoplasm. That which PENSA considers to be the cytoplasm is, therefore, nothing more than vacuolar sap. Alkaloids do indeed bring about flocculation (an aggregated state) of anthocyanin in the form of granules showing Brownian movement. The granules are precipitates of tannin absorbing the pigment as they form. These precipitates may assemble in little chains or in a network, and were erroneous- ly likened by PENSA to the figures observed in meristematic cells. In the meristematic cells it is a question of small elements shaped like granules or filaments, resembling the chondriosomes, made up of a concentrated colloidal solution quite different from the vacu- oles known at that time. DANGEARD later gave these small ele- ments their true title of young vacuoles. There is nothing in common between these small vacuoles and the precipitations of anthocyanin obtained by PENSA in mature cells. Credit must go to P. A. DANGEARD for having oriented this study in a new direction. We have already said that in studying the origin of the vacuoles in very diverse plants by means of vital stains, among others cresyl blue, this investigator found in the meristematic cells of plants and in the growing tips of fungal hyphae that vacuoles always appear as numerous and minute ele- ments in the form of granules, isolated or united in little chains, or of filaments which often anastomose in networks, staining very deeply and homogeneously with vital dyes. These very closely resemble the chondriosomes and are composed of a very con- centrated colloidal solution. They are elements which by hydra- tion become transformed little by little in the course of cellular differentiation into fluid vacuoles. Later, taking up our observations on the origin of anthocy- anin, P. A. DANGEARD (Cf. p. 130) demonstrated that, just as ARTHUR MEYER had predicted, the elements which we had described as chondriosomes in reality represented young vacuoles with very Chapter XIV — 149 — The Vacuolar System concentrated colloidal contents, the enclosed pigment giving them a natural color. Consequently, the formation of anthocyanin, from its beginning, is associated with the manner in which the vacuoles are generally formed. Struck by the resemblance between young vacuoles and the chondriosomes, as we ourselves had been, DANGE- ARD was led at the beginning of his research to liken them to chon- driosomes and to think that the forms described under this name in animal cells corresponded to certain aspects of vacuolar develop- ment analogous to those forms encountered in plant cells. He believed, furthermore, that he could demonstrate that the colloidal substance of which the vacuoles are formed, corresponded to a special substance found in all vacuoles, regardless of the type of cell, which he identified with metachromatin (volutin of MEYER). Consequently, in spite of current opinion, there could be no rela- tion between the chondriosomes and the plastids. Hence DANGE- ARD gave the name vacuome, or vacuolar system, to all the vacuoles contained in a cell in the various phases of its development. This expression was destined, in his own mind, to replace that of chon- driome and the term mitochondrium that of mitochondrial sub- stance. “The greatest error of cytologists,” he said, “is to have confused the chondriome and the metachromatin with the plastids. This, at any rate, is what I am going to try to demonstrate. The chondriome, which has been the object of so much investigation, must, in my opinion, be considered otherwise than it has been to the present time. It may be defined as the whole vacuolar system in its various and successive aspects.” Starting from the fact that vacuoles are stained by dyes and are capable of taking up almost the entire amount of dye in a solution, DANGEARD made his vacuome play an essential rodle in the phenomena of nutrition of the cell. According to him, meta- chromatin, the substance specific for the vacuome, plays at one and the same time an osmotic and a selective role. It fixes the nutri- ents as it, in turn, is fixed by the vital stains. In this way DANGE- ARD explains the formation of anthocyanin pigments. These, ac- cording to him, arise in the cytoplasm and are fixed by the metachromatin of the vacuome by virtue of its selective power. Thus DANGEARD transfers to the vacuome, the hypothesis which REGAUD had proposed to explain the réle of the chondriome. Lastly, P. A. DANGEARD and his son, P. DANGEARD, think that the vacuoles are permanent elements of the cell and multiply only by division, thus agreeing with the conception of DE VRIES, but with this difference that, for the DANGEARDs, the vacuoles by de- hydration of the metachromatin may become solid in certain phases. This is true for aleurone grains and vacuoles of dormant spores of fungi. This notion of taking the chondriome for the vacuome, which rests exclusively on observations made with vital stains, was inad- missible, it being true that at that time it had already been demonstrated that the chondriosomes are not stained by the dyes used by P. A. DANGEARD, but only, and then with difficulty, by Guilliermond - Atkinson — 150 — Cytoplasm other dyes which do not have a predilection for the vacuoles. More- over, the chondriosome-shaped vacuoles are of temporary nature and, even beginning with the first stages of cellular development, these vacuoles take in water and become transformed into large vacuoles, whereas it is known that the chondriosomes persist dur- ing the entire life of the cell. The only question that could be asked was whether, as we had supposed, the vacuoles were not derived in some cases from the chondriosomes. It was not logical to say that the chondriome and the vacuome were one and the same thing. The interpretation of DANGEARD has therefore been abandoned for a long time now, even apparently by its author, although without explicit statement to that effect. 9 Fic. 94. — Barley root. Stages in development of the vacuolar system. 1-5, meristem; 6-8, adjacent region of differ- entiation; 9, mature cells of cortical parenchyma. Vital staining with neutral red. Our investigations, beginning with that period, gave more details and indeed confirmed the observations of P. A. DANGEARD in so far as the development of the vacuoles is concerned, but they showed that the chondriosome-shaped vacuoles and the chon- driosomes have in common only their shape, and that the vacuome is a system completely independent of the chondriome. They showed, furthermore, that the vacuoles are not characterized by a specific substance corresponding to metachromatin and that the colloids which the vacuoles contain are substances of very diverse nature, having as a common property only their ability to absorb vital stains. These facts were afterwards verified by a large num- ber of cytologists and are today definitely accepted. The vacuoles do not, in general, stain by mitochondrial techniques and it is the very complex case of the leaflets of the rose which has caused all the difficulty. It has been seen that in the cells in the young teeth of Chapter XIV — 151 — The Vacuolar System these leaflets (Fig. 92) there are chondriosome-shaped vacuoles con- taining a substance which, when preserved with mitochondrial methods, becomes yellow, but which may be stained by iron haema- toxylin and which, if the destaining is not carried far enough, ap- pears black. This would make one think that these bodies corre- spond to chondriosomes impregnated with anthocyanin. In reality, as we were able to demonstrate by later work, they are vacuoles containing tannin with which anthocyanin is associated. Now, tannin, like the lipide substance of the chondriosomes, is rendered insoluble with fixatives containing potassium bichromate, and may be stained by iron haematoxylin. On the other hand, the study of plants in which anthocyanin is not associ- ated with tannin, makes it possible to observe that this pigment is not preserved by mitochondrial techniques and that the young chondrio- some-shaped vacuoles in which pigment forms do not stain with mitochondrial methods. Therefore, DAN- GEARD, instead of correcting a partial error committed by us, made the mistake of gen- eralizing it. Let us examine in more de- tail the development of the vacuoles in the phanerogams. : f In the barley root (Fig. 94), rant steces in the development of the vacuolar for example, the phenomena ora ie ee at of sepals. Vitally stained studied by DANGEARD and y then by us, are particularly clear. If a very young root of a seedling is studied in a solution of neutral red by crushing the root gently, in such a way as to dissociate the cells without injuring them, numerous minute elements are seen in all the cells of the meristem. They are scattered about in the cytoplasm and are stained deeply and homogeneously by the dye. In the very young- est cells these elements are all small granules but they soon elongate to undulous filaments which often afterwards anastomose into a network. By their form and their dimensions these ele- ments show a striking resemblance to the chondriosomes and an observer not forewarned would easily take them for such. Never- theless, they are distinguishable at first sight from the chondrio- somes by a much greater diversity of appearance and by the fact that they are reticulate. They seem to be composed of a very con- centrated colloidal solution of semi-fluid consistency whose refrac- tivity is such that they can easily be seen without vital staining. In the course of cellular differentiation, it is observed that these elements swell by absorbing water and then coalesce. They are of Guilliermond - Atkinson — 152 — Cytoplasm most diverse appearance: dumb-bell-shaped, granules arranged like a string of beads, club-shaped, spindle-shaped, networks of monili- form filaments. They then become transformed into small, spher- ical vacuoles which always stain uniformly, but as they continue to take in water, they soon appear only faintly colored. They some- times contain a few deeply stained precipitates, which show Brown- ian movement and are caused by the precipitation of some of the colloidal contents of the vacuole under the influence of the dye. These vacuoles fuse and finally, in mature cells, form a single large vacuole which occupies the major part of the cell, pushing the nucleus and cytoplasm to the periphery. The cytoplasm is now reduced to a thin layer around the vacuole which appears faintly colored and shows only a few precipitates. Fic. 96. — Anagallis arvensis. Stages in the formation of a glandular hair on the corolla; anthocyanin pigments present from the first (in vivo). The development of the vacuolar system in the root of the wheat may be quite as easily observed and the phenomena take place in the same way. In most of the phanerogams and the pteridophytes!, moreover, an analogous development of the vacu- olar system is recognized. Excellent examples are furnished by the epidermal cells of very young leaves of Iris germanica, by the cells of very young hairs from the sepals of the same plant (Fig. 95), by the glandular hairs on the leaflets of the walnut (Fig. 93), by the leaves of Anagallis arvensis and others. In the glandular hairs, the vacuoles, like those in the teeth of the rose leaflets, con- tain from the very beginning an anthocyanin pigment which makes 1In the apical cell of the pteridophytes, however, there are found only large liquid vacuoles and in the cells of the meristem which are derived from the apical cell there are no small filamentous vacuoles (EMBERGER). It seems as if the apical cell had already passed through a stage in which the vacuoles were filamentous and semi-fluid, after which these filamentous vacuoles had taken in water and coalesced. Chapter XIV — 153 — The Vacuolar System it possible to follow their entire development without using vital dyes. The large vacuoles of mature cells, instead of staining dif- fusely without precipitation or forming only a very few precipi- tates, may behave differently. Very often, as for example, in the epidermal cells of Iris germanica, the vital dyes do not stain the vacuolar sap and only produce in the vacuole deeply stained bodies showing Brownian movement or else bring about, at the same time, both a diffuse coloration of the vacuolar sap and the pro- duction in the sap of colored precipitates. 6 vis Fic. 97. — Pea root vitally stained with neutral red. 1-4, meristem; numerous small filamentous vacuoles, uniformly stained. 5-7, differentiating cells; swelling and fusion of small vacuoles, whose colloidal substance is precipitated by the dye as deeply stained bodies showing Brownian movement. So the vacuoles seem to be at first small elements composed of a very concentrated colloidal solution which, by taking in water, gradually swell and coalesce during the period of differentiation of the cells, and become large vacuoles containing an extremely dilute colloidal solution. This progressive dilution of the vacuolar colloidal solution is made evident by the fact that neutral red, which at first gives the vacuoles a deep homogeneous color, stains them only weakly when the cells have attained a certain degree of dilution but generally brings about a precipitation of the colloid as deeply stained bodies which show Brownian movement. The precipitation is more or less copious depending upon the nature and concentration of the colloid. When the vacuoles have come to the end of their development, they may, in the presence of vital stains, stain weakly and homo- geneously without showing any, or at most very few, precipitates, as in the roots of barley and wheat (except for the cells of the root cap). In other, more frequent cases, vital dyes bring about the formation of numerous deeply stained precipitates, which show Brownian movement, at the same time that they stain the vacu- olar sap diffusely (vacuoles in the epidermal cells of Iris germa- nica). In still other cases, the vital dyes at first cause only colored precipitates. These later fuse and may dissolve in the vacuolar sap which then becomes diffusely colored. Guilliermond - Atkinson — 154 — Cytoplasm Rather often, there are normally found in the vacuoles more or less large, spherical bodies, or small granules united in mul- berry-shaped masses, which are the result of a partial precipita- tion of the colloidal solution within the vacuoles. These bodies stain with vital dyes which, at the same time, bring about other precipitates of the same or of different natures. The normal pres- ence of these bodies in the vacuole could be explained by the fact that the colloidal micelles contained in the vacuoles in some cases do not possess a power of unlimited imbibition, so a time seems to come when they cease to take in water. A disturbance in equi- librium occurs and this leads to the production in the vacuolar sap of a coacervate. It may be added that rather frequently the large vacuoles of mature cells, especially when they contain tan- nins, continue to enclose a very concentrated so- lution and in living material are exceedingly refractive. These vacuoles which seem to be in the state of a jelly do not form precipitates with vital dyes or else form them with great difficulty. In the latter case they show an intense homo- geneous color. There are even cases in which the jelly is almost solid and in such cases it becomes very difficult to plasmolyze the cells, as is seen in the vacuoles of the pericarp of Ilex Aquifoliwm (GUILLIERMOND, CHAZE). Vacuoles behave differently according to the nature of their contents. The development of the vacuolar system which Fic. 98. — Bud of We have just described is of very general occur- ae canadensis v’ rence and has been observed in very widely sep- neutral red. Vacu- arated plants (phanerogams: P. A. DANGEARD, oles globular and univ GyILLIERMOND, P. DANGEARD, BAILEY, ZIRKLE and formly stained; or seal pind (colorless others; pteridophytes: EMBERGER). Based ieect Meats All the vacuoles of the higher plants however, do not follow exactly this development. Thus, in studying the formation of the vacuoles in the bud of Hlodea cana- densis, it is observed that in all the cells of the meristem of the stem and of the youngest foliar primordia, there are numerous, very small vacuoles which are always globose and never filamentous. These generally stain uniformly and deeply with neutral red, but in certain cases there is seen in their interior a single, deeply stained, corpuscle showing Brownian movement. This corpuscle has been produced by a precipitation of the colloidal contents of the vacuole. These vacuoles, which are not visible in living mate- rial without being stained, seem to be constituted of a less concen- trated colloidal solution than are those in ordinary cases. They later swell up by taking in water and then gradually coalesce to form, in mature cells, a single vacuole which neutral red stains diffusely while also causing the production of numerous precipi- tates. There are, therefore, no filamentous or reticulate formations Chapter XIV — 155 — The Vacuolar System but only spherical vacuoles to be found at the beginning of develop- ment of this type of vacuolar system. Among the fungi, the Saprolegniaceae constitute particularly favorable objects for the study of the vacuolar system, to which we will have to return later. When the mycelium of a species of Sapro- legnia is examined in a solution of neutral red, there are observed at the growing extremities of the plant, vacuoles which at first are generally small globular elements. These elongate and appear as thin, tenuous canaliculi, more or less oriented in the direction of the longitudinal axis of the hypha. These canaliculi anastomose and form a delicate and complicated network. In this region they appear more fluid and with too little difference in refractivity be- Fic. 99. — Saprolegnia vitally stained with neutral red. Development of the vacuolar system. 1-3, tips of growing filaments; anastomosing canaliculi. 4, older filaments; fusion and swelling of canaliculi. 