BIOLOGY LIBRARY 6 PROTISTS AND DISEASE i r 1 . ,,,, PROTISTS AND DISEASE VEGETABLE PROTISTS; ALGAE AND FUNGI, INCLUDING CHYTRIDIINEAE ; VARIOUS PLASSOMYXINEAE, THE CAUSES OF MOLLUSCUM CONTAGIOSUM, SMALLPOX, SYPHILIS, CANCER, AND HYDROPHOBIA; TOGETHER WITH THE MYCETOZOA AND ALLIED GROUPS Being the Sixth Book published for the Author on the same Subject BY J. JACKSON CLARKE, M.B. LOND., F.R.C.S. SENIOR SURGEON TO THE HAMPSTEAD AND NORTH-WEST LONDON HOSPITAL, AND SURGEON TO THE ROYAL NATIONAL ORTHOPAEDIC HOSPITAL LONDON BAILLIERE, TINDALL AND COX 8, HENRIETTA STREET COVENT GARDEN 1922 (.All tights resetted) C15 BIOLOGY LIBRARY G FEINTED IN GREAT BRITAIN EEKATA. On p. vi. of Preface in line 5 "nucleus " should be "cell." On p. 109 mention should have been made that in 1892 Ivanovsky demonstrated the filtrability of mosaic disease in the tobacco plant. PREFACE A CHANGE of title from "Protozoa and Disease" of four preceding books to "Protists and Disease" has become necessary by reason of my having found that the pathogenic organisms dealt with are not protozoa, but protists allied to Synchytriaceae. In other words, in making comparative study of alien bodies that abound in the lesions of molluscum contagiosum, cancer, and other diseases, we have examined protozoa with great care when we should have given the closest attention to every detail of the life-history of certain vegetable protists, especially Synchytriaceae and some Olpidiaceae. But in spite of our having searched in the wrong depart- ment of biology the nature of 'cancer, smallpox, and other human diseases would before now have become plain to all, had there not been some weakness in the foundations of biology. The nature of this weakness is not obscure: it consists in a serious defect in the cell- theory, a defect the bearing of which is shown in chapters of this book, and which may also be briefly outlined here. If we bear in mind that the word "nucleus" denotes a morphological conception, the cell- theory in its present v a 2 582150 vi PREFACE accepted form may be defined as consisting of Virchow's " Omnis cellula a cellula" (1855) -plus Flemming's " Omnis nucleus a nucleo " (1882). The original theory comprised two propositions; firstly, that the nucleus is the unit of structure and function in both plants and animals; and, secondly, that new nuclei and new cells arise in an amorphous basis. The first part was at once recognized by Virchow as a great advance in science, but he did not attach importance to the second part, which indeed was later found to be untrue for the forms of life to which it was applied. In its present state as defined above the theory has proved to apply to nearly all known living things, and it has been the instrument by which so much imperishable biology has been built up that the fact that it is contradictory of dominant phases of well-known groups of organisms has not yet received due attention. It applies as far as is known to all Metazoa, and to all plants above the Thallophyta, and to most of these, and to many Protozoa ; but it is patently at fault when it is applied to dominant phases of certain protists, some thallophytic and others protozoan. That no qualifying clause has been added to the cell- theory to meet this position is strange seeing that game- togenetic chromidia are now commonplaces in biology, and that these vital processes translate terms such as "free nucleus-formation' 5 into prophecies fulfilled. PEEFACE vii In Nature variation is so great that misstatements concerning reproductive processes of some forms of life have proved to be accurately descriptive of what occurs in other forms. Denial of the existence of non-nucleated phases of some protists renders complete comprehension of many diseases impossible. In the following pages I show once again how the plasson state plays a leading part in the lives of the parasites which cause molluscum contagiosum, cancer, smallpox, &c. Had English pathology been conversant with Synchytrium from the year 1892 to the year 1895, the immediate causes of cancer, smallpox, and syphilis might have been agreed upon during that period, and, incidentally a new province of biology would have been then acquired. The first practical aim of this book is once more to beg those who in this country direct investigations into the immediate causation of disease to arrange for systematic examination of molluscum contagiosum. This minor malady, which shows in the skin like dimpled pearls, deserves more consideration than all the pearls in the world for the light it sheds on other far more serious diseases. If unprejudiced and willing examination of molluscum bodies is made in the simple way described in this book, it will be recognised in the course of a few days that these bodies are parasitic organisms. In molluscum we have an easy key to smallpox, the filtrable organisms, and to cancer. viii PREFACE Another aim has been to establish the biological relation- ships of the pathogenic organisms dealt with; and I trust that readers who are trained in the biology of cryptogams will condone the verbal and ideal stumblings of a beginner ; and that others, to whom the matter of this part of the book may be as new as it was to the author two years ago, will follow with sympathy and some profit the progressive, if limited, illumination that has been attained. In order to make this attempt possible, it has been necessary for me to consult literature outside that contained in our professional libraries, and I am under particular obligation to the officers of the library of the Linnaean Society of London, and of the Cryptogamic Botany Depart- ment of the British Museum. Both for direct information and for guidance in the choice of literature Mr. J. Rams- bottom's articles in the " Transactions of the British Mycological Society " have been indispensable to me. My indebtedness to colleagues and others who have assisted me with gifts of material and specimens is acknow- ledged in the text, but I must make additional reference to the skilled help generously given me by Dr. J. Herbert Perkins, pathologist to the Hampstead and North-West London Hospital. J. JACKSON CLARKE. LONDON, September, 1922. CONTENTS PAGE PREFACE v CHAPTER I. FOUNDATIONS. Meaning of the term protist The cell-theory Chroniidia Plasson Filtrable viruses Protoplasmic motion Culture of metazoan cells and tissues Chromatin and nuclei Intra vital staining Medicine a branch of biology Taxonomy Terminology Nomenclature CHAPTER II. SOME ALGAL AND FUNGAL PROTISTS. Algae Ulothrix zonata Vaucheria sessilis Cystococcus humicola Bhodochytrium spilanthidis Fungi Saprolengia Besting conidia Biologic forms of fungi Parasites of Saprolegnia Peronosporeae Pythium Parasites of Pythium Pythium debaryanum Nuclear processes in Pythium Phytophthora infestans Cystopus candidus Details of structure in oospores Nuclear processes of Cystopus Equivalence of a gerni-hypha to a brood of zoospores Zygo- mycetes Gemmae Parasitic mucors Nuclei of Mucor Some features of parasitic fungi Symbiosis Mitochondria 22 CHAPTEE III. CHYTRIDIINEAE. The genus Chytridium Bhizidiaceae Polyphagus englenae Cladochytriaceae Olpidiaceae Plasmodium-formation Sorus-formation Course of the disease Olpidiopsis Nuclear processes in Olpidiopsis Sphaerita endogena Synchytriaceae The life-cycle of synchytrians Synchytriurn stellariae ibid, succisae ibid, endobioticum . ... 54 ix x CONTENTS CHAPTER IV. PAGE THE SYNCHYTRIAN NUCLEUS. Nuclear divisions in the sorus Sorus formation in S. endobioticum The gametic stages in S. endobioticum A species of Synchytrium in a thistle The nuclear cavity The synchytrian nucleolus Plasson ; pycnoplasson, chasmatoplasson . . . . >, . . .78 CHAPTER V. CANCER-BODIES" IN SYNCHYTRIUM AND IN SYPHILIS. Bird's-eye nuclei in Synchytrium ibid, in cancer Living bird's-eye bodies in a syphilitic lesion " Cells of endogenous origin " . . . . . , . 91 CHAPTER VI. PLASSOMYXINEAE AND MOLLUSCUM CONTAGIOSUM. Definition of name Plasso- myxineae Plassomyxa contagiosa, cultures Water cultures Enumeration and details of vital changes in Plassomyxa contagiosa Not a Coccidium Microhenads Kinds of filter used for virus filtration Historical notes of virus filtration The size of microhenads The molluscum body not a symbiotic cell Anaerobic cultures The histology of the tumour Further cultural results Reaction to iodine and sulphuric acid Incubation period of filtered virus The identity of Plassomyxa with the filtered virus Synchytrium and Plassomyxa contagiosa compared The geo- graphical distribution of molluscum Avian molluscum or bird-pox Flagellate- diphtheria in pigeons Details of culture method . . .99 CHAPTER VII. PLASMODIOPHORACEAE. Subdivisions of the group Plasmodiophora brassicae Sorosphaera veronicae Spongospora scabies . . . . . . 