5, later stage; precipitated bodies in sap. 6, still later stage; single canal containing precipi- tates. tween them and the cytoplasm for them to be observed without staining. A little further from the tip of the hyphae, these canali- culi are observed to swell, little by little, then to converge, so that in differentiated regions of the hyphae, there is only a single canal running from one end of the siphon to the other. This canal occupies the major part of the hyphae and in it the vital dyes pro- duce both numerous, semi-fluid, precipitates and diffuse staining of the vacuolar sap. The cytoplasm, accordingly, now constitutes only a thin layer about this canal and the nuclei are appressed to the wall of the siphon. This vacuole, therefore, is of a special type; namely, a single canal running from one end of the filament to the other and obvi- ously adapted to the siphonate structure of the Saprolegniaceae. This form can be found in Vaucheria but it is a rather rare type. Guilliermond - Atkinson — 156 — Cytoplasm In most fungi, except for the Phycomycetes, the vacuoles appear in the tips of the growing hyphae as very numerous, small, globular elements which sometimes stain uniformly and deeply with the vital dyes and sometimes remain uncolored but contain a deeply stained corpuscle showing Brownian movement (Fig. 100). These vacuoles swell in regions farther away from the tip, then coalesce, until, in the regions still farther away, they form large colorless vacuoles filled with deeply stained corpuscles. These bodies swell after a time, then may dissolve and give the vacuole a diffuse and homogeneous color. This is also true in the yeasts in which there exist in the bud several small, spherical vacuoles, sometimes uni- formly stained with neutral red, sometimes not stained at all, but containing colored corpuscles. These small vacuoles fuse during the growth of the bud until there is present only one large vacu- ole or a few large vacuoles filled with stained cor- puscles showing Brownian movement. (Fig. 101). In some of the lower plants, the vacuoles de- velop quite differently. In many algae, the Con- jugatae for example, they are always in the form of large liquid vacuoles. In other algae they may, on the contrary, appear during the entire cellular development as very small, semi-fluid, usually globular, vacuoles, scattered about in Fic. 100. — Endo- myces Magnusti. Vital staining with neutral red of filaments whose vacuoles (V) contain intensely colored gran- ules (CM) which show Brownian move- ment and which cor- respond to bodies of metachromatin ob- tained with Bouin’s fixative stained with haematein. Gl, lipide granules around vacu- oles. the cytoplasm and never undergoing hydra- tion. These vacuoles, formed of a very concen- trated colloidal solution, stain homogeneously and deeply with neutral red, usually without precipita- tion. This is the type of vacuole generally found in the Phytoflagellates (Euglenas, Peridinieae and Volvocales observed by the DANGEARDs), in cer- tain land forms of the Chlorophyceae (Pleuro- coccus, Pleurastrum, Prasiola reported by PUy- MALY). The Cyanophyceae also contain similar vacuoles which are found localized in the parietal cytoplasmic layer surrounding the central body, i.e., the region which corresponds to the nucleus (GUILLIERMOND) . The bacteria seem to belong to this category. In them, by vital staining or after fixation, there are observed, especially at the poles of the cell, metachromatic corpuscles which seem to corre- spond to small vacuoles with very concentrated metachromatin (GUILLIERMOND, Mlle. DELAPORTE). Vacuoles of this type are en- countered in the conidia, in the spores and in the zoospores, of fungi and algae (zoospores of Saprolegniaceae and Ulothrix (Fig. 103), for example), and in pollen grains (P. DANGEARD, Mlle. Py).! The data which we have just reviewed, confirm, therefore, the observations of P. A. DANGEARD. These data show that vacuoles 1Mlle. Py has shown that in dehydrating pollen in a vacuum, a solidification of these vacuoles is obtained. The vacuoles become comparable to aleurone grains within the pollen grains, although the pollen grains do not lose their viability. Chapter XIV — 157 — The Vacuolar System seem to be encountered in all cells in all phases of development and that, in general, they appear in embryonic cells as minute elements composed of a very concentrated colloidal solution and that they sometimes show a great resemblance in form and dimensions to the chondriosomes. These chondriosome-shaped elements swell and are transformed into large liquid vacuoles containing very dilute colloidal solutions. These facts do not, however, confirm DANGE- ARD’s interpretation in which he identifies the vacuolar system with the chondriome. Although in some cells the young vacuoles present forms almost identical with those of the chondriosomes, there are other very numerous cases in which the young vacuoles, on the contrary, have an appearance which does not permit of any confusion with the chondriosomes. For example, in some algae, the vacuoles remain constantly in the state of large liquid inclusions. Nevy- CY Ud ertheless, since it is evident that these two categories of elements may sometimes be easily mistaken for one another, it is advisable to examine here the characteristics which make it possible to distinguish between them. Chondriosome-shaped vacuoles and chondriosomes. Characteristic dif- ferences:- The vacuoles, even in their chondriosome-shaped state, are essentially distinct from the chon- es driosomes. “im their histochemical | -yact scuins wie acetal a Ls. characteristics. An inherent differ- flamentay ‘cue oe oes og es ence rests in the fact that although vacuole by the dye; small vacuoles in the vacuoles stain deeply with vital a intal ge eae dee ree eet nN stains (neutral red, cresyl blue, Nile oles in germination tube seem to blue, etc.), the chondriosomes, on the *™ % 72% contrary, have no affinity for these dyes and are not colored in the living state except with special stains (Janus green, Dahlia violet, methyl violet), which usually show no marked affinity for the vacuoles. Besides, as has been seen, staining of the vacuoles is essentially a vital phenomenon and ceases when the cells are killed. On the contrary, the chondriosomes stain only temporarily; their coloration is stable only in dying cells and is then always accom- panied by vesiculation, a state soon followed by the death of the cells. The coloration persists, even after the death of the cell. Meristematic cells may be found in which chondriosomes are vis- ible in the living state and the independence of these from the vacuoles may be made certain by vital staining, since the chondrio- somes remain unstained. The same observation may be made for the Saprolegniaceae in which the chondriosomes are always clearly visible in the living state in all stages of development of the plant. We have obtained, furthermore, in these and other fungi (Hndo- Guilliermond - Atkinson — 158 — Cytoplasm myces Magnusti) a double vital staining of the chondriome and the vacuolar system by means of using a mixture of a solution of neutral red and Janus green or Dahlia violet. In this way, we were able to follow the simultaneous development of the two systems during the entire growth of the plant. The chondriosomes by this method are colored green or blue and the vacuoles red. (Fig. 104). These initial forms of the vacuoles which look like chondrio- somes, are very fragile, just as the chondriosomes are, and they swell and fuse into larger spherical vacuoles during prolonged observations with vital staining, but this alteration of shape has nothing in common with the cavulation of the chondriosomes (cf. p. 101). Solutions of osmic acid preserve and heavily blacken the chondriosome-shaped vacuoles whenever they contain phenolic compounds; otherwise it may preserve them in a greatly swollen condition without blackening them. It is known that the chondriosomes are, on the contrary, very well preserved with osmic acid but are not darkened by it. ; In preparations treated by mitochondrial tech- niques, the chondriosome-shaped vacuoles usually appear as uncolored canaliculi, sometimes anasto- mosing in a network in the midst of the faintly colored cytoplasm. Sometimes the contents of the vacuole are colored, but this is rare. When it does occur, however, they are always found condensed by the action of the fixatives in the middle of the colorless canaliculi so that it is not possible to confuse the elements of the vacuolar system with the chondriosomes. Sometimes also the colloidal contents of large liquid vacuoles, de- Hie” stontum. “"'w. Yived from the chondriosome-shaped vacuoles by moe ve ane hydration, show bodies precipitated by the action DANGEARD). of the fixatives which stain with mitochondrial technique. (Figs. 105, 106). In all cases in which the vacuoles contain tannins, the chon- driosome-shaped vacuoles appear as filaments, or as a network, somewhat dilated by fixatives and colored yellow by potassium bichromate, or blackened by osmic acid, according to the method employed. The large liquid vacuoles resulting from their fusion show, with the same methods, either granular precipitations or large corpuscles stained yellow by potassium bichromate or black- ened by osmic acid. But there is no method, in most cases, which makes it possible to stain the colloidal contents of the vacuoles after fixation, when they do not contain tannins, unless, perhaps, the Golgi methods which will be discussed later (Fig. 107 and 108). In most fungi and some algae, however, we have seen that the colloidal substance of the vacuoles, or the metachromatin, made insoluble and precipitated in the form of corpuscles by formalin Chapter XIV — 159 — The Vacuolar System or alcohol, is stained deeply red by aniline blue or violet basic dyes, as well as by haematein. It consequently shows a whole series of histochemical reactions which are very well known and are very different from those of the chondriosomes. It is, therefore, well established that there does not exist the slightest relation between the chondriome and the vacuolar sys- tem; they are two independent systems which are coexistent in the cell. There is between the young stages of the vacuoles and the chondriosomes merely a coincidence of form. This close re- semblance exists, however, only in a very limited phase in the development of the vacuoles and seems to be explained by the fact that the vacuoles are at that moment in a semi-fluid physical state which is closely allied to the physical state of the chondriosomes. Physical characteristics of the es vacuoles:- The chondriosome- a shaped vacuoles seem to be of semi- : fluid consistency and in a physical state which approaches that of the chondriosomes. Ultramicro- (E73 (it scopic examination shows that in he \Y/ a general the chondriosome-shaped g y vacuoles are no more visible than é the chondriosomes (Fig. 109). In Pag, q certain cases, however (teeth of s\ \ st young rose leaflets, barley and ea a ) wheat roots), it is possible to dis- 5 if tinguish them. They appear optic- ally empty and are visible only be- Fic. 103. — a, Cladophora; vital staining eases (Of them stamtiny tuminouss ee ee eon loeee: contours, 1.€., when they can be arrangement of vacuoles in Bb vegetative cells and in ec zoosporangium. ds. ¢; seen, they show the same charac- Cladophora; zoospores which have ceased feristics. in this Tespeeteasedouthe | 2°2tms: (7, Brvopsts piemosa, | Z00spare- - g, Ulwa Lactuca; zoospore which has just chondriosomes and the plastids. come to rest. st, stigma. (After DAN- With the chondriosomes and the ““"”): plastids, they might be classified as a coacervate system. The liquid vacuoles, derived by swelling from these chondrio- some-shaped vacuoles, also appear optically empty even in cases in which they enclose an abundance of colloidal substances (tan- nin, metachromatin). Rather infrequently they are visible by reason of their faintly luminous contours. Certain indirect meth- ods, however, often make it possible to locate their position. Thus in the yeasts, there exist in the cytoplasm bordering on the vacu- oles, numerous lipide droplets which appear very luminous and because of them the position of the vacuoles may be easily detected (Fig. 110). There sometimes appear within the vacuoles strongly lighted granules which show Brownian movement, but these are al- ways visible in direct lighting and are not of the order of micelles. The vacucle, then, in its liquid state seems to be composed either of a colloidal solution whose micelles are very small and not Guilliermond - Atkinson — 160 — Cytoplasm visible, or of a very fluid hydrogel. Nevertheless this solution is very unstable and easily precipitable, as we have seen in studying the action of vital dyes on the vacuoles. Sometimes, however, the large vacuoles of mature cells remain, as has been said, in the state of a very concentrated colloidal solution, a sort of jelly. In this case, they are generally visible in the ultramicroscope because of their luminous contours. The filamentous vacuoles seem, like the chondriosomes, to have a specific weight rather like that of the cytoplasm, although often it is higher. For example, AKER- MAN found by use of the centri- fuge that the filamentous vacuoles existing under certain conditions in the tentacles of Drosera are heavier than the cytoplasm. H. CLEMENT, by the same process, was not able to displace the chon- driosome-shaped vacuoles contain- ing anthocyanin in the teeth of young rose leaflets. In recent work, MILOVIDoV has shown that, in the youngest cells of the teeth, the chondriosome-shaped vacuoles con- taining concentrated solutions of tannin and anthocyanin become oriented in the direction of the cen- trifugal force and are therefore heavier than cytoplasm. The large vacuoles derived from them, and containing a more dilute solution of tannin, become oriented in the opposite direction, 7.e., centripetal- fis ly, and are lighter than the cyto- Fe ee ating “plasm, \ MILQVIDOV obtained jsinus with perhe ea whieh stains he ens lar results with barley roots. In ices ake eae. eatoles (Gil, V), or in cells of the youngest part of the older vacuoles CV): causes precipitation of meristem, the vacuoles are heavier their metachromatic substances as deeply ; colored bodies. Gl, lipide granules. than the cytoplasm and are easily displaced in the direction of cen- trifugal force. In regions situated just a little above, the vacu- oles which are beginning to take in water have about the same density as the cytoplasm and are no longer displaced. In the region in which the cells are already differentiated, the vacuoles are much lighter than the cytoplasm and are displaced in a centripetal direction. (Fig. 111). Thus the small vacuoles which look like the chondriosomes are semi-fluid or sometimes almost solid elements. They seem to be Chapter XIV — 161 — The Vacuolar System composed of a jelly or of a coacervate. One is therefore led to believe that the development of the vacuoles described in the pre- ceding pages may possibly be nothing more than an unlimited imbibition of small elements, shaped like granules or filaments, which are in a jellied or coacervate state. This imbibition would involve the transformation of the original gel into a very dilute solution represented by the liquid vacuoles. Chemical nature of the colloidal substance of vacuoles:- The histochemical characteristics of young vacuoles, which we have enumerated to establish a distinction between the chondriosome- shaped vacuoles and the chondriosomes themselves, prove that the Ne Fic. 105. — Ricinus root. Meristem fixed by Regaud’s method. Black, filamentous or granular chondriosomes (Ch) and vacuoles (V), the latter distinguished by an external hyaline region due to a contraction of their colloidal contents during fixation. colloidal substances of the vacuolar sap have an essentially variable constitution. Vacuoles are present in all plants but there does not exist any substance characteristic of them, as is the case for the chondriosomes. They contain diverse substances having noth- ing in common except their property of fixing vital stains. Vital staining alone reveals differences in coloration between vacuoles. Cresyl blue, for example, changes color in vacuoles which contain metachromatin (majority of fungi, certain algae). It stains them a diffuse red and the enclosed corpuscles are colored dark red. In the cells of phanerogams, the staining of the vacu- oles is extremely variable. Whenever the vacuoles contain phe- nolic compounds, they take a pure blue color with cresyl blue be- cause of their acid pH. This same blue color is sometimes observed when the vacuoles contain lipide substances (phytosterol or phos- Guilliermond - Atkinson — 162 — Cytoplasm pholipides reported by REILHES). In other cases the vacuoles take on colors toward the violet. DANGEARD used the characteristic shown by blue vital dyes, of staining red or violet those vacuoles which contain metachromatin, as the only basis for his theory of the universal presence of meta- chromatin in vacuoles. But this staining reaction (Fr. métachro- masie) is not a characteristic belonging alone to the substance called metachromatin. Metachromatin has been so named because it Fic. 106. — Root of pea. A, meristem; chondrio- somes (m) stained, vacuoles (P.V.) colorless. B, C, meristem of central cylinder; contents of filamentous vacuoles (P.V.) clearly distinguished from the chon- driosomes by a peripheral hyaline region caused by contraction of the contents of the vacuole by the fixative. D, differentiated cortical parenchyma; single large vacuole with densely stained bodies (P.V.). m, chondriosomes. V, vacuole. Regaud’s method. changes all blue and violet aniline dyes and haematein to a red color after fixation. Now this still unexplained phenomenon has no rela- tion to the color change obtained with vital staining. One can not, as do the DANGEARDs, relate the colloidal contents of all vacuoles to a single substance and call it metachromatin, since metachromatin is a substance present in fungi, so named by reason of a color change which it shows after fixation, not when vitally stained. Be- sides, it has been seen that metachromatin is preserved by alcohol and formol, whereas the colloidal substance of the vacuoles is usu- ally destroyed by all fixatives, even those of mitochondrial tech- niques. If the blue coloration which the vacuoles sometimes take with cresyl blue is often an index of an acid reaction, as is the case for Chapter XIV — 163 — The Vacuolar System the vacuoles containing phenolic compounds, the red or violet coloration which they take in other cases is difficult to explain, for it is known that cresyl blue in solution in pure water shows only a single change in color: it takes on an orange tint for a pH value of 11.2. Nevertheless, according to MANGENOT and Mlle LAURENT, cresyl blue at a pH which is not accurately measured, shows a change to violet, on condition that the medium contain diverse colloidal substances (sodium silicate, dextrine, protein, etc.) or even sugar (saccharose, according to unpublished work). This fact has been confirmed by CHADEFAUD. ei | Ee un 8&6 ae = + “se s — ae TE (s ft ag Fic. 107 (left). — Dematiuwm. Bouin’s method, stained with hemalum. 1, filament. 2, germinating conidium. VCM, vacuole containing metachromatin precipitates. mn, nucleus. Fic. 108 (right). — Saprolegnia. Chondriome and vacuolar system. 1-8, Vital staining with neutral red. 1, tip of filament; reticular vacuole (RV); other elements omitted. 2, older filament; tendency of network to become a diffusely stained canal (V) containing deeply stained bodies (CM), other elements visible but unstained. 3, still older filament; vacuolar eanal (V) loses its stain, other bodies as in (2). 4, mature filaments. Regaud’s method with iron haematoxylin; vacuolar canal unstained, lipide granules dissolved, chondriosomes (Ch) strongly stained. 5, 6, mature filament. Meves’ method with acid fuchsin; chondriosomes (Ch) red, lipide granules (Gg) brown. Ch, chondriosomes. WN, nucleus. It must be added that the change to red in the vacuoles may also depend on the chemical constitution of colloidal substances which the vacuoles hold in solution. Mucilaginous substances, agar, for instance, change cresyl blue to a color toward the red, no mat- ter what the pH. According to LISON, this color change in vital staining is a histochemical reaction characteristic of all sulfuric esters of high molecular weight. Now, mucilages or polyholoside esters are the most important among them. From these considerations it follows, therefore, that the change toward red of the vacuoles, which is due to the most variable causes, and which may be obtained in vitro by the addition of the most diverse substances, can not possibly serve to characterize a chemical substance. In general, the vacuolar colloids appear in the higher plants to be protein substances, perhaps proteins soluble in alcohol, and Guilliermond - Atkinson — 164 — Cytoplasm are often associated or combined with tannins and perhaps with mucilages. LLOYD and various other authors have shown that tannin is often combined with mucilages in the state of a com- plex, and it is thought that the vacuoles containing raphides en- close mucilages. Furthermore, recent work has shown that in certain cells the vacuoles contain a colloidal solution of phytosterol or of phosphoaminolipides. These substances may become par- tially solid in the epidermal cells of the Liliaceae (Fig. 112). These cells ordinarily contain a large inclusion, formed of a complex of phytosterol and of phosphoaminolipides, which MIRANDE described for the first time under the name of sterinoplast. This, he considered Fic. 109. — Rose. Cells from a tooth of a leaflet under the ultramicroscope, showing faintly luminous contours of the vacu- oles (V) and of the nuclei (N). 