128 CHAPTER VIII. MYCETOZOA. Broad features of Didymium and Stemonitis-The plasmodium Granules Protoplasmic motion The sclerotium Nuclear processes in Mycetozoa Fertilization Chromidial elements Bionomics A parasitic Mycetozoon Culture of a Mycetozoon Sporangia Morphological details 131 CONTENTS xi CHAPTER IX. PAGE ACEASIBAE, AFFINITIES AND PHYLOGENY OF MYCETOZOA 155 CHAPTER X. NOTES ON SMALLPOX, SYPHILIS, CANCER, ETC. Cystic ureteritis Olpidiiforma cobboldi A false analogy Smallpox Guarnieri's Experiment Mitoses in epithelial cells Variations in the parasites Cytoryctes, the cause of vaccinia Spirochaetes in vaccinia Intranuclear phases of Cytoryctes Nucleophaga and Karyorcyctes Parasitic forms occurring in Syphilis The parasite of syphilis and Synchytrium Spirochaetes Gummata Syphilis and cancer Cytoryctes luis Sarcoma and cancer Cancer in pathology Connective-tissue cancer Parasites that contain chromatin Spirochaetes in cancer Subdivision of the parasites A choriocarcinoma and some parasitic nuclei Conclusions and comparisons Epithelial cancer Spor- angium-formation in cancer Alternative modes of reproduction of cancer parasites Spirochaetes in cancer Culture of cancer-parasites Plassomyxa f orma-maligna ] mitation cancer Metastasis Conclusions Adenoma of the breast Hydrophobia Pasteur Nucleoli Vaccines and pro-vaccines Specific remedies for specific fevers ? Virchow Tumours in present-day pa,thology 164 CHAPTER XI. CHROMIDIA AND NUCLEI OF A PROTOZOON. Arcella Vulgaris, Life-history of ibid., Cytology of The cell-theory adjusted to facts 212 LIST OF ABBREVIATIONS, ETC 220 BIBLIOGRAPHY 222 INDEX . . ' . 227 PROTISTS AND DISEASE CHAPTER I FOUNDATIONS CIVILISATION still expects accredited instruction upon the real nature of cancer and other tumours ; smallpox and other fevers. If these capital diseases were but fugitive discomforts, it would still be of interest, even of importance, to inquire into their causes for the purpose of adding to existing biological knowledge. These facts demand that we review the rudiments of our biological information, and that we convert as much of it as circumstances require or allow into knowledge by personal practical work. Protists. We need to know some very primitive forms of life, some of those that are conveniently grouped in our minds under the designation of protists. This name is not now used in its original sense as invented by Haeckel, synonymous with " Monera," to apply to a proposed new kingdom consisting of organisms devoid of nuclei ; but as one designation for the two overlapping sub-kingdoms or 1 2 \ ^ t&OTISTS AND DISEASE phyla, the Protozoa and the Thallophyta, the latter term applying to the algae and fungi, including the diatoms, the desmids, and the bacteria. With Haeckel the Third King- dom was to be that of the Monera, and any non-nucleated cell was to be termed a cytode. The question of the Monera was touched upon in 1877 by Huxley, but at that date the chromidial state of living organisms was unknown and the data for even a partial solution of the problem were wanting. In 1896 the position was summed up judicioiisly by Delage and Herouard : " For a long time great importance was attached to the cytodes and the Monera of Haeckel . . . but it has been found that this supposed absence of a nucleus is explained in many cases by defective methods. Since nuclei have been discovered in the most of the Monera, of the cytodes, and even in bacteria, some people, by rather hasty induction it seems to us, have denied the existence of non-nucleated organisms." Remembering that filtrable organisms, chromidia, and akaryote phases of protists such as Sorosphaera (see Chapter VII) were unknown when the passage just quoted was written, we cannot but admire the moderation of thought and expression shown by the French zoologists. The Cell-Theory. A study of protists must include an examination of the cell-theory of life as it exists at present in biology. The nucleus was recognised as a normal feature in the epidermal cells of orchids by Robert Brown in 1833. FOUNDATIONS 3 In 1839 Schwann's view was stated thus : " The Cytoblas- tema, or amorphous substance in which new cells are to be formed, is found either contained within cells already existing, or else between them in the form of intercellular substance. ... In cartilage it is very consistent and ranks among the most solid parts of the body ; in areolar tissue it is gelati- nous ; in blood quite fluid. ... In plants, according to Schleiden, the nucleolus is first formed, and the nucleus around it : The same appears to be the case in animals." In 1841 Kemak described direct division of nuclei, and in 1873 Anton Schneider described indirect division (karyo- kinesis or mitosis). Coming to a more recent date I will refer, and I do so