1, tip of tooth. 2-4, differentiated cells. to be a cytoplasmic inclusion, a sort of plastid, elaborating phy- tosterol. The work of MIRATON and of EMBERGER has demon- strated that the sterinoplasts are not simple vacuolar concretions. The recent work of REILHES has established the fact that the vacu- olar sap of these cells contains a solution of phosphoaminolipides and of phytosterol which, in mature cells, becomes partially solidi- fied in the vacuoles in the form of large bodies composed of a phos- phoaminolipide-phytosterol complex. Inclusions, apparently of the same nature, have been cited in the vacuoles by other authors: in the epidermis of Iris, especially, in which they absorb anthocyanin, when the vacuoles contain the pigment, and have been described under the misnomer of cyanoplasts (POLITIS). Similar inclusions have also been described in the epidermis of the flowers of Del- phinium cultorwum (SCHARINGER). We have shown as well that in the cells of the root cap of barley and of wheat, there are phosphoaminolipide solidifications and, by cultivating barley roots in media very rich in sugar, GAUTHERET succeeded in making a great number of lipide concretions appear in the vacuoles of most of the cells. The vacuoles of Monotropa according to WEBER contain large Chapter XIV — 165 — The Vacuolar System quantities of lipides. Furthermore, BUVAT reported in the vacu- oles of many roots, the existence of phosphoaminolipide concretions sometimes combined with proteins. In the algae, the colloidal substances of the vacuoles are also very diverse. Proteins, tannins and mucilages seem to exist in them but metachromatin and volutin are also frequently found. This last substance, which is also found in the bacteria, especially characterizes the fungi, in which, with the exception of some of Fic. 110. — Saccharomycodes. (A), under the ultramicroscope; (B), with direct light. A, 1-6 S. Ludwigit. Vacuoles (V) usually invisible; position marked by strongly lighted lipide granules (Gl; A8, B5) surrounding them and corpuscles of metachromatin (C; A2, B2) which they contain. 4, contour of vacuole visible. 6, cytoplasm coagu- lated. 7-9, S. pastorianus. B, S. Ludwigii. the Phycomycetes (Saprolegniaceae and Peronosporaceae), its presence is of general occurrence and in which it appears to play the role of reserve product. In fact this substance accumulates in the vacuoles of the epiplasm of the asci of yeasts and of the higher Ascomycetes and is absorbed by the ascospores at the same time as the glycogen and the lipides which are coexistent with it in the epiplasm. Volutin offers histochemical characteristics which as we have seen make recognition of it easy. Its chemical constitu- tion is still not well determined but there are good reasons for supposing that it is formed by a combination with nucleic acid (ARTHUR MEYER). From very recent work of Mlle. DELAPORTE and Mlle. ROUKEL- MAN on yeasts, it seems that metachromatin corresponds to a zymonucleic acid compound which has been extracted in great quantities in yeasts. According to these investigators, the nuclei of these fungi, as well as those of other plants probably, are com- Guilliermond - Atkinson — 166 — Cytoplasm posed of thymonucleic acid, like those of animal cells, which ex- plains why the Feulgen reaction is obtained just as well in plant, as in animal, nuclei. In the Saprolegniaceae, finally, we have found sphaerocrystals which appear to have some relation to the phosphoaminolipides. The vacuoles contain, as well as the colloidal material just discussed, numerous crystalloid substances. The most wide-spread of these are organic acids, halogen salts, nitrates and phosphates, sugars (saccharose, glucose, fructose, etc.), heterosides and pig- ments. Among the halogen salts we must mention the presence of iodide in a dissolved state in the vacuo- lar sap of numerous marine algae (Rho- dophyceae and Phaeophyceae). Its local- ization in the vacuole may be demon- strated in vivo by cresyl blue. This pig- ment in the presence of iodized solu- tions forms red crystals (Fig. 113) ar- ranged in the shape of a bouquet (SAU- VAGEAU and MANGENOT). Among the pigments may be mentioned the oxy- flavanol pigments which are very pale yellow in color and the anthocyanin pig- ments which are red, violet, or blue. Both types are extremely widespread in green plants. Anthocyanin pigments, when found in high concentration in the vacuole, may crystallize there in BiG a iw ae) Phe orm on meedie-shaped crystalsjor red and centrifuged. The chondrioe sphaerocrystals (Fig. 114). These two pome shaped jyacucleaic ec ue types; ofa pigment show. histochemical stem, heavier than cytoplasm, are eee ae) those tak- characteristics closely allied to tannins By tee aes oe are eg (darkening with ferric salts, blackening those still higher in the root, lighter with osmic acid). The flavins may also than cytoplasm, are displaced cen- : i tripetally. (After MrLovipov). be cited. These are yellow pigments playing at the same time the role of hydrogen carriers and the rédle of Vitamin B.. We have recently found them in great abundance in the vacuoles of a fungus E'remo- thecium Ashbyii where they crystallize in the form of needles or sphaerocrystals. The alkaloids are very wide-spread in the vacuoles of the phanerogams and may be recognized by testing with iodine- potassium iodide (Bouchardat’s reaction), which precipitates the alkaloids in the vacuoles as brown granules. In bringing this inventory to a close, there must be mentioned asparagine crystals and leucine crystals (lozenge-shaped or sphae- rocrystals) and especially calcium oxalate crystals. These latter are found in the vacuoles of a great number of phanerogams, some- times as long needles (calcium oxalate monohydrate or acid cal- cium oxalate) belonging to the monoclinic series; sometimes in the state of octahedral crystals, isolated or twinned—quadratoctahedra, in the nature of crossed twins (calcium oxalate trihydrate) —be- Chapter XIV — 167 — The Vacuolar System longing to the tetragonal system; sometimes as crystal dust. The crystals of calcium sulphate are also very frequently found (Clos- terium, Spirogyra). Fic. 112. — Lilium candidum. Lipide concre- tions within the vacuoles. A, in epidermal cells of the bulb; small granules (1) agglomerate into mulberry-shaped masses (2), these, B, in older scales become large bodies which are globular with a denser center (4) or of radiating crystalline structure (8). C, in drying outer scales, these break up and myelin figures (5) appear which finally, in dead cells, form masses of wound up threads (6). Vacuolar pH and rH::- It is very difficult to obtain an exact idea of the vacuolar pH. It has been seen indeed that the change of color of the vital dyes has only a very questionable value. Then, too, the indicators of »H do not penetrate into the vacuoles and are besides very toxic. However, evaluations have been attempted by CROZIER, HAAS and IRWIN, who sought to extract the vacuolar sap of certain algae, such as Valonia, in which the articulations are oc- cupied by an enormous vacuole. These evaluations have shown that the vacuolar pH in Valonia has an acid reaction (5.0-6.7). CROZIER, HAAS and SCHMIDT have used an interesting method by making the anthocyanin pigments serve as indicators. These pig- ments can be extracted from the petals and in mixing them with buffer solutions of known pH, all their different possible colors may be obtained and consequently the coloring which the petals show naturally may be related to a known pH. The results obtained by this method inform us as to the vacuolar pH. It scales from 3.0-8.0. We have been able with GAUTHERET to find a way to evaluate the vacuolar pH but only qualitatively. CLARK and PERKINS have Guilliermond - Atkinson — 168 — Cytoplasm shown that neutral red reduced in an alkaline medium (pH 8.2 for example) gives a leucoderivative which, by acidification of the medium (pH 5.2 for example), is transformed into a second deriva- tive, yellow in color and fluorescent, which is distinct from the Fic. 114. — Epidermal cells of sepals. Fic. 1138. — Cells of stipe of Pigment in the vacuoles, partly in Laminaria flexicaulis. Bouquet solution, partly crystallized. 1-2 Del- crystals formed by the pre- phinium. 1, D. Ajacis; pigment blue, cipitation of iodide by cresyl clusters of needle-shaped crystals. 2, blue in the vacuoles. jee hort. var. with dark blue flowers; long, phaeoplast. lo, fat globule. entangled needle-shaped crystals. 3, (After MANGENOT). Verbena hybrid; large sphaerocrystals. Fic. 115. — Mycelium of Hremothecium Ashbyii. a, vacuole with flavin in solution; b, vacuole with flavin crystals; gl, lipide granules. first. This latter is also obtained when neutral red is reduced in an acid medium. Finally, although oxidation of the leucoderiva- tive is rapid in contact with air, that of the fluorescent form is very slow. Now, in treating wheat roots, or the epidermis of Iris rich in phenolic compounds, with the leucoderivative of neutral red, we have found that the vacuoles take a yellow coloration which, in contact with the air, reddens only slowly. On the con- trary, the cells of the bean root which are lacking in phenolic com- Chapter XIV — 169 — The Vacuolar System ee i ee eee pounds remain uncolored with the leucoderivative, and the leuco- derivative which they have absorbed becomes Oxidized instantane- ously in contact with air. It may therefore be believed that the vacuoles of wheat and Iris are acid whereas those of the bean are not. Vacuolar rH has been evaluated by means of vital stains (Ma- TILDA BROOKS) or by micro-injection of indicators in algae such as Spirogyra. These measurements have given an rH of 16-18. Fic. 116. — A, Petiole of Begonia; octahedral crystals, isolated (lower cell), or twinned (upper left), in the shape of a sea urchin (upper right). B, Leaf of Alog succotrina; raphides. Chapter XV ORIGIN AND SIGNIFICANCE OF THE VACUOLES Aleurone grains: their formation. — Aleurone grains are found in every seed. Their significance has been discussed for a long time. These grains are to be found in large numbers in all cells of the embryo and are especially voluminous in the cells of the coty- ledons or of the endosperm. In the Gramineae they are localized in the protein layer of the endosperm. Aleurone grains vary in structure. In many cases (Ricinus, Cucurbita Pepo) they are composed of an amorphous protein mass enclosing a crystalloid of the same nature and one or several spherical bodies called globoids, formed of phytin. Sometimes twinned crystals of calcium oxalate are found in the protein mass or in the globoids. In other cases the aleurone grains do not con- tain crystalloids and enclose in their protein mass only numerous globoids (Gramineae). Lastly, there are cases in which the aleurone grains are composed entirely of amorphous protein (Legumes). Two opinions have been formulated as to the origin of aleurone grains. Some authors considered them to be plastids in which were formed pro- tein and the globoids. Fic. 117. — Aleurone grains in seeds. a, b, Ricinus; Others contended that one erystalloid and 1-2 globoides. c, Oenanthe Phel- they resulted from sol- eee eae cease of calcium oxalate in protein idification of the vacu- oles during dehydration of the seed. This latter opinion is now demonstrated to be true. If, during the maturation of the seed, the vacuoles from any part of the embryo or endosperm are examined using vital stains, liquid vacuoles are found which are more or less large, according to the type of cell and its state of development. These vacuoles contain protein substances in solution and neutral red causes a precipitation of these proteins within the vacuoles as deeply stained bodies. In the course of development of the seed, the vacuoles be- come much richer in protein. In the stages immediately preceding maturation, 7.e., at the time when dehydration takes place in the seed, it is observed that the vacuoles sometimes have a tendency to break up as they lose water, becoming smaller and smaller, and less and less fluid. They stain deeply and homogeneously and are now semi-fluid. They often at this moment become filamentous or reticulate, analogous in form to those observed in meristems. Later when the seed has reached maturity and passes into the dormant stage, the semi-fluid vacuoles by more marked dehydra- Chapter XV —- 171 — Origin of Vacuoles SPL ee eee ree 2 a ee tion take on the appearance of solid, spherical, very refractive bodies. These bodies can, by crushing, be expelled from the cell and then will not take up neutral red unless they have been im- mersed in water for a long time. The vacuoles are thus transformed into bodies of protein nature whose dimensions are variable. They are very small in some cells and very large in others. They are aleurone grains and are formed by the solidification of the protein contained in solution in the vacuole. As a result of losing water, a grain of protein has there- fore been substituted for a vacuole. It is not astonishing, then, to Fic. 118. — Ricinus. Endosperm. 1, young cell, starch (A) forms in chondriconts. 2, just before maturation; starch is absorbed, the large vacuole is fragmented into small vacuoles con- taining protein crystals (C) and protein precipitates (P). 3, oil globules (H) blackened by osmiec acid, protein crystals (P). 4, detail of (3). 5, dormant seed; A, aleurone grain, G, globoid. 6, during germination of the seed; aleurone grains (P) beginning to dissolve. M, chondriosomes. 1, 2, 5, 6, Regaud’s method. 3, 4, Meves’ method. find along with the protein in these vacuoles, inclusions of phytin and crystals of calcium oxalate, products encountered in many vacuoles. Some aleurone grains may even contain oxyflavanol pig- ments or anthocyanin. Investigations of SPEISS and CHAZE have shown that aleurone grains of some varieties of maize contain an anthocyanin pigment which gives them their characteristic black coloration. At first red, this pigment appears in the vacuoles which will later be transformed into aleurone grains, then remains ab- sorbed by the protein of which the aleurone grain is composed. At germination, when the aleurone grains are again transformed into vacuoles, the anthocyanin pigment changes to red. Furthermore CHAZE has found oxyflavanol pigments in the yellow or white ker- Guilliermond - Atkinson — 172 — Cytoplasm nels of other varieties of maize as well as in the seeds of other grains (barley, wheat, rye, oats). Aleurone grains stain deeply with mitochondrial techniques which do not at all stain the liquid vacuoles from which they are derived, except in periods preceding solidification when the stains bring about flocculation of the col- loidal solution, and the formation in the vacuoles of precipitates stainable with iron haematoxylin. At the time of germination, when the seed again takes up water, the aleurone grains absorb it and become semi-fluid. At this period they often show again a tendency to elongate into filaments capable of anastomosing in a network. At the beginning of hydration, the aleurone grains stain deeply ana homogeneously with vital dyes. Then ) the filamentous and semi-fluid, reticulate r) oH ny d vacuoles derived from them swell as water continues to be taken in and appear as spherical liquid vacuoles which by coales- cence gradually become transformed into yee Ohb Bernie large vacuoles in which the vital dyes cause ‘eg strongly colored precipitates. In sections prepared with mitochondrial rar techniques, the forms which were reticulate doer and filamentous, at the beginning of ger- &e mination, show a heavily stained, compact ee contour which is filamentous, or granular, °% and which is surrounded by a clear zone. &. The spherical liquid vacuoles, which succeed Py them, still enclose stained corpuscles but : these corpuscles become less and less abun- Fic. 119. — Tulip. Epic dant, as more and more water is taken into oo eel wution Pees. the vacuoles and, finally, in‘the large vacu- mentation of large vacuole, oles, no stained contents are found. containing anthocyanin, into small, sometimes reticulate, or filamentous, _vacuoles Reversibility of form in the vacuolar sys- caused by plasmolysis in a ; é 5 S 5% solution of NaCl. tem. — PIERRE DANGEARD’s investigations, and our own, describing this development show, therefore, that there exists a certain reversibility between the two vacuolar forms. It has previously been shown that vacuoles ordinarily appear under very different aspects according to the age of the cells: 1. As numerous, minute, semi-fluid vacuoles which have more or less the shape of the chondriosomes. 