with affection and gratitude, to Edmund B. Wilson's " The Cell in Development and Inheritance," 2nd edit., 1904. It is to be remembered that when Wilson wrote generative chromidia had not been discovered. This premised, one passage may be quoted, p. 294 : " It may now be taken as a well-established fact that the nucleus is never formed de novo, but always arises by the division of a pre-existing nucleus." Now, many chromidia, such, for instance, as some of those of the protozoon Mastigella vitrea, arise for the very purpose of making nuclei de novo, which is the same thing as free nucleus -formation. Disagreement between facts such as this and the generali- sation just quoted are considered in Part III, 1912. Some points call for restatement. Let us glance again at pictured PROTISTS AND DISEASE examples of two modes of origin of new nuclei, and we see a great contrast. In Fig. 1 are seen three stages of regular mitosis in a protist : chromosomes, centrosomes, spindle, etc. In Fig. 2 new nuclei are being formed from a chromidium, a segregated mass of akaryote germ-plasm that has been extruded from the nucleus of its parent protist FIG. 1. STAGES OF MITOSIS IN ACANTHOCYSTIS ACULEATA, A HELIOZOON : a, metaphase ; b and c, anaphases. (From Doflein, after Schaudinn.) From Part III. and lies in the cytoplasm, equivalent to a metazoan ovotestis. In the vegetative part of its life Mastigella undergoes mitosis quite like that of Acanthocystis, Fig. 1. Chromidia. An akaryote structure destined to produce gametes illustrates one kind of chromidium, the generative ; the other kind is the vegetative. Generative chromidia have been recognised (H. Wager) as of two kinds ; firstly, those that replace nuclei, and secondly, those that are capable of being re-formed into nuclei. Of the latter the garnet o- genetic chromidium of Mastigella is an example ; in this FOUNDATIONS 5 case the origins of the new cells, Fig. 2, 2, Chr., are not differentiated into nucleus and cytoplasm. Vegetative chromidia are again of two kinds ; firstly, portions of a nucleus extruded diffusely into the cytoplasm, and secondly, chromatin accumulated in nucleus-like structures. FIG. 2. MASTIGELLA VITREA : 1. Formation of the chromidium (Chr.). 2. The chromidium increased in size. 3. The chromidium alone more highly magnified, to show free nucleus -formation. N, nucleus. G, gamete. (After Goldschmidt, from Doflein.) From Part III. Once it is fully developed the chromidium, Fig. 2, Chr., is an independent organism much as is the prothallus of a fern. If we could suitably subdivide it, each segment would doubtless live and be able to produce gametes. The foundation of all biology and pathology, the cell- theory, as it exists to-day, is unsound in that it is contra- dictory of akaryote states which are dominant in the lives of certain protists. Plasson. If we look at Goldschmidt' s drawing, Fig. 2, 2, of the origins, or primordia, of the gametes of Mastigella, 6 PROTISTS AND DISEASE when first they appear in the matrix of the chromidium, we can see in them no differentiation of structure ; each gametoblast is a particle of apparently structureless material. For such substance Haeckel used the term " plasson." This totipotential form of living matter has been defined in an article on the Proteomyxa by S. J. Hickson : " It seems probable then that the protoplasm of the Proteomyxa really represents the protoplasm of the higher Protozoa and Metazoa plus the substance of the nuclei. It is the substance which van Beneden, in 1871, proposed to call the ' plasson,' that is, the formative substance which is capable of becoming, either in ontogenetic course or in phylogenetic course, monocellular elements, after that the chemical elements of the plasson have been separated to constitute a nucleus and a protoplasmic body. '' Haeckel was right in that non-nucleated organisms exist, but those we know have also phases in which they have nuclei ; therefore he was mistaken in assuming that his cytodes were of necessity devoid of nuclei at all stages of their life-history ; indeed, many of them have been found to have nuclei at all stages. In estimating Haeckel' s work we should remember that he was the first to note the inception of foreign particles by leucocytes ; the bearing of this function on immunity was first indicated by Carl Roser in 1881, and later named phagocytosis by Metchnikoff. FOUNDATIONS 7 Filtrable Viruses. The living virus of foot-and-mouth disease was found by Loeffler and Frosch (1898) to pass through filters which arrest any known bacteria. Since that date a number of other living poisons have been found to be filtrable, including those of measles, smallpox, and molluscum contagiosum. Many pathologists now write with facility of " invisible micro-organisms," so it is of value to examine this very important field. M. Arthus, in a most interesting book published last year, writes thus : " We admit that the causal microbe exists, but suppose that it is so small that it eludes microscopic search, and this hypothesis for it is nothing more than a hypothesis is supported by the facts elicited in the study of pleuro-pneumonia of cattle." The author goes on to say that in the serous exudation of this disease fine granules can be seen with the highest powers of the microscope ; they can be stained, though with difficulty, and they can also be cultivated on very special media. Arthus asks whether we may not see in the virus in question a link between the visible and cultivable organisms on the one hand, and the invisible organisms on the other hand. We find no word here of these most minute of known living units having any phases other than the filtrable. And this is the usual view, though it is proved to be an error by what is seen in simple water cultures of molluscum bodies : see Chapter VI. 8 PROTISTS AND DISEASE It is as if a new " Third Kingdom " were being erected to replace that of the Monera. Just as there are cytodic (plasson, chromidial) stages of some protists which have obvious nuclei at other stages, so there are filtrable stages of parasites such as molluscum corpuscles, which are relatively large bodies; and, just as some living organisms may con- ceivably be persistently akaryote, so others may possibly exist only in the filtrable phase. Protoplasmic Motion. " The colloidal nature of proto- plasm is manifested in many of its properties. Its power of adsorption which lies at the basis of many cell reactions and certain staining processes, is similar to that of other colloids. . . . The permeability of a vacuolate cell is in general the resultant of the permeabilities of the ectoplast, cytoplasm, and tonoplast" (L. W. Sharp, 1921). The chief cause of stability in colloids is Brownian movement (Bayliss, 1920, q.v.). This movement is seen in many living cells, e.g. in the protoplasmic strands of Spirogyra. Streaming is probably present in the protoplasm of all active cells. It was first recorded in Chara by Corti in 1774. In some cases, as in the cells of the stamina! hairs of Trad-escantia, the stream does not carry the nucleus with it ; in other cases, as in the leaves of Vallisneria, the nucleus moves with the rest of the protoplast. Most impressive are the to-and-fro currents of the plasmodium of Mycetozoa : in these the outward current lasts over a minute, the return a little under a minute. In FOUNDATIONS 9 Fig. 38 is copied de Bary's instance of a rotatory movement replacing the alternate currents in a mycetozoan plasmodium. In a subsequent chapter an account is given of a streaming or circulation of protoplasm in the bodies peculiar to the disease molluscum contagiosum. This streaming may last for several days and may be accompanied by the presence of bud-like protrusions and of separate minute spheres with a similar streaming. Apart from other evidences in molluscum bodies this streaming is a strong witness to life : and it is a phenomenon any one who may have the material can reproduce. Besides such movements of protoplasm of a cell there are currents in channels or linear vacuoles : watching a large fungus hypha growing in a slide-and-cover culture I have seen in minute parallel channels steady currents pass some in the direction of growth, others the opposite way ; one such hypha was undivided when first seen, later septa were formed, but these did not appear to affect the circula- tion. By fixing root-tips in a mixture of neutral formalin (10 c.c.), K 2 Cr0 4 (2-5 g.), HgCl 2 (5 g.), H 2 (90 c.c.), Bensley demonstrated the channels he named canaliculi, the presence of which is an indication that intracellular circulation of liquids exists. In mitosis the centrosomes and the two spindle poles have been found to be alike electrically, and, if so, they cannot represent an ordinary electromagnetic field. The filament that connects a nucleus and the 10 PROTISTS AND DISEASE blepharoplast at the base of many flagella suggests that the movement of these is effected by the same apparatus as mitosis. As is shown in Fig. 22, 6, zoospores of synchytrians penetrate their