2. As a small number of large, spherical, liquid vacuoles, always containing colloidal substances, but in very dilute solutions, i.e€., vacuoles corresponding to the classical definition of vacuoles and capable of fusing into a single enormous element. The first of these is found in embryonic cells, the second in mature cells. If the cells are greatly dehydrated, the spherical liquid vacuoles may lose their water, become concentrated, and may again take on a semi-fluid consistency and look like chondriosomes. A more com- Chapter XV — 173 — Origin of Vacuoles plete dehydration leads finally to the transformation of these semi- fluid vacuoles into solid bodies (aleurone grains) by a solidification of their colloids. The aleurone grains, by taking up water anew, are capable of again assuming the semi-fluid consistency and ap- pearance of chondriosomes and, by a continuance of this process, may finally become liquid vacuoles. The filamentous appearance of the vacuoles seems to be the result of their semi-fluid state, for in the semi-fluid state, the vacuoles are generally filamentous or reticulate and stain uniformly and deeply with vital dyes. In the liquid state, the vacuoles are generally spherical and are stained only weakly with the vital dyes, which bring about a precipitation of the enclosed colloids as deeply stained granulations showing Brownian movement. The vacuoles are then composed of drops of a very dilute colloidal solution. In 8 Fic. 120. — Saprolegnia. Modifications in form of vacu- olar system in a single branch, cultivated on 1% peptone bouillon with 0.001% neutral red, in a van Tieghem and Le Monnier cell. 1-6, spherical vacuoles fuse to form a single canal which then, 7, 8, is transformed into a net- work (After Mile. CASSAIGNE). the solid state, they appear as globular bodies, which do not stain with vital dyes unless imbibition has previously taken place, but, on the contrary, always stain after fixation. The vacuoles, there- fore, may pass from one form to the other depending upon the water content of the cell. This reversibility has been obtained experimentally, further- more, in various cells, among others, in the epidermal cells of peri- anth parts of red tulips (Fig. 119). In the open flower, these cells contain a large vacuole, occupying almost the entire volume of the cell, and containing a concentrated solution of red anthocyanin pig- ment. Now, by plasmolyzing mature epidermal cells with a strongly hypertonic solution, we have observed that these vacuoles, as they lose water, may break up into small vacuoles which become semi- fluid and appear granular, filamentous and reticulate. Similar re- sults have been obtained in the Saprolegniaceae (GUILLIERMOND) and in the epidermal scales of Allium Cepa (KUSTER). Guilliermond - Atkinson — 174— Cytoplasm If one observes more closely the changes in appearance of the vacuoles brought about by hydration and fusion of small vacuoles into one large one, and, inversely, if one observes the fragmentation into very numerous, small, semi-fluid, filamentous or reticulate vacu- oles—a sort of splitting up of the large vacuoles—one is led to admit that in these phenomena the vacuoles play only a passive role. Their contraction and division are brought about by the degree of water taken into the cytoplasm which causes movements of the latter, the consequences being felt by the vacuoles. The cytoplasm, under some influences, may extract a part of the water contained in the vacuole and may swell. This swelling is therefore produced by movements of the cytoplasm, particularly by emission into the vacuoles of lamellate prolongations which finally partition off the vacuole into multiple vacuoles. These, in losing their water, be- come very viscous and look like chondriosomes. In the reverse process, the cytoplasm is capable of restoring to the vacuoles a part of their water of imbibition, bringing about a hydration and increase in volume of the latter. These then fuse into a single large vacuole. It is phenomena of this type which must take place at the beginning of the maturation of the seed and which must take place during the process of plasmolysis; some of the water from the vacuole passes into the cytoplasm and must cause a swell- ing, to which may be attributed the fragmentation of the vacuoles, and it is not until later that the cytoplasm in turn gives up its water to the exterior. In germination, the contrary phenomena must take place. The vacuole absorbs the water at first accumu- lated in the cytoplasm, and, as the latter continues to dehydrate, the vacuoles progressively swell and again fuse to form a very large vacuole. This view of the matter seems, moreover, to be confirmed by the fact that the viscosity of the cytoplasm increases as the plant grows old, correlative with the development of the vacuole which ends by occupying almost the entire cell. This reversibility of vacuolar form is to be compared with a remarkable phenomenon in the tentacles of the leaves of Drosera rotundifolia, described long ago by CHARLES DARWIN, and desig- nated by him as aggregation. While studying the modifications which occur in the pedicel of the tentacles as a result of the stimulus produced by an insect, DARWIN saw in each cell that the cytoplasm, which was colored red by pigment before stimulation, broke up before long into an aggregate of deeply stained corpuscles appear- ing as granules, clubs, rods or filaments showing amoeboid move- ments. The study of this phenomenon, taken up by various authors (GARDINER, DE VRIES, GOEBEL, and AKERMAN) has shown that in reality the phenomenon observed by DARWIN consists of a multiple fragmentation of the vacuole and not of the cytoplasm. The cells of the tentacle contain a single, very large vacuole filled with antho- cyanin. At the moment of stimulation this vacuole undergoes a great fragmentation. It splits into a large number of small chon- driosome-shaped vacuoles. Immediately after stimulation, these minute vacuoles fuse to constitute again a single large vacuole— Chapter XV — 175 — Origin of Vacuoles the cell returns to its initial state. This is therefore a phenomenon entirely comparable to that observed during the formation of aleu- rone grains and during the process of plasmolysis. AKERMAN’s work has shown that this phenomenon consists of a modification in volume of the vacuoles as a consequence of a great deal of water being taken in by the cytoplasm. The result is a fragmentation of the vacuole, caused by swelling of the cyto- plasm, and at the same time an increase in osmotic pressure. This pressure increases by 5 atmospheres. Centrifuging revealed, as has been seen, that, in the stimulated cell, the vacuoles are more dense than the cytoplasm and, conversely, in the unstimulated cell, the cytoplasm is more dense than the vacuole. The study of these phenomena has been taken up recently by DUFRENOY, HOMES, and KEDROWSKY working on Drosera, by QUIN- Fic. 121. — Drosera rotundifolia. Glandular cells in the tentacles. Vacuolar system made visible by red pigment in cells, may change from large to small, spherical, filamentous or reticulate vacuoles. A, after DE VRIES. B, after Homés. TANILHA and MANGENOT in Drosophyllum lusitanicum. These in- vestigations show that it is necessary to differentiate two sorts of physiologically distinct cells in the tentacles of Drosophyllum and of Drosera. First, there are those which, covering the upper sur- face of the tentacles, are secretory. They excrete the complex and viscous liquid which forms a drop at the tip of each tentacle. These cells, when they are not going through a period of digestion, possess a group of small filamentous or granular vacuoles, an arrangement probably having some relation to the loss of water which takes place in the cell (QUINTANILHA, DUFRENOY, HOMES, MANGENOT, KEDROWSKY). Secondly, there are the cells which compose the lower part of the head and the pedicel. Each of these cells con- tains, except during the periods of digestion, a large fluid vacuole, colored red by anthocyanin. During digestion, this enormous vacuole becomes very finely divided into a large number of small, globular or filamentous elements, colored violet-grey (DUFRENOY, MAN- GENOT). ‘These small filamentous vacuoles are oriented parallel to Guilliermond - Atkinson == 176 — Cytoplasm the longitudinal axis, while the small vacuoles are accumulated against the proximal wall of the cell (MANGENOT). This polarity of arrangement, which becomes more marked as digestion becomes more active, clearly indicates that these cells are traversed by a continuous flow of material caused by protein digestion (proteolesis) at the level of the extremities of the tentacles. Thus, the “aggregated” state of the vacuoles, 7.e., their irregular dis- position, seems to correspond to differ- ing physiological conditions—to a secre- tion in the cells which cover the ex- tremity of the tentacle, to an absorp- tion in the cells of the stalk—but both these processes indicate the passage of a current across the cells. This same arrangement of vacuoles is found in young sieve tubes in the angiosperms in which the fragmented and polarized vacuoles are very polymorphic. In these cells, large vacuoles and small filamen- tous or reticulate vacuoles are found. They are also found in the conducting elements of the Laminariales and the Rhodophyceae (MANGENOT) (Fig. 123). Recent work seems to suggest that the vacuoles undergo a similar frag- mentation each time that the cells are in the process of active secretion (MAN- GENOT, Mlle. Py, THOMAS, GUILLIER- MOND). The reason for this is not yet known. . Another phenomenon to consider here is that of the frequent changes in form of vacuoles in many cells. The Fy % % = z BH eg Fic. 122. — Drosophyllum lusitanicum. Epidermal cells of the pedicel of a tentacle. Vacuoles in grey, arrows in- dicate the direction of the head of the gland. A, during digestion of protein. B, dur- ing inactive period. C, tip of leaf with tentacles; one gland showing pedicel, head and drop of secretion. (Af- ter MANGENOT). observation of a species of Saprolegnia in van Tieghem and Le Monnier cells, grown in a nutrient solution to which neutral red has been added, made it possible to record this phenomenon under excellent conditions (Fig. 120). In the extremities of growing filaments the vacuoles generally appear as very small elements shaped as granules, rods or filaments. These ele- ments are carried along by cytoplasmic currants which cause them to change shape constantly. They are capable of swelling and of contracting, of passing from the shape of granules to that of fila- ments and conversely. In the space of a few seconds, they are frequently seen to fuse and form rather large spherical vacuoles which, themselves, may give rise, by budding, to small vacuoles, or may be completely split up into a multitude of small elements which Chapter XV — 177 — Origin of Vacuoles then elongate and anastomose to form a network. So the trans- formation from large vacuoles to a network and the converse transformation are here again observed. It is only a little farther away from the tip of the filament that all the vacuoles fuse to form a vacuolar canal (Mlle. CASSAIGNE). A prolonged observation of growing yeast without the aid of vital stains makes it possible to see that the large vacuoles, which appear rather stable, in these fungi are themselves subject to changes in shape. After a period of stability their contours may suddenly become irregular, angular, may vary continually, contracting and dilating and finally come back to a tem- porarily stable form. Often they are seen suddenly to put out long and thin prolongations, which later contract as the vacuole returns to its spherical shape. Ultramicroscopic observation of the vacuoles of fungi (in cases where it is possible because of the faintly luminous outlines of these elements) has shown also that their contours often manifest slowly undulating movements. These phenomena seem to be caused here not only, (1) by differences of imbibition between the cytoplasm and the col- loidal contents of the vacuoles and (2) by movements of the cytoplasm, but also by modifications of surface tension. The phenomenon of vacuolar con- traction:- In addition to these phenom- ena of vacuolar fragmentation, there must be cited here another phenomenon of quite a different nature, namely, a particular state of the vacuoles which was first observed by WEBER. In the flowers of the Boraginaceae (Symphy- Fic. 128. — a, Alaria escu- lenta (Laminariales), b, Bon- nemaisonia asparagoides (Rho- dophyceae). Globular vacuoles at one end of cell; plasmodes- mic threads connect the cells in the conducting tissue. (After MANGENOT). tum tuberosum, Anchusa italica and Mertensia sibirica), this investigator observed that the vacuolar sap of cells is a jelly, capable, under certain conditions, of contract- ing “spontaneously” 7.e., quite aside from all plasmolysis. This phe- nomenon has been observed in flowers infiltrated with solutions containing from 1%-0.1% of neutral red. There is then produced a contraction of the colloidal contents of the vacuole whose con- tours are curiously parallel to those of the cell itself. WEBER pro- poses to call this phenomenon vacuolar contraction and interprets it as a syneresis, a name given by GRAHAM to the spontaneous con- Guilliermond - Atkinson — 178 — Cytoplasm traction of a gel when liquid is expelled. The phenomenon pre- supposes the existence in the vacuole, around the contracted jelly, of a liquid phase (serum) produced at the moment of contraction. This phenomenon of vacuolar contraction seems to be very gen- eral indeed. There is frequently encountered, in the epidermal cells of flowers, a colorless space enclosing a contracted intravacuolar mass and surrounded at the periphery of the cell by a thin cyto- plasmic layer. Neutral red may be superimposed upon the natural color of the vacuolar sap, when it is rich in anthocyanin compounds, and will stain the contracted vacuolar mass intensely. Careful ob- servation of the colorless space reveals that it is not empty. It is occupied by a fluid. Plasmolysis of this modified vacuole, moreover, is quite possible and water is lost from the peripheral colorless fluid, which appears at first very poor in dissolved material. Little by little, over a period of from 24-48 hours, however, the fluid reddens. The reddening never reaches the intensity of that of the contracted mass. It is evidence, however, of a slow penetration of colloidal material into the peripheral liquid. Then a second contraction is frequently produced at this time. The fluid separates into a new color- less peripheral serum, and into a deeper red region, contracted about the mass originally isolated. WEBER thinks that the same phenom- enon may explain the presence in many phanerogam cells of two categories of Fic. 124. — A, B, See- § yacuoles which are adjacent in the cell, the charomyces ellipsoideus vi- : 5 5 ; 5 tally stained with neutral one liquid and lacking in tannin, the other Sede ecules a bude San formed of a tannin jelly. These will be Cae i ee le taleen up later. WEBER underlines also the branches seem to form de = notential importance of this phenomenon in the realization of rapid changes in the turgidity of cells, perhaps in the mechanism of certain organ move- ments. He points out in this regard that the motor swellings of leaves of the Leguminosae (Mimosa), as MANGENOT has shown, have vacuoles containing a tannin jelly, side by side with small vacuoles which do not contain colloidal substance. WEBER com- pared this vacuolar contraction to the natural production of large colloidal corpuscles observed in the vacuoles of the mature cells. This contraction can not be attributed to a phenomenon of syne- resis, for it consists only in the partial precipitation of the vacuolar colloid. It seems, on the contrary, to correspond, as we have said, to the formation of a coacervate within the vacuolar sap. Origin of vacuoles:- P. A. DANGEARD and then P. DANGEARD, as a result of his own work on aleurone grains, were led to adhere to the theory of DE VRIES and WENT and to admit that the vacuoles Chapter XV ——179-— Origin of Vacuoles are never formed de novo, but always arise by the division of pre- existing vacuoles, and are transmitted by division from cell to cell. But the DANGEARDs believe that it is not the vacuole itself, that is transmitted, but the metachromatin, which they consider to be the universal substance of their vacuome. According to them, this metachromatin persists in a solid state after the disappearance of the vacuole in the seed, as well as in the spores of fungi, and re- forms vacuoles anew at germination by taking in water again. This theory is difficult to admit in view of the fact that we know that there is no single chemical substance which is characteristic of vacuoles. Actually, it is extremely difficult to study the origin of vacuoles in the phanerogams because of the great number, and the small size, of these elements in embryonic cells. It is known, moreover, from what has just been said that vacuoles exist in all cells and that they are capable of dividing and of fragmenting. It has been observed besides that during mitosis the vacuoles are distributed between two daughter cells. It is for this reason that BAILEY and ZIRKLE, without committing themselves on this subject, say that they have never seen vacuoles form de novo and that nothing proves that this phenomenon is possible. But neither the fact of the distribution of the vacuoles between the daughter cells during mitosis, nor that of their persistence in the aleurone grains of the seed and their transmission to the embryo, proves that the vacuoles can not rise de novo. Moreover, we can scarcely permit ourselves to consider them as individualities of the cell, incapable of forming de novo, when through their extreme instability of form, they may in the space of a few minutes be split up into very minute elements capable soon of fusing again. Some fungi are more favorable for the study of the origin of the vacuoles than are the phanerogams. In the mycelium of Peni- cillium glaucum (Fig. 101) or of Oidium lactis, for example, lateral branches may be observed to form from filaments already con- taining large vacuoles and in these branches, which at first do not have them, small globular vacuoles are seen to appear which can hardly have any relation to the large vacuole of the filament from which the branch arises. This is also true of the buds of the yeasts in which there are small vacuoles which do not appear to be derived from the large vacuole of the mother cell. We concluded from these very clear facts observed by means of vital dyes that vacuoles may be formed de novo (Figs. 124-126). P. DANGEARD has objected, and with reason, that vital stains can cause alterations of the vacuoles, for example, their immedi- ate fragmentation when in the process of dividing. It is certain that vital dyes stop the multiplication of cells in certain cases, par- ticularly in the fungi. DANGEARD again took up the study of the formation of vacuoles in yeasts and in following the budding of these fungi in a moist chamber without vital staining, showed that the large vacuole of the mother cell always puts out a delicate pro- longation into the bud. The extremity of this prolongation is cut Guilliermond - Atkinson — 180 — Cytoplasm off and forms the small vacuole of the daughter cell. DANGEARD has sought more recently to demonstrate, but this time with vital dyes, that the vacuoles of algal zoospores are always transmitted by means of the filament put out at germination. In Saprolegnia, growing on media to which neutral red has been added and observed in van Tieghem and Le Monnier cells, we have shown, however, that the vacuoles which in the zoospores appear as small granules, fuse at the moment of germination to constitute a single large vacuole and then, in the germination tube, small globular vacuoles appear which do not seem to be derived from the large vacuole of the zoospore. Now, if the objection of PIERRE DANGEARD is sound in regard to vital staining of the ordinary fungi which, when carried out between slide and cover soeseses a9 6¢ Fic. 125 (left). — Saprolegnia. Germination of zoospores in a van Tieghem and Le Monnier cell on 1% peptone bouillon with 0.001% neutral red. 1, 2, zoospore. 3-20, germination tube. The large vacuole extends into the germina- tion tube and may fuse (15) with small vacuoles which form at the tip. (After Mlle. CASSAIGNE). Fic. 126 (right). — Saccharomyces pastorianus. Types of formation of vacuoles during budding of the yeast grown in a van Tieghem and Le Monnier cell without vital dyes. A, 1-8, Observation during 1 hr. 1-5, successive frag- mentation and fusion. 6, 7, prolongation sent into bud. 8, separation from vacuole of mother cell. B, vacuole of bud formed de novo. C, vacuole of bud formed by a kind of budding from vacuole of mother cell. (After Mlle. CASSAIGNE). glass do not grow as long as they keep the neutral red accumulated in their vacuoles, this objection is evidently not sound in the case of Saprolegnia, which can be grown on media to which neutral red has been added. Mlle. CASSAIGNE repeated this study and ob- served the development of vacuoles, both in the germination tube and in the growing filaments. She was able to see small vacu- oles form de novo which later refused to constitute a single large vacuole, or elongated into filaments and anastomosed into a net- work. Nevertheless, since the conditions of observation were ad- mittedly abnormai because of the presence of neutral red, Mlle. CASSAIGNE repeated the work of P. DANGEARD on the yeasts in which she followed budding without vital staining. Now, she ob- served that actually the vacuole of the bud may arise from the budding of the vacuole of the mother cell, as DANGEARD indicated, but that often this vacuole also arises de novo in the bud. These observations seem therefore to furnish proof that these vacuoles form de novo. In consequence of our research, we have Chapter XV — 181 — Origin of Vacuoles proposed an hypothesis to explain the formation of vacuoles. This hypothesis is based, on the one hand, on the fact that colloidal sub- stances contained in the vacuoles are of very diverse constitutions, and, on the other hand, on the fact that the cytoplasm is constantly the locus of secretory phenomena (production of reserve or of waste products). The hypothesis assumes that among these prod- ucts, those which are in a colloidal state separate by an unknown physical-chemical mechanism from the cytoplasm in the form of colloids non-miscible with the cytoplasmic colloids and composing a distinct phase of the latter. They appear in the form of small elements. These, by virtue of their semi-fluid consistency and of their physical state, which is rather like that of the chondriosomes, are subject to the same laws which determine the shape of the chondriosomes. This explains the resemblance in form of these two elements. According to our hypothesis, these colloids possess a capacity for taking up water which is stronger than that of the cytoplasm, and when the cytoplasm has reached its maximum point of imbibition, the excess water is absorbed within these ele- ments which are gradually transformed into a true solution and constitute the vacuoles. In these vacuoles during the different stages of their development, there may accumulate by absorption, according to this theory, all the products secreted by the cytoplasm which are capable of forming solutions or pseudosolutions within the vacuoles. This hypothesis, which resembles that of PFEFFER, would apply at least to a great number of cases but probably not to all. The presence in some cells of several distinct categories of vacuoles:- Recent research by MANGENOT has drawn attention to the existence of two distinct categories of vacuoles which are ob- served in the mature cells of numerous plants. They were glimpsed and very briefly cited some time ago by WENT, KLERCKER and LLOYD. Vacuoles which are rich in tannin, and very refractive, and which reduce osmic acid instantly, are often observed in the same cell side by side with, but in reality distinct from, other vacuoles which do not contain tannin, are very slightly refractive and show no reaction with osmic acid. The respective dimensions of each are sometimes the same, or again the tannin-containing vacuoles may be much more voluminous than the others, or conversely, may be smaller, in which case they may appear as filaments or small granules scattered in the cytoplasm around the vacuoles. Vital dyes apparently stain these two categories differently. Cresy] blue, for instance, stains the vacuoles containing tannin, blue or green, and the other vacuoles, violet or rose. Cells with tannin-containing vacuoles are very widespread in plants (Legumes, Mimosa, Ber- beris, Eucalyptus, Oxalis, Monotropa, Hypopitys). These two categories of vacuoles, the one acid and rich in tan- nins, the other without tannins and seeming to have a high pH, BAILEY has found in the cambial cells of gymnosperms and arbores- Guilliermond - Atkinson — 182 — Cytoplasm bas pee lh ane nepna else DO RST el Sen ee cent angiosperms and MILOVIDOV pointed out their existence in epi- dermal cells of rose leaflets. More recent research has made it possible to show the rather frequent presence of specialized vacuoles in epidermal cells of 10 vy Fic. 127. — 1-7, Fruit of Rubus fruticosus. Two types of vacuoles. 1-4, exocarp. 1, young green fruit; large and small colorless vacuoles (V,v). 2, ripening fruit; large vacuole with raspberry-red anthocyanin pigment and small colorless vacuoles. 3, ripe fruit; three vacuoles with red pigment, numerous small vacuoles either with colloidal bodies (g) or needle-shaped crystals (c), both colored violet by anthocyanin. 4, ripe fruit; one large vacuole with red pig- ment, smaller vacuoles with crystals of dark violet pigment, isolated or in bundles. 5, mesocarp, ripening fruit; large vacuole (V), with dilute solution of raspberry-red pigment, numerous small vacuoles (v), with brick-red pigment and raspberry-red colloidal granules. 6, 7, mesocarp, ripe fruit; small vacuoles with one or more violet colloidal bodies, sometimes also with needle-shaped crystals of pigment, isolated or in groups, sometimes with the crystals only. 8, Wisteria sinensis; epidermis of petal; large central vacuole (V), with red-violet pigment and small peripheral vacuoles (v), with a concentrated solution of blue-violet pigment and crystalline needles of dark blue pigment. 9, Hibiscus syriacus; epidermis of petal; large central vacuole (V), with red pigment and dark red tannin bodies (P); small peripheral vacuoles (v), with mauve pigment. 10, Canna indica; epidermis of leaf; large vacuole (V), with red pigment and a large spherical crystal (S); one or more small colorless peripheral vacuoles. (in vivo). leaves, fruits, and flowers which contain anthocyanin pigments. One of the most curious cases is to be found in the epidermis of the petals of Wisteria sinensis, already reported by WENT, in which all the cells show two very distinct categories of vacuoles: one large central vacuole and several small peripheral vacuoles. The large central vacuole contains tannin and a reddish violet antho- Chapter XV — 183 — Origin of Vacuoles cyanin pigment. The small peripheral vacuoles lack tannin and contain a very concentrated solution of bluish violet anthocyanin pigment which is capable of partially or totally crystalizing into long needle-shaped, dark blue crystals. Another no less interesting example is seen in the exocarp and mesocarp of the fruit of Rubus fructicosus, in which all cells likewise possess two sorts of vacu- oles, the one large, solitary and centrally placed containing, at the same time, tannin and a cherry-red pigment; the other, small, spherical, extremely numerous, and scattered in the parietal layer of the cytoplasm. These latter are without tannin and form at first a brick-red pigment, but when the fruit is mature, there appear in each of these vacuoles, large colloidal bodies, dark violet in color, which show concentric zones. These are the result of the precipitation of the colloidal content of the vacuoles which has absorbed the pigment contained in the vacuoles. At maturity the vacuolar sap changes from brick-red to pale violet, then to white, whereas blackish, :violet-blue crystals shaped like needles, or sphaerocrystals, are deposited in the interior of the vacuole between the colloidal bodies. In certain parts of the epidermis of the petals of Hibiscus syriacus also, there are found in each cell a large central vacuole, enclosing tannin as well as a raspberry-red anthocyanin pigment, and small peripheral vacuoles enclosing a mauve pigment. In all the cases which we have just examined, the two categories of vacuoles contain colloidal substances and have the property of accumulating vital dyes, but this is not, however, universal. In a very great number of cases (in the epidermis of leaves, stem and petals of roses, in the petals of Lathyrus odoratus, Prunus japon- ica, Camellia japonica, Tropaeolum majus, in leaves of Canna in- dica, etc.), there are found together constantly, in each cell, two categories of vacuoles: a large central vacuole containing tannins or other colloidal substances as well as an anthocyanin pigment, and small colorless vacuoles seemingly without any colloidal sub- stances. Those of the second category sometimes contain very minute crystals showing Brownian movement. In the elongated cells of the inner portion of the fleshy pericarp of the fig there are two categories of vacuoles of very curious appearance, each vary- ing both in number and dimension in the cell. One type contains violet-red anthocyanin pigment together with colloidal substances and is very variable in shape, the larger among these having irreg- ular contours which give them an angular appearance, the smaller ones being chondriosome-shaped elements. The other type is color- less, lacking in colloidal substance and all of them, no matter what their dimensions, appear perfectly spherical. In this case, the vacu- oles which do not contain tannins or other colloidal substances never stain, which seems therefore to add further proof that vital stain- ing of the vacuole is due exclusively to the presence in them of colloidal substances. The case of the pericarp of the fig is par- ticularly interesting because it shows us that the shape of the vacuoles, whether irregular or like that of the chondriosomes, seems Guilliermond - Atkinson — 184 — Cytoplasm to be attributable to the viscosity of their contents, since the co- existing vacuoles without colloidal substances are always spherical. In all cases in which the cells contain two categories of vacu- oles, these vacuoles become distinct very early and it is very diffi- cult to determine their origin. It would seem, however, that the vacuoles lacking in colloidal substances arise by exudation from vacuoles rich in colloids, for, by plasmolysis, it is possible in some cases to obtain experimentally the formation of similar small vacu- oles in cells which do not contain any. It would be natural, therefore, along with WEBER and KUSTER, to relate this phenomenon to vacuolar contraction and to attribute it to a syneresis assuming that the vacuoles not staining with vital dyes are totally lacking in colloidal substances. But we have seen that this is not always so and in the fruit of the blackberry there exist two categories of vacuoles both of which contain colloidal substances. In this case it might be supposed that these two categories of vacuoles, which seem to correspond to small accumulation and transportation centers for various meta- bolic products, are always distinct and have no genetical connection, or else that they arise by a differentiation from a single category of vacuoles, but by the phenomenon of coacervation and not of syneresis (Cf. p. 177). However this may be, this last ex- planation does not apply to the lower plants, in particular to the algae, in which Sha ap there are encountered still more fre- coh freee the tmae p= quently, several categories of vacuoles in a ihe fesliy eects tse single cell. In the brown algae it has been waive invabe andvahape )) Knowntor a Jonge time that thene vexisp with’ colloidal, contenisand’ viscous meclusions which have been called anthocyanin pigment; the other (v), varying in size fucosan granules (HANSTEEN), or phy- but always spherical, with- _ sodes (CRATO), whose morphological sig- nificance has been the subject of numerous discussions. These inclusions stain vitally like the vacuoles and yet are present at the same time with other large vacuoles whose contours are more fluid and which also take the vital dyes but stain differently. Cresyl blue, for example, gives the inclusions a greenish blue tint, whereas the vacuoles take a violet-blue color. These inclusions contain in fact catechin tannins, showing the phloroglucinol-hydrochloric test, which explains the greenish blue color which they give to cresyl blue. Although these phenolic in- clusions are always separated from the other vacuoles even in the beginning, it seems logical to consider them, as does MANGENOT, Chapter XV — 185 — Origin of Vacuoles as corresponding to specialized vacuoles, for the same reason that we consider as specialized vacuoles, those encountered in the phanerogams in which group of plants phenolic compounds are always localized in vacuoles, often having forms corresponding to those of the physodes. CHADEFAUD, who recently described simi- lar physodes in the Phaeophyceae, thinks, on the contrary, that these inclusions are chondriosomes which elaborate mucilages and phenolic compounds. This opinion does not seem plausible to us. The same peculiarities are found in Vaucheria in which the re- cent work of MANGENOT has shown, apart from the central vacu- olar canal previously discussed, the existence of numerous small, peripheral rod- or granule-shaped vacuoles formed of a very con- centrated colloidal substance. These small vacuoles which P. A. DANGEARD confused with the chondriosomes, take a blue color with cresyl blue whereas the vacuolar canal stains violet. MANGENOT thinks these small vacuoles are composed of mucilages with which, in a great many cases, tannins are associated. In Euglena viridis, as well as small vacuoles composed of a concentrated solution of metachromatin, there are found in the sub-cuticular cytoplasmic layer, spheres colored purple-violet by cresyl blue and appearing as small vacuoles. According to CHADE- FAUD, these are specialized vacuoles containing mucus (Fr. corps muciféres). In this same region in other Euglenas, there are ob- served elements shaped like bacteria, colored blue with cresyl blue, rarely violet, which are capable of ejecting their contents as a long filament. They seem to correspond to trichocysts such as are ob- served in certain ciliates. In Cladophora (Fig. 103), P. A. DANGEARD cited two categories of vacuoles, those centrally placed which are large and liquid, others at the periphery which are small, semi-fluid elements shaped like rods. The existence of specialized vacuoles (with the exception of those lacking in colloidal substances whose existence appears to be connected with a phenomenon of syneresis), shows us that it is not possible to consider the vacuome as a morphological entity in the sense of DANGEARD or to adhere to the theory of DE VRIES. It is difficult, moreover, to keep for the term “vacuole” its classical meaning and to limit it to liquid inclusions of the cell, since it is now established that the well characterized vacuoles of the majority of plants are themselves derived from semi-fluid inclusions whose consistency is often greater than that of the cytoplasm itself, vacu- oles which during the development of the cells again pass through semi-fluid and even solid phases. Furthermore, we have seen that in some lower plants the small inclusions, which, by the nature of their contents and their predilection for vital stains, correspond un- questionably to the vacuoles of more evolved plants, may remain constantly in the semi-fluid state. One fact, however, stands out very clearly from the investiga- tions just reviewed. It is that the protoplasm itself, 7.e., living mat- ter, is incapable of being stained with vital dyes except in a transi- Guilliermond - Atkinson — 186 — Cytoplasm tory way. Either it excretes them into the vacuole or else it dies, poisoned by them. It is only in products resulting from its metabo- lism that the stains accumulate. We are thus brought back to the idea expressed by many cytologists, VON MOLLENDORFF among others, that vital dyes normally stain only that which we call the deutoplasm, or paraplasm, in which are grouped all the products arising from protoplasmic elaboration. The vacuoles belong in this category and perhaps it would be suitable to include under the general heading of vacuolar system (a term preferable to that of vacuome, which involves the idea of morphological entity) all the paraplasmic colloidal inclusions of the cytoplasm which are not of a lipide nature or, at least, in which the lipides do not consti- tute the essential element. These inclusions are composed of aque- ous solutions of colloidal substances elaborated by the cytoplasm, not miscible with it (doubtless forming a coacervate system sepa- rate from the cytoplasm) and are characterized by a more or less high concentration. They are however capable under certain physi- cal conditions and in certain cells, by reason of their capacity for taking in water which is stronger than that of the cytoplasm and often unlimited, of becoming dilute and of taking on the aspect of liquid inclusions or true vacuoles. In a word, the liquid vacuole according to this interpretation may be formed each time that there is deposited in the cell a product of secretion in a colloidal state more capable of absorbing water than is the cytoplasm. So the vacuolar system expresses a physical state, an aqueous phase, separated from the cytoplasm, and containing various more or less concentrated, colloidal and crystalloid substances of paraplasm which may, according to the nature of these substances and the conditions of the cell, have a rather high viscosity and which are able to pass from the liquid to the semi-fluid or solid state. If, in the great majority of plants, the vacuolar system appears to us as a morphological entity, it is undoubtedly because plant cells undergo a considerable hydration, and because, from the first stages of their development, the paraplasmic inclusions whose en- closed colloids take up water, are transformed into liquid vacuoles, which run together very quickly and become a single and enormous vacuole in differentiated cells. It follows logically that all the products of metabolism capable of forming solutions or pseudo- solutions with water would collect in this single vacuole. In some lower plants and in animals, on the contrary, the cells would not undergo this hydration and the paraplasmic inclusions would gen- erally remain in the cytoplasm as concentrated colloidal solutions, making a distinction among them more easy by reason of the chemical contents characteristic of each. According to this hypothesis it is possible to see, as does MANGE- NOT, a similarity between the vacuolar system and the lipide inclu- sions — paraplasmic formations capable occasionally of being stained in the living state because the vital stains are soluble in lipides. In these inclusions, which are formed of neutral fats and which are found in practically all cells, there accumulate all the Chapter XV — 187 — Origin of Vacuoles products of secretion of the cytoplasm capable of being dissolved in them (phytosterol, lecithins, oils, carotinoids, etc.). They may remain scattered in the form of small inclusions in the cytoplasm, or may fuse together, as in the spores of certain fungi and in the adipose cells of animals, to constitute a single enormous fatty globule occupying the entire cell. A distinction may therefore be made in the paraplasm between hydrophilic inclusions (vacuoles) and hydrophobic inclusions (lip- ide inclusions) . Be that as it may, these investigations, taken all together, show that vacuoles are present in all cells, just as are the chondriosomes. Although both are present, the vacuoles cannot, in any way what- ever, be considered similar to the elements of the chondriome. There is reason to think that they have no permanence, no indi- viduality which is transmissible from generation to generation, or as PARAT says, “Only the group is significant, only the vacuome is an entity, the expression of a cellular equilibrium, the bond in metabolism, an ‘aqueous phase’ whose elements disappear and are replaced by others.” Digestive vacuoles:- The theory which we have just formulated in regard to the significance of the vacuoles permits us to incorpo- rate the vacuoles of the Myxomycetes in the vacuolar system. It is known that in this group as well as in the Amoebas, there do not seem to be any digestive vacuoles which take up vital dyes but there are vacuoles which are distinguished from ordinary vacuoles by their exogenous origin. These vacuoles arise from food particles surrounded by a little water in the cytoplasmic mass. If the hy- pothesis which we have formulated on the origin of vacuoles be admitted, it follows that in spite of their exogenous origin, these vacuoles are in the same category as the others, contrary to the opinion of VOLKONSKY who definitely separates them under the name of gastriole. There are other vacuoles, present in the flagellate algae which are pulsating vacuoles. Their significance is still unknown. Chapter XVI THE ROLE OF THE VACUOLAR SYSTEM AND HYPOTHESES CONCERNING IT One of the most important functions of vacuoles is to regulate the exchange of water which takes place in the cell by osmotic phenomena. This was brought out by the classical research of DE VRIES. We have already mentioned this function (p. 125). Now we must show the applications of it made by DE VRIES. From his experiments, this investigator thought out a method by which the osmotic pressure of a cell might be determined. This consists in placing fragments of living tissue (for example, stami- nate hairs of Tradescantia, which have been mentioned before as particularly favorable for these experiments) in solutions of a known substance such as sugar, arranged according to concentra- tion. There may then be found a limiting concentration at which plasmolysis is just