host by a nuclear structure which resembles a blepharoplast with its connecting filament ; other ehytri- dians and, as Waterhouse found, the sporidia of Puccinia graminis, appear to effect penetration in a similar way. Simulation of Life. In his book, " The Protozoa," 1901, Calkins gave a good account of the life- simulating states of dead matter : amoaboid movements simulated by a drop of olive oil in water ; Buetschli's mock-protoplasm, olive 011 mixed with salt or sugar in water ; Rhumbler's shellac- coated spicule of glass incepted by a drop of chloroform in water and ejected when the shellac is absorbed, with many other instances. I have made Buetschli's experiment both with salt and oil, and sugar and oil. A few crystals that project from the oil into the water are seen to dissolve, causing active currents of very brief duration ; and slow local passive currents are seen in both oil and water ; there was nothing that should be mistaken for active living matter. Culture of Metazoan Cells and Tissues. Every cell of a living metazoon is an animalcule, and with minute care to exclude bacteria (see Lee's " Microtomist's Vade Mecum," 1921) such cells may be kept alive and cultivated artificially. Nerve-cells from the embryo frog were first grown by Ross FOUNDATIONS 11 Harrison in 1907, the medium being lymph from adult frogs. Next Burrows grew tissues from embryo chicks on blood plasma in plate cultures. Using the same method Carrel and Burrows cultivated tissues (kidney, spleen, bone- marrow, etc.) of adult cats and dogs. Connective-tissue from the embryo chick has been kept in culture for as long as eight years by Ebeling. An incuba- tion of three days was observed by Busse before the cells of fragments of rabbit's heart or aorta developed new cells. The latter are branched and anastomose, but when bacteria are placed near them they become detached from one another and round ; they are attracted to the bacteria (chemiotaxis) (see the Lancet, April 29, 1922, p. 856). All these features are in keeping with what is familiar in the study of granulation-tissue by other methods. When the infecting organisms of molluscum contagiosum and kindred affections are more generally appreciated, such cell-cultures in practised hands may be useful in the study of some details of the stages of infection. Cellulose and Chitin. Chemically isomeric with starch, cellulose is one of the most abundant of the products of life. Like starch it grows by intussusseption, and from it are made a number of degradation products such as the jelly so freely produced by many algae, etc. In the form of cotton- wool it responds readily to the usual tests, turning blue with iodine solution followed by sulphuric acid diluted with water ; equal parts (Bower) or one of iodine solution to two of 12 PROTISTS AND DISEASE acid (Chamberlain), also with chloro-iodide of zinc and iodine ; and dissolving in saturated solution of copper hydrate. Where fungi are present one might expect these reagents would reveal them by colouring their cellulose, but the re- actions are of value only when positive. It is rare to find fungi that respond. Fungal cellulose often requires long maceration in 15 per cent, potash solution before it may react. In Mycetozoa de Bary obtained the reaction but once in the innermost layer of the sporangium wall of Trichia varia, and had many negative results. The capillitia of Didymium and Stemonitis changed to violet with the acid alone. Woody fibre is composed chiefly of lignin, a modification of cellulose, but does not react in the same way. In pro- tozoa, chitin, a nitrogenous substance akin to keratin, takes the place of cellulose, though one Rhizopod, Chlamydomyxa, is partially enclosed in a capsule of cellulose. Cellulose also forms the tunic of Ascidians, animals near the vertebrates in organisation. Chromatin and Nuclei. The living nucleus can be studied easily in many protists and cells. It contains refracting material which stains readily ; hence called chromatin (Flemming, 1879). When this is not diffused through the whole nucleus we see in fixed preparations that it lies in a net which is continuous as a rule with the cyto- plasm through the nuclear membrane. This supporting net is called linin, doublet of linen (Schwarz, 1887). FOUNDATIONS 13 In mitosis spindles are formed by re-arrangement of linin fibres. Spindles are usually formed inside, but some- times outside the nuclear membrane. The substance that separates two nuclei after mitosis comes from the linin spindle. In the