beginning, 7.e., in which separation of the proto- plasm from the angles of the cell wall is first detected. This limit- ing phenomenon is considered as a criterion and it is recognized that it corresponds to an equality in osmotic pressure: the solution is therefore isotonic with respect to the vacuolar sap. By this method DE VRIES made known one of the fundamental laws of osmosis. By a series of progressive comparisons of dif- ferent substances, it is demonstrable that they are isotonic when each produces incipient plasmolysis of cells. By this biological method, DE VRIES was able to show that isotonic solutions are equi- molecular, 7.e., equal osmotic pressures are developed by an equal number of molecules. The method is so sensitive for sugars that DE VRIES was able to determine the molecular weight for raffinose about which chemists disagreed. Electrolytes, however, are ion- ized in solution and each ion, acting as a molecule, increases osmotic pressure. Consequently DE VRIES had to introduce into this law a coefficient of correction (isotonic coefficient). Osmotic pressure of the cell sap varies according to the con- ditions of the life of the plant: the osmotic value is 4-5 atmospheres for aerial parts of aquatic plants, 12-14 atmospheres for cells in the root of the bean, almost 100 atmospheres for the chlorophyll- bearing parenchyma of various plants. Normally, cells are always distended by their vacuolar sap. This rigidity is called turgidity. The cells are entirely comparable to a blown up balloon: the internal pressure manifests itself if the membrane is pierced by a microdissecting needle and the proto- plasmic and vacuolar contents escape with force just like the air of a punctured balloon. Turgidity plays a considerable role in the life of plants in maintaining their rigidity. When it is lacking, the plants lose their rigidity and wilt. Cells have, moreover, the means of regulating the concentration of their vacuolar sap in Chapter XVI — 189 -—— Role of Vacuoles such a way that it is always hypertonic with regard to the sur- rounding medium. As soon as the concentration of the latter increases, the cells hydrolize their reserve starch and the resulting sugar goes into the vacuolar sap whose osmotic capacity increases. This phenomenon has been given the name anatonosis. The pres- ence of colloidal substances in the vacuole suggests that their réle is not reduced merely to osmotic actions but that they intervene also in the processes of the passage of water in and out of the cell. It is, in fact, this inward and outward passage of water, inter- vening alternately between vacuolar colloids and cytoplasmic col- loids which explains the reversibility of form of the vacuolar sys- tem discussed earlier. The vacuoles are accumulation regions, the reservoirs of a large number of metabolic products or of reserves, and are particularly regions of excretion of toxic substances, as the action of vital dyes tends to indicate. In the vacuoles, there accumulate all the products secreted by the cell which can be dissolved in water, forming true or pseudosolutions (proteins, holosides, heterosides, tannins, fla- vins, oxyflavanol and anthocyanin pigments, organic acids, alka- loids, certain lipides, mucilages and so on). These various prod- ucts may appear in the meristematic cells at the very beginning of development of the vacuolar system, or at any stage whatever, during the development of the system. It has been possible to demonstrate, notably by microchemical reactions, that in the seed- ling of tobacco, alkaloids appear in the cells of the meristem of the root, in the chondriosome-shaped vacuoles formed by the hydra- tion of aleurone grains (CHAZE). There have been localized also in the chondriosome-shaped vacuoles of the meristematic cells, cer- tain heterosides, such as the saponarosides (POLITIS). This is true for tannins (GUILLIERMOND, P. DANGEARD), the oxyflavanol compounds and anthocyanin pigments (GUILLIERMOND). The vacu- olar system is certainly more than a locus for the accumulation of these various products. The presence of colloidal substances in the vacuoles and their predilection for vital stains lead us to sup- pose, as do the DANGEARDs, that the vacuoles can exercise a role in absorption phenomena because of these very properties of ab- sorption, imbibition and combination which bring about the pene- tration of the dyes. DEVAUX believes the vacuolar system to be the site of chemical affinities of the cell and explains that the vital dyes penetrate the cell without staining the protoplasm and accu- mulate exclusively in the vacuoles, 7.e., in the non-living parts of the cell, because the chemical affinities of the living substance are masked by reciprocal saturation. So by his theory of polarized (catalytic) membranes (Cf. p. 121) only the non-living inclusions of the cytoplasm, such as the vacuoles, are capable of fixing the dyes, and there is localization of protoplasmic activity on the surface presented by the protoplasm and the vacuole. It has been seen that this opinion is not justified. If, actually, some dyes, like neutral red, traverse the cytoplasm without ever staining it and accumulate only in the vacuole, this is not true for other dyes which Guilliermond - Atkinson — 190 — Cytoplasm can stain the cytoplasm in living cells. Nevertheless this staining is only transitory and the dye moves from the cytoplasm into the vacuole. It is only after the dye has been localized in the vacu- oles that the cells are capable of growing and no staining other than vital staining of the vacuoles is compatible with growth. The hypothesis of DEVAUX seems to be confirmed by the works of GENEVOIS and GENAUD, who have shown that absorption of salts by cells occurs exclusively along the cellular and vacuolar membranes. It is necessary, however, to make reservations in regard to the absence of chemical affinities from the cytoplasm, since it has been seen that certain stains may, under some con- ditions, be retained by the cytoplasm (pp. 18, 142). It has often been supposed that the vacuolar system is not a simple center of accumulation of metabolic products but that it is at the same time the seat of phenomena of hydrolysis and of synthesis. According to KEDROWSKY and VOLKONSKY, the vacuoles are the secretion ap- paratus of the cell and the seat of enzymes, particularly of pro- teases, but this view seems to be exaggerated. There is reason to believe that in the chemical phenomena which take place in the vacuole, it is the cytoplasm which plays the active rodle, the vacu- ole having only a passive role. Let us add that PARAT considers that in animal cells, methylene blue is always reduced in the cytoplasm and in the chondriome (rH <12), and that it is, on the contrary, re-oxidized by the vacu- oles (rH <16), which does not seem to be true in plant cells. Going back to the hypothesis of ROBERTSON (p. 122), PARAT thinks that the pair: chondriome plus vacuole, presides over the synthesis of proteins, which, according to ROBERTSON, calls for a lipide phase and an aqueous phase and thus gives a morphological basis for this hypothesis. PARAT considers further that the vacuome is the crucible in which are completed the operations begun in the chon- driome, but these points of view are very hypothetical and lack a solid foundation. It has been seen that this hypothesis of PARAT is no longer tenable, now that it is demonstrated that the chondriosomes do not of themselves have a reducing roéle, contrary to what had been supposed, and that the vacuoles may in certain cases be just as capable of reducing actions as the chondriosomes. Chapter XVII GOLGI APPARATUS, CANALICULI OF HOLMGREN AND OTHER CYTOPLASMIC FORMATIONS Golgi apparatus and the canaliculi of Holmgren in animal cells:- By using methods of silver nitrate impregnation, GOLGI (1898) brought out in the cytoplasm of nerve cells (Purkinje cells and invertebrate ganglia of Strix flammea) a network of very fine filaments to which has been given the name internal reticular ap- paratus of Golgi. This formation was made the object of impor- tant studies by CAJAL. KOPSCH showed later that the Golgi apparatus can also be brought out by osmic impregnation at 40°C. This later method has the advantage over the preceding that it is much easier to use, for, unlike impregnation with silver, it does not result in so many failures. For this reason, it has been the starting point for a great deal of research which has revealed in most animal cells, formations which osmic acid blackens, just as it does the Golgi apparatus described by GOLGI and CAJAL. These formations, in spite of their widely differing morphological aspects, have been grouped with the Golgi apparatus on the single basis that they stain like it. These formations are not generally repre- sented by a network but by small elements scattered in the cyto- plasm, appearing as spherical or ovoid bodies, composed of a chromophobic substance surrounded by a chromophilic substance which is thicker on one side than on the other. They are known as dictyosomes or Golgi bodies. Many cytologists today think that the Golgi apparatus is a permanent feature of cytoplasm in the same way as is the chondri- ome, and there has been described, during mitosis, a division of the Golgi bodies between the daughter cells which has been called dictyokinesis (PERRONCITO). Finally, scientists are coming to the belief that this apparatus is the center of elaboration of metabolic products. In brief, it is thought to play the réle formerly attrib- uted, first, to the ergastoplasm and, later, to the chondriome. So the Golgi apparatus may be said to have supplanted the chondriome for those who adhere to this view. The Golgi apparatus, however, is not, like the chondriome, a well defined system. It is not visible in living material nor can it be revealed by microdissection (KITE and CHAMBERS). It can be demonstrated only by methods which, as we shall see, are in no wise specific. Morphologically, it is so imperfectly character- ized that BOWEN said “The Golgi apparatus is above all a sub- stance, a cellular apparatus, whose modelling has only a secondary interest.” Such a definition could only be acceptable if the Golgi apparatus were composed of a well-defined substance. Now its chemical nature is completely unknown and it does not even have definite characteristics of fixation and staining. It is not certain, Guilliermond - Atkinson — 192 — Cytoplasm moreover, that the images obtained by osmic acid correspond always to those produced by silver methods and there has been no proof whatever that the dictyosomes can really be homologized with the Golgi network. One can not, therefore, suppress the thought that under this name have been grouped very diverse formations. One is forced to admit that there is reason to distin- Fic. 129. — Barley root. 1, meristem vitally stained with neutral red; vacuolar system deeply stained, more or less reticulate. 2-11 Bensley’s method; 2-9, meristem; vacuolar system has the appearance of Holmgren’s apparatus. 10, 11, differentiating cells; Holmgren’s apparatus transformed into large vacuoles. guish between formations which are perhaps entirely different: the Golgi network of GOLGI and CAJAL obtained by means of silver methods and the dictyosomes later brought out by osmic methods. HOLMGREN, on the other hand, by the use of special methods, described in certain animal cells a network of hyalin and colorless canaliculi appearing as clearly as if punched out of the dense and stained cytoplasm. This apparatus, called the canaliculi of Holm- gren or fluid canaliculi or trophospongium, has been found in a great number of animal cells. HOLMGREN at first considered it as Chapter XVII — 193 — Golgi Apparatus a system of intracellular canaliculi opening freely to the exterior and serving for the entrance of nutritive juices as well as for the excretion of metabolic products from the cell. Nevertheless, after further research, this worker was led to deny all communication of these canaliculi with any part of the pericellular space and considered them to be formations completely separated from the lymphatic circulation and probably comparable to the network of Golgi. It is certain, however, that, of the formations described by HOLMGREN, some correspond to canaliculi communicating with the exterior as this investigator at first thought. But in these pages we reserve this term exclusively for the formations described by CAJAL under the name which has been currently used since then Fic. 180. — Pea root. Vacuolar system. Method of da Fano. 1-4, network strongly impregnated with silver. 5, fusion to uni- formly stained vacuoles, which later (6) appear like dictyosomes, then (7) become larger with silver-impregnated precipitates. by numerous cytologists, viz. Golgi-Holmgren apparatus. These canaliculi do not seem to us to be comparable to the structures now called dictyosomes. Possible relationships of the vacuolar system with the apparatus of Golgi and of Holmgren:- The facts concerning the vacuolar system in plant cells have given the question a new orientation. Well before the origin of vacuoles and their property of accu- mulating vital dyes were known, BENSLEY (1910) had succeeded in bringing out the canaliculi of Holmgren in the cells of the meri- stem of the root of Alliwm Cepa and had proved that they are transformed into vacuoles in the course of cellular differentiation. Having been struck by the resemblance of the young filamentous and reticular vacuoles in embryonic cells to the formations known as the Golgi apparatus in animal cells, we had formulated, in our Guilliermond - Atkinson — 194 — Cytoplasm very earliest investigations, the hypothesis that the Golgi apparatus might well correspond to a vacuolar system analogous to that of plant cells. Moreover, we had shown that, by means of Regaud’s method, the young filamentous and reticulate vacuoles may be seen as a network of colorless canaliculi within the grey-tinted cyto- plasm and present absolutely the aspect of the canaliculi of Holm- gren. That led us to think that the apparatus of Golgi and that of Holmgren might perhaps be one and the same formation, corre- sponding to certain phases of the vacuolar system analogous to that of plant cells. A little later, with MANGENOT, we tried to verify this hypothesis in the meristem cells of the barley root (Fig. 129) which, as we have seen, contain small filamentous vacuoles, very characteristic and easy to bring out by vital staining with neutral red. Treating these cells by the method recommended by BENSLEY for detection of the canaliculi of Holmgren, we succeeded in obtaining images very comparable to those of an apparatus of Holmgren formed of colorless canaliculi, appearing as if punched out against the grey cytoplasmic background. These, in differentiat- ing cells, swell and coalesce and, in the mature cell, are transformed into large vacuoles. Moreover, in treating the same root with the sil- ver impregnation methods which a ar eee ub eyed Loe nne On a sui atioale ida i Fariots wiethod #Alearenelm LICUlaly wapparacus, wwe, Obtained sam See rete oe ai some aP- the cells of the meristem a network like that of Golgi, corresponding ex- actly to the apparatus of Holmgren, obtained by the methods of Bensley, and to the filamentous and reticulate phases of the vacu- olar system, as they appear after vital staining with neutral red. These observations which seemed to verify our hypothesis, were later confirmed in animal cytology by the work of A. CortT1, then of PARAT and of his collaborators. CoORTI proved, in fact, that the apparatus of Golgi and the apparatus of Holmgren constitute a single formation, corresponding to a system of lacunae, which the author called lacwome and which he compares to the vacuolar system of plant cells. Furthermore, in entirely independent re- search, PARAT and his collaborators showed that the Golgi appa- ratus and that of Holmgren correspond to a single formation— positive and negative images, respectively, obtained by different methods and comparable to a vacuolar system like that in plant cells and capable of being stained vitally by neutral red. The re- search of PARAT and his collaborators have, however, proved that many of the formations assigned to the Golgi apparatus are images of the somewhat altered chondriome, or are a superposed chondri- ome and vacuolar system, or else are differentiated chondriosomes. Chapter XVII — 195 — . Golgi Apparatus As a result of these investigations, we extended our research to cover a large number of plants, belonging to the most varied groups, which confirmed and completed our earlier findings. The study of the vacuolar system, notably in the seedling of the pea, gave us particularly suggestive results. In the meristem cells of the root (Fig. 130), there is obtained by silver methods an entirely characteristic reticulate apparatus and it is observed that during Fic. 132. — Saprolegnia. Vacuolar system. 1-8, vitally stained with neutral red; reticulate, tending to fuse into a vacuolar canal. 4-6, Bensley’s method; system appears as canaliculi of Holmgren. 7, 8, da Fano’s silver impregnation method; system impregnated with silver, resembles Golgi’s network. 9, 10, Kolatchev’s osmic impregnation method; system strongly blackened. cellular differentiation this network swells and is transformed into small spherical vacuoles, each containing a precipitate heavily blackened by the silver and often arranged as a crescent on the bor- der of the vacuoles, thus appearing like the Golgi elements or dic- tyosomes. Finally, in differentiated regions, the vacuoles are seen to swell and run together to form large vacuoles containing a more or less large number of corpuscles, blackened by a deposit of metal- lic silver. There are, therefore, obtained with silver methods, images which can be perfectly superimposed on those furnished by vital staining with neutral red. By means of these methods of impregnation, metallic silver is deposited on the filamentous and reticulate elements of the vacu- Guilliermond - Atkinson — 196 — Cytoplasm olar system, which gives them a homogeneous black coloration, and in the vacuoles derived by hydration of these elements, the same methods bring about the precipitation of the colloidal contents in the form of corpuscles on which the metallic silver is deposited. The silver methods also permitted us to bring out aleurone grains during their transformation into vacuoles which swell, at first assuming filamentous forms having a tendency to anastomose, Fic. 133. — 1-6, Saccharomyces ellipsoideus. 17, Ashbya gossypii. 8-10, Saccharomyces pastorianus. 1-10, da Fano’s method; Metachromatin bodies, produced by flocculation from the colloidal solution in the vacuole, strongly blackened by a deposit of metallic silver on their surfaces. 11-18, Geotrichum lactis; capricious impregnation with Kolatchev’s method. 11, 18, metachromatin bodies within vacuole. 12, 16, 17, metachromatin in vacuoles (v) ; chondriosomes (c) are swollen and vesiculated. 14, 15, chondriosomes are swollen and vesiculated. 18, chondriosomes are well preserved. 19-22, Saccharomyces pastorianus; Kolatchev’s method; metachromatin bodies blackened by osmium. then later appearing as large spherical vacuoles containing nu- merous corpuscles which take up the silver. Bensley’s method, applied to the meristem cells of the root, brings out canaliculi entirely reminiscent of those of Holmgren. In differentiated regions of the root, these are gradually trans- formed into large vacuoles. Results just as diagrammatic were obtained in Saprolegnia. In the extremities of the filaments, Bensley’s method produced reticulate figures of the vacuolar system in the form of the appa- ratus of Holmgren and silver methods gave them the appearance characteristic of the reticulate apparatus of Golgi. Farther away Chapter XVII — 197 — _ Golgi Apparatus from the tip, they become transformed into a vacuolar canal con- taining numerous corpuscles which take up the silver (Fig. 182). Silver methods gave us similar results in other fungi (Endo- myces Magnusiti, yeasts) whose vacuoles are not filamentous but begin as small spherical elements filled with metachromatin. The silver methods make these elements appear as small vacuoles, each containing one silver-staining body, whereas in the larger vacuoles, arising from the coalescence of the smaller elements, these methods bring about the precipitation of numerous silver- staining corpuscles which correspond to metachromatic corpuscles. The images obtained are here again similar at all points to those & Fic. 134. — Pea. Various cells from the same _ root. Kolatchev’s method. 1, central cylinder; blackened cytoplasm appears reticulate. 2, cortical parenchyma; chondriosomes fairly well preserved, heavily blackened by the osmium, a few are vesiculate. 3, adjacent cells; at left, only the chondriosomes and plastids are blackened and strongly vesiculated; at right, only vacuolar precipitates are impregnated. 4, Differentiated cells above the meristem; only vacuolar precipitates are blackened. produced by neutral red. Silver impregnations also bring out the metachromatic corpuscles of some algae (Tribonema) and bacteria. Research carried out by means of silver methods, regularly controlled by vital observation in cells very favorable for this, es- tablish the fact, therefore, that the vacuoles, whatever their con- tents, have the property of reducing silver nitrate and of bringing about in their interior the production of particles of metallic silver, giving images analogous to those produced by vital dyes. On the other hand, silver methods, although bringing out the vacuoles very clearly, are not specific for them and sometimes the chondri- ome (chondriosomes and plastids) may be impregnated also and even the chromosomes (giving in that case superb mitotic fig- ures). There is no alteration of the chondriosomes and plastids, however, so that it is not difficult to recognize them when they are blackened by the silver. Guilliermond - Atkinson — 198 — Cytoplasm Relationships between the Golgi apparatus and the chondrio- somes and plastids:- The above results have been a subject of much debate in animal cytology, and various authors, among others BOWEN and GATENBY, DUBOSCQ and GRASSE have not been able to confirm the observations of A. CORTI and PARAT. It must be noticed that all these authors abandoned silver methods and used only osmic methods. There was therefore the question as to whether osmic methods produce the same results as the silver methods. Work which we have done using these methods on plant cells has shown us that the osmic methods are much less specific than the silver methods. If the impregnations have lasted only a week, there is a blackening of the vacuolar system only when it encloses Fic. 135. — Vicia Faba. Cells in the seed before maturation. Silver impregnation of da Fano. 1-6, parenchymatous cells of the cotyledon; 1-4, Golgi net- work. 5-6, deprived of oxygen, the Golgi network breaks up into vacuoles containing silver-impregnated precipitates. 7, epidermal cells of the integument of the seed; Golgi network. (After SANCHEZ). tannins, which instantaneously reduce osmic acid. Otherwise, it is the chondriome which is impregnated and it may be well pre- served but is often vesiculated. If the impregnation is prolonged to two weeks, there is a more profound alteration of the plastids and chondriosomes, which become large vesicles and sometimes even anastomose into a fine network, much like the network of Golgi, but now the vacuolar system may also be impregnated. These impregnations are then very irregular and it is not rare to ob- serve, side by side in the same section, cells in which the chondri- ome alone is blackened, sometimes well-preserved, sometimes strongly vesiculated, and other cells in which the chondriome and the vacuolar system are both blackened, still other cells in which only the vacuolar system is affected (Fig. 183, 11-18). These re- sults demonstrate therefore that osmic methods constitute a dangerous technique, a constant source of gravest error. Without having the pretension of entering here into a field which is not our own, we will confine ourselves to saying that Chapter XVII — 199 — Golgi Apparatus what we have observed in plant cells, under as accurate condi- tions as possible, leads us to think that the so-called Golgi forma- tions observed in animal cytology have not been well characterized. Indeed, they have been observed most often by the use of methods far from specific without recourse to other techniques and with- out confirmation from living material. Our research on plant cells would seem to indicate that the Golgi apparatus (apparatus of Golgi, Holmgren, Cajal) 7.e., the network, might often correspond to a vacuolar system like that in plant cells, whereas most of the dictyosomes obtained by osmic methods must be put into the cate- gory of vesiculated chondriosomes. (An opinion recently formu- lated by FILHOL is that some dictyosomes correspond to differenti- ated chondriosomes, doubtless destined to play a role in the secretions of the cell). In reading the reports of some cytologists, one has the impression that they are re-discovering the chondriome under the name of Golgi apparatus. The so-called Golgi appa- ratus in plant cells:- It is clearly demonstrated, at any rate, that the Golgi apparatus does not exist in plant cells. If we pass in review the vari- ous work on cells carried out with the idea of finding a Golgi apparatus, we see that ne ne oe eet cot all that has been described as Osmic impregnation. (After Miss Scorr). such corresponds either to the vacuolar system or to the chondriome (chondriosomes and plas- tids). Thus SANCHEZ, LUELMO, GONCALVES DA CUNHA, ZIRKLE by silver methods and Miss F. M. Scott by osmic methods, obtained superb Golgi networks which correspond to the vacuolar system in its filamentous and reticulate phases and which, more- over, are considered as such by these authors. DREW, on the contrary, figured in the root of Alliwm Cepa under the name of Golgi apparatus, elements obtained by silver methods which it is easy to classify with the chondriome (chondriosomes and plastids) and which correspond exactly to that which is ob- tained by mitochondrial methods. On the other hand, BOWEN, then BRONTE GATENBY and his collaborators, described in a variety of plant cells, treated with osmic methods, certain elements in the form of rings. These they consider to be distinct both from the _ chondriosomes and from the vacuoles. These authors give to these formations the name of osmiophilic platelets (Fig. 138) and con- sider them to be Golgi apparatus. BEAMS and KING claimed that they had demonstrated the existence of the osmiophilic platelets of BOWEN and GATENBY by the following process: they subject the tip of the root of Vicia Faba to ultra-centrifuging by means of the ap- paratus of Beams, then, immediately after this process, they im- Guilliermond - Atkinson — 200 — Cytoplasm pregnate it with osmium. After this treatment, the cells show at one of the poles corresponding to the direction of centrifugal force, an accumulation of chondriosomes and starch-bearing plastids. At the opposite pole are accumulated in order, the lipides, the vacuolar sap and the osmiophilic platelets which consequently seem to be lighter. But the figures given by the authors are not very con- vincing and it seems that the substance which was affected by the centrifugal force corresponds only to starch-containing plas- tids and that the so-called osmiophilic platelets represent vesicu- lated chondriosomes. It has been seen that with osmic methods KIYOHARA obtained analogous figures (vesicles) which he interprets as normal forms of the plas- tids. This author, starting with a false premise in the form of a defective ob- servation of living material in which he saw only vesiculated chondriosomes, concluded that mitochondrial methods alter the chondriosomes, whereas the Golgi methods preserve them in their vesiculaneaorm (Cf... p: 91); Our work, however, has furnished proof that these so-called osmiophilic plate- lets are none other than vesiculated chondriosomes and plastids. Finally, WEIER, not being able to find in plants a Golgi apparatus independent of formations already known, and having _ succeeded, in Polytrichum commune, figures of the Geet armurauue im in impregnating large plastids by Golgi the root. 1, 2, meristem; only methods, came to the conclusion that chondriosomes differentiated. 3, 4, : - ° differentiating cells with Golesi ap. the Golgi apparatus is represented in aoe Cees plant cells by the plastids (Cf. pp. 91, reality plastids. 92). This opinion is however, inad- missible, for the reason that the plas- tids are a variety of chondriosomal elements, belonging to chloro- phyll-containing cells, and in direct relation with photosynthesis which characterizes these cells, and they are not found in fungi. Besides, the ordinary chondriosomes are impregnated by Golgi methods just as well as the plastids. It must be added that GICKLHORN, studying large spherical bodies which are found localized in the vacuoles of epidermal cells of Iris, noticed that under the influence of osmic acid they blacken and become a spongy structure, then are transformed into a net- work which looks like Golgi material. This worker thinks, there- fore, that it is to structures of this nature that the Golgi formations must be attributed. The work of one of our students, REILHES seems to have demonstrated that these bodies are composed of a phytosterol. LIEBALDT has recently supported this same opinion. It is possible that analogous formations have been described in Chapter XVII — 201 — _ Golgi Apparatus animal cells as the Golgi apparatus, but in exceptional cases only. Therefore the opinion of GICKLHORN is not a solid basis for generalization. It is therefore demonstrated that all the formations described as Golgi apparatus in plant cells are dissimilar elements, belonging either to the vacuolar system or to the chondriome (chondriosomes and plastids) and that consequently there is no Golgi apparatus in plants. Other cytoplasmic formations:- In the cytoplasm of many cells and espe- cially in that of the Protista, granules of chromatin have been reported which were supposed to have orig- inated as emissions from the nuclei. For several years, great importance was attached to these’ granules called chromidia. In reality the chro- / midia, as a group, have never been | characterized histochemically. It has been proved, on the contrary, that they represent dissimilar elements which can be stained with iron haematoxylin and which correspond, either to chon- Fic. 138. — Vicia Faba. Osmiophilic driosomes altered by the fixatives, or eh ape tere wae beat ie a to vacuolar precipitates. Sreeatiant i ear PEC a There is reason, also, to mention here the formations described for the first time in animal cells by the BOUIN brothers, and by GARNIER, then by PRENANT, as ergastoplasm and later found as well, in some plant cells. These are in reality rather undefined formations and appear as superposed lamellae, or as spiral filaments, which have a strong affinity for nuclear stains. They have been observed in glandular cells and an important réle in secretory phenomena has been attributed to them. All cytologists are in agreement today in recognizing that ergastoplasmic formations have no separate existence. They are most often simply artifacts — alteration fig- ures of the chondriosomes, plastids, vacuolar colloids, or para- plasmic inclusions, produced by fixatives. Perhaps they corre- spond also to the differences in chemical composition of certain regions of the cytoplasm. Chapter XVIII LIPIDE GRANULES, MICROSOMES AND OTHER METABOLIC PRODUCTS Observation of living material in most, if not all, plants shows that there exist in the cytoplasm in addition to the elements dis- cussed above, certain small granules, spherical in shape, which we call lipide granules. These granules have often been confused with mitochondria. In living cells observed in direct illumination, they are the most clearly visible of all the cytoplasmic inclusions because of their high refractivity. They are still more distinct with lateral illumination, under which circumstances they are usually the only visible elements of the cytoplasm. They appear strongly lighted against the black background (the cytoplasm) on which they can very distinctly be seen to move. With the Zeiss micropolychro- mar they can very clearly be seen to have a color different from that of the cytoplasm and much more accentuated than that of the chondriosomes. The lipide granules are distinguished very sharply from the mitochondria by their high refractivity, by their very rapid displacements in the cytoplasmic currents and by the variability of their size. The smallest have a size in- ferior to that of mitochondria and the largest may greatly exceed it. These Fic. 189. — Tulip. Epidermal granules are distinguished from the color lest under we wine ianitochondriayeaiso by their @smium= seope. Only the lipide granules (G) carried about in the _cyto- reducing properties. plasmie trabeculae are _ visible. O, fatty body. In some cases they may agglomerate in mulberry-shaped masses or in little chains and fuse to become huge globules. It seems that most of the bodies described as oleoplasts or elaioplasts correspond to ag- glomerations of granules of this nature formed under influences as yet unknown. In each epidermal cell of the leaf of Vanilla planifolia, WAKKER first called attention to a voluminous body which he called an elaioplast. It is generally larger than the nu- cleus and is localized in the cytoplasm and in the neighborhood of the nucleus. This body is irregular in shape and composed of very numerous small lipide droplets which according to WAKKER are enclosed in a protein film. These bodies, which were thought to be plastids elaborating lipides, have also been found in the epidermis and other tissues of many plants, especially of the Monocotyledons (Fig. 142). They cannot however be considered as plastids. In the epidermis of tulip, each cell encloses a large fatty body which Chapter XVIII — 203 — . Lipide Granules arises from the fusion of numerous small globules and appears to correspond to the elaioplasts of WAKKER. On the other hand, the bodies comparable to the elaioplasts which are encountered in the hepatics have a constitution much more complex. They are much more difficult to interpret and are still little known. The quantity of lipide granules varies a great deal from one cell to another, according to the state of cellular development. There are cells in which they are very rare but usually they are very numerous. These granules give lipide reactions. As well as reducing osmic acid (Fig. 141), they stain with Sudan, scarlet R, tincture of Alkanna and indophenol blue. They seem to have a vari- able chemical constitution and the microchemical characteristics either of simple lipides or those of compound lipides, according to Fic. 140. — Elodea canadensis. 1, Cell from the leaf fixed with Meves’ method, stained with acid fuchsin, which colors the chloroplasts (P) and chondriosomes (M) red; lipide granules (GL) colored dark brown by the osmic acid. 2, detail; some chloroplasts are dividing. the type examined. In paraffin sections, Regaud’s method does not preserve them and the osmic acid used in the method of Meves turns them brown. Sometimes they stain in frozen sections with Dietrich’s and Regaud’s methods. (Fig. 143). These granules, which correspond to the microsomes of other writers, represent simple products of metabolism and perhaps in many cases they are also the product of a transitory, or final breaking down, of the lipides from the lipo-protein compounds (phenomenon of lipophanerosis). AMIN has recently shown that their quantity increases, especially when the cells are submitted to high temperatures. These granules attracted the attention of P. A. DANGEARD, who in his observations of living plant cells described them suc- cessively as microsomes, spherosomes, cytosomes and liposomes. This investigator made of them a permanent system of the cell which he designated first as spherome, then as cytome and then Guilliermond - Atkinson — 204 — Cytoplasm as ergastome. The terms cytosomes and cytome were first created by P. A. DANGEARD to replace those of spherosomes and spherome, which were applied to the lipide granules in question here. This writer, perceiving later that, as well as these granules, there also exist chondriosomes, whose reality up to then he had refused to admit, substituted the above terms for those of chondrio- somes and chondriome and made the distinction, from that time on, between the cytome corresponding to the chondriome and the ergas- tome which includes all the lipide granules of a cell and for which he reserved the term lipo- some. Here is the description which he gives of them (1919): “The spherome is composed of all the microsomes. The microsomes are small, very refractive sphero- somes of a fatty appearance which blacken more or less with osmic acid.” P. A. DANGEARD, contrary to our judgment, maintained that it is the microsomes, with the ele- ments of the vacuome, which rep- resent what the cytologists had for a long time been calling chondriosomes. At the present time, DANGEARD seems to have re- nounced this opinion. KOZLOW- SKI also confused these same granules with the chondriosomes. After observing only living mate- rial, he maintained that plastids arise by simple agglomeration of these granules. Reserve lipides which are Fic. 141. — Endomyces Magnusii. A-c, found in many cells appear, in the Raseda| method; DL Mew! mibel: ovtoplasm, like the microsomes drioconts; Gr, lipide granules, brown which we have just been discuss- eet LMC to dae aoc) ane pub-are present. in miteRyerear er quantity. In endosperm cells of the castor bean for example, at the period immediately preceding the maturation of the seed, there are seen to form abruptly in the cytoplasm, numerous small granules comparable to those which normally exist in every cell and which present the same histochemical characteristics. These finally fill the cytoplasm com- pletely and then fuse into large lipide globules. In the cytoplasm of some filaments of Saprolegnia, especially in the extremities of the filaments which will form the zoosporangia, numerous granules are also seen to appear which are comparable to the small microsomes encountered in the other filaments. These fuse later into larger globules which accumulate in the zoospores and serve as reserves. Chapter XVIII — 205 — Lipide Granules Fatty degeneration:- Fatty degeneration which is exhibited by many cells at the end of their development, especially in the fungi, is also brought about by the increasingly large production of granules, similar to the microsomes, which run together into large globules. This process seems to correspond to a lipophanero- sis 7.e., a breaking down of the lipides, of the lipoprotein com- pounds which comprise the cytoplasm. Essential oils and resins likewise appear as small globules in the cytoplasm and their very refractive appearance and their histo- chemical reactions greatly resemble those of the lipide granules, from which they are only with difficulty distinguished. Other metabolic products:- The cytoplasm of plant cells may contain a great number of other substances arising from cellular metabolism. These substances, however, are not constant like those mentioned above and are de- termined by certain physiological states of the cell. Some of these products are inclusions in the cytoplasm. Among them must be men- tioned that which is called florid- ean starch or starch of the Rhodo- phyceae. It differs essentially from ordinary starch by the fact that it is not formed in plastids but appears in the cytoplasm. This starch becomes visible we the Fic. 142. — Philodendron. Secretion cytoplasm as granules of variable canal of an aerial root. C, lumen of the dimensions, some extremely small, Sa, Gr. serine, cls: By fats, a fraction of a by others of a diam- driosomes; N, nucleus; 12) plastids. “Meves’ eter which may exceed 20-30n. Beare acid fuchsin. (After Miss These variously shaped granules (ovoid, plano-convex, bi-concave, discs etc.) are doubly refractive, if they are large enough, and stain mahogany-brown to violet-red with iodine. The chemical nature of this “floridean starch’ is not yet fully determined. It seems however that various substances have been described under this name. Some of them, appearing as rather large granules, correspond to a special variety of starch (VAN TIEGHEM, KYLIN). Others exist in the cytoplasm as a clus- ter of minute granules capable even of emigrating into the vacu- oles. Among these, some seem to be composed of a substance related to the glycogens (ERRERA, COLIN); others are products (alkaloids) still imperfectly known (GUEGUEN). In the cytoplasm of the Euglenas are likewise found granules of paramylum characteristic of the Phytoflagellates. These gran- ules appear in the cytoplasm as discs, prisms, rods, stars and so forth, showing alternately dark and light concentric layers like the starch grains. They do not stain with iodine but have been compared to starch. Mention must also be made of para-glycogen, Guilliermond - Atkinson — 206 — Cytoplasm a substance related to glycogen which is sometimes encountered in bacteria and Phytoflagellates as granules which stain brown with iodine. Fic. 143. — Iris germanica. Chondrio- somes (M) and plastids (P) in the form of chondrioconts in epidermal cells of the leaf. 1, living material. 