following pages where my own observations are referred to and it is not otherwise stated, cells, plasmodia, or tissues were stained with acid haematoxylin after fixation by Foa's solution: see note at the end of this book. With this stain pure chromatin is of a deep blue colour. Ehrlich divided dyes into basic and acid according as the colouring matter plays the part of an acid or a base the compound employed (Wilson). Ehrlich's statement only applies to cover-glass preparations dried and fixed by heat without the use of reagents (Lee's " Microtomist's Vade Mecum"). Heidenhain termed the chromatin of Flemming " basi- chromatin " to distinguish it from nucleoli, cytoplasm, and other parts of cells which take acid dyes, such as acid fuchsin and eosin. He concluded that basichromatin is rich in nucleic acid, which Bayliss defines as a compound of phosphoric acid with a 5-carbon sugar and a purine derivative. The staining reactions of nuclei of the same organism may vary : thus Barrett found in the chytridian Olpidiopsis Saprolegniae that after the gonoplasm has passed into the oogonium the nuclei in the resulting oospore stained more deeply with safranine than they did before fertilisation. 14 PROTISTS AND DISEASE As Wilson points out, there is no specific stain for chromatin. Similar staining reactions do not necessarily mean chemical identity ; both the nuclei and matrix of cartilage stain alike with methyl green. Staining reactions have their value and we should be able to make use of every technical resource ; at the same time we must not forget that a process which reveals one feature brilliantly will conceal others of equal importance ; nor that technique is a means, not an end. Bodies suspected of being protists should invariably be first carefully examined unstained in conditions as nearly as possible like those in which they are discovered, and then in water. In this way alone can we let them show by their own activities if they are living organisms. They should also be tested by attempting cultures in water as described below in Chapter VI. Intravital Staining. Methylene blue, neutral red, and other dyes are used to stain living tissues and organisms. Their applicability is limited ; it is generally agreed that when a nucleus takes a stain it is dead. Neutral red stains granules, and it has the advantage of being a test of acid or alkaline reaction, becoming brighter red with the former, yellow with the latter. It is a basic dye, its name referring to its tint. Janus green (Hoechst) has been found to stain the mitochondria of living human lymphocytes ; mitochondria, being soluble in alcohol, chloroform, and ether, are destroyed by the ordinary processes. FOUNDATIONS 15 Medicine a Branch of Biology. Members of the medical profession have direct access to a wide field of biological experience and material from which the academic biologist is debarred. Our daily study of our patients is a biological occupation. Each advance a doctor makes in skill is a biological achievement. If such advance is a discovery new to his science, and is published, it is a contribution to the humane applied biology we call Medicine. " Pathology is by no means the smallest branch of that beautiful science biology," Virchow wrote in 1855. To have in the foundations of our consciousness the well-established principles of general biology is necessary to every medical practitioner. But principles cease to guide if they are not revived from time to time by personal experi- ence ; to spend an occasional hour or two watching with the aid of a microscope living algae, fungi, Mycetozoa, etc., brings real refreshment. In order to be able to share any new knowledge we may gain in matters that pertain to general biology we must use terms that cannot be misunder- stood ; for this purpose we must learn the rules of technical biological conversation, and by so doing we shall obtain for ourselves the key to the treasures stored in systematic biological literature. The responsibility of instructing the rest of the community on the subject of disease must be ours. Ours also is the duty of investigation ; lay biologists have voluntarily come to our aid from time to time ; and too often from being untrained to know or to counter the 16 PEOTISTS AND DISEASE dangers that they ran, their zeal has cost both them and us their lives. Classification or Taxonomy. Any collection of objects can be classified in various ways ; e.g. by size, shape, or colour. Classification is abstract, artificial : as Delage and Herouard