2, Meves’ method with acid fuchsin. 38, Regaud’s method. Lipide granules (Gl) very refractive in 1, blackened by osmium in 2, not preserved in 3. Protein crystalloids are sometimes encountered also in the cyto- plasm. In the tubers of white potatoes, for example, a crystalloid of protein is found in the cytoplasm of most of the cells. It is rather large and cubical in shape and is deeply stained by iron haematoxylin. Similar variously-shaped crystalloids (spindle- shaped, cubical etc.) often exist in the plectenchyma of the carpo- spore of the Agaricaceae and in the mycelium of Spermophthora gossypii, as well as in the Mucors, in which they have been de- scribed under the name of crystalloids of mucorine (VAN TIEGHEM). It is suitable also to mention a voluminous spherical inclusion which is present in the cytoplasm of certain special cells of vari- ous Rhodophyceae. This is called an iodine reservoir (Fr. ioduque), is very refractive and occupies the major part of the cell. Its nature is still unknown but it seems to contain iodine which is free or in the state of an unstable compound released when the reservoirs are broken. Attention has been called in the same algae to cells called bro- mine reservoirs (Fr. bromuques), which rather analogously contain bromine. Let us mention furthermore that there exist in the proto- Chapter XVIII — 207 — _ Lipide Granules plasm of the Thiobacteriales, very refractive granules which appear to be sulphur. Many metabolic products are diffuse in the cytoplasm and cannot be detected, but others can be brought out by microchemical re- agents. Among these is glycogen which is so widely distributed in fungi (Fig. 145) and which can be detected by the iodine-potassium >. —— ms < One ~S _ Sy =~ \< es e —— “ee Fic. 144 (left). — Spermophthora gossypii. 1, 2, Filaments with crystals of protein. 3, Detail of crystals. Fic. 145 (right). — Endomyces Magnusii. Living oidia and tip of a filament treated with iodine-iodide. Small acacia-brown areas of glycogen (GL) are sometimes near the nuclei (N) but are not related to the chondriosomes (C). L, lipide granules. iodide reagent, giving a mahogany-brown color. This product ap- pears directly in the cytoplasm, generally around the vacuoles, or the nuclei, in small areas which later run together and fill the entire cytoplasm. At times when it accumulates in too great quantity in the cytoplasm, the glycogen may even spread out into the vacuoles, where it often is precipitated as small slightly refractive globules. Lastly there are the amyloids, substances colored blue by iodine which are diffused in the cytoplasm of some bacteria. Chapter XIX CYTOPLASMIC ALTERATIONS It is impossible to discuss fully the vast and, moreover, incom- pletely known, question of cytoplasmic alterations. We shall con- sider very briefly: (1) the disturbances which accompany the nat- ural death of cells; (2) the morphological alterations which vari- ous physical agents provoke in cells; (3) the reactions shown by the cytoplasm and its morphological constituents under the influ- ence of parasites. Alterations produced in dying cells:- When living cells of any tissue are examined, even in Ringer’s solution, it is always seen that they more or less quickly manifest those signs of alteration which, sooner or later, end in their death. Such alterations seem inevitable. They are explained by the artificiality of the medium in which the cells are placed, by the pressure of the cover glass, by the lack of air, and by the too intense lighting which is, never- theless, necessary for observation. This is what makes the study of living material so difficult. One manages to retard these altera- tions by examining leaves or bracts which are protected by their cuticle but are so thin as to be transparent. The cells are altered, however, in the region where the organ has been severed from the plant and the alteration is then transmitted, more or less rap- idly, to all the cells. The complicating factor is that one can not know exactly when the alteration of the cell begins. By the move- ments of the cytoplasm, it is possible to determine whether a cell is living. As long as cytoplasmic movements continue, the cell is living and does not present any important alteration. Neverthe- less, cytoplasmic movements do not prove that the cell has not already manifested alterations, for, in general, all wounding causes marked acceleration of the cytoplasmic currents and there are cells in which these movements are to be induced only by lesion. The alteration of the cytoplasm is manifested by a thickening of the chondriosomes and of the plastids, soon afterward accompanied by their transformation into vesicles and later, by interruption of cytoplasmic circulation and its replacement by Brownian move- ment. It is difficult to know where the first alterations of the cell begin. We possess relatively accurate data as to the moment at which death of the cell occurs. A first sign of death in a cell is shown when the vacuole, stained with neutral red (the only ele- ment stained in the living cell), abruptly loses its color and the dye is taken up by the nucleus and cytoplasm. This is a universal phenomenon which seems to be brought about by a modification of the perivacuolar membrane, permitting diffusion of the dye accu- mulated in the vacuole. This phenomenon may be compared to Chapter XIX — 209 — Cytoplasmic Alterations that of nuclear autochromatism reported in certain cells whose vacuoles contain anthocyanin (P. A. DANGEARD, MOREAU, GUILLIER- MOND). These writers observed that at the moment of death of these cells, the vacuoles lose their color and the pigment becomes localized in the cytoplasm and especially in the nucleus. This phe- nomenon is observed particularly in the final stages of plasmolysis. BECQUEREL advocates the use of a mixture of neutral red and methylene blue. Neutral red stains the vacuoles of living cells and the less penetrating methylene blue colors only the cytoplasm of dead cells. The examination of the cells with the ultramicro- scope, as has been seen, makes it easily possible to determine the moment when the cell dies. As soon as death occurs, 2.e., the coagulation of the cytoplasm, one witnesses a series of phenomena, designated as autolysis, which consist of an autodigestion of the protoplasm under the action of intracellular proteo- lytic enzymes. The enzymes, whose ac- tion is no longer inhibited, induce modi- fications in the cell, characteristic of degeneration, 7.e., cellular necrobiosis. This consists essentially of an autoly- Sis, 2.e., of a digestion, starting in the interior of the cell itself and instigated by the enzymes. These enzymes, al- though present during life, do not act on living material because of some still unknown mechanism. i The; modifications. generally }pro-\ cae er sel aad ccs woe duced in the cell take the form of more _ living and plasmolyzed. 2, dead, and more marked vesiculation of the Pisstids vesiculated. 4 myelin ne chondriosomes and plastids, bringing A seein sige Se eens about the alveolar structure described anthocyanin. ” by BUTSCHLI. The mitochondrial vesi- cles, which are often enormous, sometimes finally burst. Their wall then breaks up into an infinity of small refractive granules which are scattered about in the cell. In its turn, the vacuole ceases to exist when the perivacuolar membrane is destroyed. This leads to a contraction of the cyto- plasm which becomes detached from the cellulose wall as if plas- molyzed, and appears as a granular-aveolar coagulum immersed in the liquid of the cell cavity. The degeneration of the epidermal cells of Iris during the fad- ° ing of the flower, involves curious phenomena associated with the chondriosomes and plastids. The plastids (leucoplasts, chloro- plasts and chromoplasts) fill with an infinity of small granules which reduce osmium. These swell and take on the aspect of enormous vesicles. Then the contour of the vesicles gradually loses its distinctness and finally becomes invisible. There remains of the vesicle, therefore, only a mass of lipide globules which have Guilliermond - Atkinson — 210 — Cytoplasm a tendency to fuse into large globules, while the substratum of the plastid is reduced to a great number of small granules which soon disappear. At the same time, the chondriosomes swell and become vesicular, as do the lipide granules. Eventually there persist in the cytoplasm only large lipide globules, produced by the disorganization of the plastids. In the chlorophyll-containing tissue, these globules appear green and in cells enclosing xanthophyll they appear yellow, the pigments having dissolved in the lipides during degenera- - tion. These modifications seem to be due to a breaking down of the lipides in the lipoprotein complex comprising the plastids (lipophanero- sis). It is apparently phenomena of this same order, produced in the cytoplasm itself, which bring about the fatty degeneration so often found in animal cells and in fungi. In other flowers (Gladiolus, tulip, Clivia), only the vesiculation of the plastids and chondriosomes is observed. The vesicles burst and disintegrate into small refractive granules which are scattered in the cytoplasm and do not reduce osmium. There is no fatty degeneration. TS germanica. Epidermal cell of a leaf plas- molyzed in a 5% Fic. 147. Alterations produced by various physical agents:- Radiations of short wave length, ultra- violet rays and X rays, as well as a particles NaCl solution with 5 : : . Peiee emitted by radioactive bodies, destroy living cells, neutral red added. Cell contents form a large ball in the cen- ter of the cell con- nected to the wall by slender threads which are enlarged here and there. Cv, bodies pre- cipitated from the colloidal contents of the vacuole by the dye; V, vacuole stained with neutral red; chondrio- somes; M, mitochon- dria; N, nucleus; F'm, structures resembling myelin figures pro- duced from the cyto- plasm. but this destruction, especially by X ray and a particles is not instantaneous. It is, on the con- trary, preceded by very complex phenomena con- cerning which we have, as yet, very little pre- cise knowledge. The action of ultraviolet rays and X rays on the morphological constituents of the cytoplasm is recorded largely in the work of NADSON and his students RoCHLIN and STERN. These investi- gations have used as subjects, various yeasts and the epidermal cells of Alliwm Cepa. These work- ers report in all cases that radiation produced first an excitation of cytoplasmic activity. The currents become more rapid, the cytoplasm forms amoeboid pro- longations in the direction of the vacuole, causing it constantly to be deformed. In the second phase, the vacuoles return to their previous shape. At the same time, lipide droplets form in large numbers in the cytoplasm, in the chondriosomes and in the plas- tids. Finally, plasmolysis occurs and the cells soon die. Radium and its salts produce similar effects, as shown by NAD- SON and his followers, as well as by MiLovipov. These workers have also studied the action of the radioactive salts on the chondri- Chapter XIX — 211 — Cytoplasmic Alterations ome. Now this action seems to be very slight. The chondriome, so sensitive to most exterior agents, is only very gradually injured. The lesions consist of a vesiculization of the chondriosomes and of the plastids, accompanied by their fatty degeneration (lipophanero- sis). In short, the effect of radium salts is to accelerate the de- generation which would normally be produced much later on. Plasmolysis leads to interesting modifications of the cell. My- elin figures appear in the cytoplasm (Figs. 146-148) in the form of pediculate buds on the border of the vacuole, which are capable of being detached and of emigrating into the vacuoles. These fig- ures do not seem to be doubly refractive. They may perhaps be attributed to a sort of unmasking of the cytoplasmic lipides. Dur- ing plasmolysis, there is also often observed a fragmentation of the vacuole into small filamentous or reticulate elements. The chondriosomes and plastids are not modified as long as the cell remains living, but they become vesiculate as soon as the cell dies (GUILLIERMOND, KUSTER). Let us Len “. add that plasmolysis of living but injured cells MZ produces the curious phenomenon described by KUSTER as plasmoschism, in which the permeabil- os ity of the ectoplasmic membrane is so greatly increased that it no longer inhibits penetra- tion. The perivacuolar membrane, however, re- x tains its semi-permeability and so the vacuole con- , tracts but the irremediably injured cytoplasm is : stretched, taking very irregular shapes and breaking apart. Bs The study of the death of cells by freezing F gave rise to the work of MATRUCHOT and MOLLI- ia qae

181, 1st eas Wetzel, K., 104, 240 Wieler, A., 52, 538, 243 Wilson, Ed. B., 29, 82, 85, 243 Wurmeser, R., 38, 223, 240 131, YAMAHA, G., 243 ZIRKLE, C., 51, 52, 140, 154, 179, 199, 224, 2438 Zopf, F. W., 8 INDEX of PLANT and ANIMAL NAMES ACETABULARIA, 42 Achlya, 59, 61, 62 Achyranthes, 46 Adiantum, 103, 104 Aethalium, 240 Agaricus, 62, 92, 240 Alaria, 177 Allium, 7, 17, 81, 91, 99, 100, 101, 102, 108, SBS AY), bres IB aIGR I Ps Palle parle 225, 2381, 239, 241 Allomyces, 59, 60, 215, 233 Aloé, 169 Anagallis, 152, 236 Anchusa, 177 Anthoceros, 44, 86, 87, 107, 110, 112, 218, 280, 241), (243 Antithamnion, 248 Arum, 79 Ascoidea, 61, 63 Ashbya, 196 Asparagus, 71, 79, 80 Asphodelus, 101 Asplenium, 105 Athyrium, 97 Azolla, 129 Azotobacter, 224 BACILLUS, 226 Badhamia, 9, 15 Basidiobolus, 224 Begonia, 169 Bellis, 52 Berberis, 181 Blepharospora, 227 Bonnemaisonia, 177 Brefeldia, 235 Bryopsis, 159, 235 CALANTHE, 52 Camellia, 183 Canna, 46, 182, 183 Catharinaea, 243 Caulerpa, 42 Cephalocereus, 49 Ceratium, 158 Cerinthe, 45, 47 Chantransia, 43 Chara, 11, 110, 237, 241 Chelidonium, 11 Chlamydomonas, 41 Chlorophytum, 88 Chondrioderma, 8, 128 Cladophora, 42, 159, 185 Clivia, 79, 80, 210 Closterium, 48, 44, 52, 131, 167, 234, 235 Conopholis, 238 Coprinus, 59, 62, 227 Corticium, 9 Cosmarium, 42, 44, 234 Cucurbita, 91, 94, 95, 96, 170 Cytoplasm Cystosira, 43 Cytinus, 111 DELPHINIUM, 164, 168 Dematium, 163 Derbesia, 42 Didymium, 139 Diospyros, 235 Draparnaldia, 42 Drosera, 160, 174, 175, 224, 227, 228, 233, 242 Drosophyllum, 175, 176, 240 ECHINOCEREUS, 49, 50 Elodea, 10, 17, 44, 52, 72, 78, 75, 86, 87, 89, 90, 92, 94, 95, 96, 101, 133, 154, 208, 242 Endomyces, 58, 62, 63, 66, 156, 157, 160, 197, 204, 207 Equisetum, 225, 235 Eremothecium, 64, 166, Eucalyptus, 181 Euglena, 40, 185 168, 231, 232, 238 FIcARIA, 101 Fossombronia, 225 Fritillaria, 127 Fucus, 11, 12, 48, 108 Fuligo, 13, 23, 24, 25, 60, 236 GaGEA, 234 Galactinia, 64 Geotrichum, 196 Gladiolus, 47, 210 Grimmia, 110 HELLEBORUS, 83, 238 Helodea, 228, 234 Hemitrichia, 60, 236 Hibiscus, 182, 183 Hyacinthus, 82, 84, 241 Hyalotheca, 44, 239 Hydrophilus, 11 Hypopitys, 181 ILEX, 154 Trisn 14ti woe 40 Do itis Gs 195 Sh, 91,, 100, 101, 136, 151, 152, 1538, 169, 206, 210, 211, 229 LAMINARIA, 168 Lathyrus, 183 Lemanea, 108, 109 Lemna, 129 Leptomitus, 59, 62, 230 Lilium, 47, 52, 81, 82, 838, 118, 167, 234 Limodorum, 111 Lonicera, 46, 47 Lunularia, 243 Lupinus, 104 MARCHANTIA, 226 Maxillaria, 45 Mertensia, 177 Mimosa, 178, 181, 235 Mnium, 224 Monotropa, 111, 164, 181 Mougeotia, 42, 52 Musa, 225 NeortTia, 46 Nephrodium, 241 Nicotiana, 233 Nitella, 233 Notothylas, 235 OEDOGONIUM, 42 4047 Plant Names Oenanthe, 170 Oenothera, 123 Oidium, 140, 179 Orobanche, 111 Oxalis, 181 PELLIONIA, 243 Pelvetia, 43 Penicillium, 58, 134, 135, 157, 179 Phajus, 45, 47, 49, Tl, %6, 77, 229 Philodendron, 205 Physarum, 60 Phytomastigoda, 232 Plasmodiophora, 60, 237 Pleurastrum, 156 Pleurococcus, 156 Polygonatum, 52 Polypodium, 240 Polytoma, 115, 118, 242 Polytomella, 115, 240 Polytrichum, 91, 200, 2381, 243 Prasiola, 156 Prunus, 183 Psaltiota, 59, 95 Pteridium, 105 Puccinia, 224 Pustularia, 58, 60, 62, 68, 71, 91, 92 Pythium, 240 RHEOSPORANGIUM, 227 Rhizopus, 11, 59 Rhodochorton, 43 Rhopalodia, 44, 234 Ricinus, 161, 170, 171, 238 Rosa, 48 Rubus, 182, 183 SACCHAROMYCES, 38, 130, 134, 178, 180, 196, 232 Saccharomycodes, 62, 136, 165 Saccorhiza, 54, 55 Salvinia, 107, 226 Saprolegnia, 17, 34, 59, 62, 63, 64, 67, 97, 98, 140, 141, 142, 100, 101, 102, 108, 132, 135, 139, 140, 141, 145, 155, 168, 178, 176, 180, 195, 196, 204, 226, 231, 232, 233, 237 Selaginella, 106, 107, 112, 218 Spermophthora, 206, 207 Sphaeroplea, 238 Spirogyra, 10) 135, 175) $8) 415 7435 44,525, 53; 86, 129, 167, 169, 226, 236, 242 Sporobolomyces, 59, 64 Sternbergia, 79 Strix, 191 Symphytum, 177 TRADESCANTIA, 10, 76, 125, 188 Tribonema, 197 Tropaeolum, 183 ULOTHRIX, 42, 156, 159 Ulva, 159 VALONIA, 38, 167, 225, 233 Vampyrella, 14, 63 Vanilla, 202 Vaucheria, 11, 15, 42, 52, 86, Verbena, 168 Vicia, 198, 199, 201, 241 107, 155, 185 WISTERIA, 182 ZEA, 131, 238, 243 Zygnema, 42, 44, 52, 226 Zygosaccharomyces, 134 8 posh ds | is pet © i hg Y | - ; Pal z_ oe \ J = reed i et Yk ee ey or eee ORE Ted ste rai wherr MOPARS ornse vie ; fait’ 8 8 nea, aise ty) TOE Oe SEE TES ' Aad tphcesere ates Lh ert present goaded ale le a be 3r} es $ 4 . yy eae ee SET cer) ‘ A A- Fr » us oars: verere Feeney ears o 837 »>, -* t YY Pee $a) Pa, DP) 6. 2 » iF) Dida yee ‘at : Yee TE Th Sesh Sev en Pil tear} ere : years aobiet , ry 4 4 8 a eieratl ies Ald ra heiee eee ot Pee ey fapabarutatats, & AIDI Se Siew mien ys aber sey : apete rR tars: ‘ i 3 Aft « etet es corel es Sy terete ees sats eip ee & PR adhe thes Viaberereis® seers ents Te ore Hitreket sarees eae yg 4 tals aiaiy x a Pm rare ar) wiring S sta OP tele eErce O18 jada) Dod bay 7 ao “INP E ele bk bree ar ph thee PSE EAE SEY He Wy Ba : cer seyrs Peas dat rind woe bates Piay Eee Pes vEar 24 o 8) Rd TEP wiogera ve Pe tt eee eee weet ere ee ere Selet <= rarer ss 4 M819) 0: 9/% ' Pewee eg tie es = Seem iacere ley eee . 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