have written, " Only individuals exist." Ai] the same we know that without systems of grouping there could be no natural science. On this subject Huxley may be quoted : " Each such assemblage is in fact a ' natural order ' in the sense in which that word is used by botanists, and although the number of these natural orders may be increased by the discovery of new forms, or diminished by the ascertainment of closer bonds of union than are at present known to exist between the orders already dis- criminated, yet the morphological types which they repre- sent will always remain, and therefore the knowledge of their characters, once acquired, will be a permanent possession.'' As far as knowledge allows organisms are grouped according to the stock they come from ; their stirpes, as John Ray (1628-1705) called them. Ray was the first to distinguish in flowering plants the two groups, mono- and dicotyledons. After the publication of Darwin's " Origin of Species " in 1859, classification became more consciously guided by phylogeny. The immense array of living organisms is first broadly FOUNDATIONS 17 separated in our minds into the two kingdoms of plants and animals respectively. The animal kingdom is divided into two sub-kingdoms or phyla, the protozoa and the metazoa. Botany has no group that corresponds exactly to protozoa, indicating a division in which the greatest structural differentiation is reached in unicellular bionts. The term protophyta was used by Sachs to denote a joint group of algae and fungi ; blue-green algae (Cyanophyceae) like Chroococcus, Nostoc, &c., and green algae (Chlorophyceae) such as Pleurococcus ; together with the bacteria and yeast-fungi. The Thallophyta in botany are a section of the cryptogams, and include all plants lower in organisation than the mosses. The body of such plants is termed the thallus (Gr.^green bough). The vegetable protists include Sachs's Protophyta and filamentous algae such as the Ulotricheae, the Conjuga- teae (Zygnemieae, desmids, and diatoms), and others such as Vaucheria, which produce oospores, together with the corresponding fungi, Phycomycetes, and a number of other groups of algae and fungi. The commoner grouping of organisms in descending scale is into classes, orders, genera, and species ; secondary groups being intercalated as affinities require. The terms genus and species as defined by Linnaeus are unchangeable ; as regards the remainder custom varies with different authors, but the terms class and order are fairly constant in application. In botany names of orders are Latin adjectives ending 2 18 PROTISTS AND DISEASE in -ae with " Plantae " understood ; e.g. Rosaceae^ompositae. For cryptogams the ending -ales is also used. The word family was first used as equivalent to order ; so by J. H. Balfour (1855), and so in the British Museum ' Handbook of British Lichens,' 1921 ; but in Bennett and Dyer's Sachs (1875) we read " Smaller groups which are called families," and some writers still follow them. The class is always a wide group. All the Protozoa are made into only 4 classes : Rhizopoda, Flagellata, Sporozoa, and Ciliata. All the plants fall into 4 great groups : Phanerogamae (flowering plants), Pteridophyta (ferns &c.), Bryophyta (mosses &c.), and Thallophyta (algae, fungi, lichens &c.). The secondary groups of Thallophyta: algae, fungi, lichens, Mycetozoa, &c., constitute each a class. As to the proper term to apply to the next subdivisions sub- class is obvious ; between it and the order groups are variously named ; series (desinence -eae) and sub-series (desinence -ineae) are convenient terms. In grouping the protists the Mycetozoa must be given a central position as being about equally animal and vegetable in constitution, and we may arrange on one side of them a series of vegetable, and on the other side a series of animal protists. Terminology. The names used for different phases and organs of protists are complicated by some groups being included both in botany and zoology. The well-known Volvox is an alga in botany, a phytoflagellate in zoology. FOUNDATIONS 19 In botany its motor organs are termed cilia, in zoology flagella. There is no essential difference between the organ indicated by cilium (eye-lash) and flagellum (whip-lash), but it is useful to use words to distinguish the single or few motor organs of the Flagellata from the relatively smaller and more numerous organs of the Ciliata. In this book the word flagellum will be used for motor organs of the zoo- spores of algae, and Phycomycetes,